CN107058339A

CN107058339A - Soybean GmCIB1 genes and GmCRY2 genes and its regulation and control bloom and aging effect

Info

Publication number: CN107058339A
Application number: CN201710260476.2A
Authority: CN
Inventors: 李宏宇; 孟颖颖; 刘斌; 赵涛; 刘军; 林辰涛
Original assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Priority date: 2013-10-12
Filing date: 2013-10-12
Publication date: 2017-08-18
Also published as: CN104561023B; CN104561023A

Abstract

The invention discloses soybean GmCIB1 genes and GmCRY2 genes and its regulation and control bloom and aging purposes.In particular it relates to have SEQ ID NO:Sequence soybean GmCIB1 genes and GmCRY2 genes, the DNA molecular comprising the gene, carrier, the protein of the gene code and related transformed cells and genetically modified plants shown in 1 or 3, the method for further relating to application and cultivation related transgenic plants of the gene in regulating and controlling plant leaf aging and blooming.

Description

Soybean GmCIB1 genes and GmCRY2 genes and its regulation and control bloom and aging effect

The application is the applying date on October 12nd, 2013, Application No. 201310490925.4 and entitled " big Beans GmCIB1 genes and GmCRY2 genes and its regulation and control bloom and aging effect " application for a patent for invention divisional application.

Technical field

The present invention relates to biological technical field, more particularly to soybean GmCIB1 genes and GmCRY2 genes and its application.

Background technology

In nature, plant senescence research has turned into one of major issue that Botanic life science is studied.Thimann et al. Propose that plant senescence is a series of degenerative process (Thimann, 1978) in growth and development process within 1978.After more than ten years Roach proposes that aging is growth with plant age, physiology course that existence and fecundity are gradually reduced (Roach etc., 1993).1997, Nooden this concept has been carried out it is perfect, propose aging be the orderly of plant soma programmed death Degradation process (Nooden etc., 1997).As plant other aging events, leaf senile is the final stage of development, finally Blade is dead, therefore limits life cycle and the life-span of blade.

Illustrating for leaf senile regulation and control process contributes to us to understand aging phenomenon and the biology original of plant life cycle Reason.The exploration of the molecular mechanism of plant leaf blade aging influences great in application of biological engineering, for example, improve after plant products, receipts Storage, stress tolerance.

So far, substantial amounts of SAGs is found in different plant species by chip analysis.Many genes may encoding regulator The factor, these regulatory factors are probably signal inductor and the part of conductor, such as transcription factor and receptoroid kinases. The further research of these controlling genes can be some important senescence regulatory genes and be found while also regulating and controlling for leaf senile Mechanism Study provides foundation.In WRKY families, function related to leaf senile with WRKY6 WRKY53 is furtherd investigate. WRKY53 is raised in aging early expression but declined in later stage experssion amount, shows that WRKY53 plays regulating and controlling effect in aging early stage (Hinderhofer K,2001).The target gene that WRKY53 includes other SAGs is that stress-related genes and transcription factor include Other WRKY factors.The aging of WRKY53 knockout mutations bodies is delayed by, and is induced WRKY53 to be overexpressed and then produced early ageing, and this shows WRKY53 is the positive regulator (Miao Y, 2004) of aging.Visited by the research of the Senescence manipulation path mediated to WRKY53 Rope WRKY53 direct downstream effects target gene.Another WRKY family transcription factor WRKY6, is invaded in leaf senile and pathogen The expression quantity of its in dye significantly raises (Robatzek S, 2002).WRKY6 is combined come controlling gene by the W-box with promoter region. The receptoroid kinase gene SIRK that many WRKY6 related genes include aging induction is relevant with aging and pathogen response.Although WRKY6 is worked in pathogen defence and aging, but SIRK is only expressed in ageing phase.WRKY6 knockout mutationss can change SAGs expression but the influence to aging is not obvious.Because the change that SAGs is expressed in knockout mutations body may be not enough to pair Leaf senile change plays a significant role.It is also likely to be the functional redundancy produced due to WRKY family members.

NAC albumen is one of distinctive maximum transcription factor family of plant, the family of this in arabidopsis just have more than 100 into Member.NAC family genes are responded in plumule and the meristem development of young shoot, the formation of lateral root, Auxin Signal Tranducation and defence In work.The expression of 20 NAC transcription factors dramatically increased in the aging of naturally-aged and dark induction (Guo Y, 2006).NAP encodes a NAC families transcription factor, and T-DNA knockout mutations bodies AtNAP shows senescence-delaying patterns.It can be seen that AtNAP is the positive regulator of leaf senile.AtNAP homologous gene also up-regulated expression in ageing leaves in paddy rice.NAC families Homodimerization and Heterodimerization are the important molecule mechanism that the transcription factor regulates and controls growth course between member, and these mechanism The leaf senile regulation and control of NAC transcription factor mediation are also assisted in.The signal that the functional characteristics of these genes includes their participations leads to The research of road and controlling gene is very valuable for the annotation of leaf senile complicated molecule path.

Another typical SAGs is autophagygene, and its function has been studied in vivo.Arabidopsis autophagygene T-DNA is inserted Enter mutant, AtAPG7, AtAPG8, AtAPG18a show early ageing phenomenon (Doelling J H, 2002；Hanaoka H, 2002；Xiong Y,2005).The mesotrophic utilization rate of aging course is than the component needed for relatively low or aging in these mutant Do not provided effectively, so as to cause aging to shift to an earlier date.

The signal way of the history in flowering of plant regulatory pathway research existing more than 100 years, at present at least four regulation and control of blooming Footpath is determined, i.e. Photoperiod pathway, vernalization approach, autonomous pathway and gibberellin pathway (Amasino, 1996；Bernier, 2005)。

Influenceed in growing process by many external environments, just one of important environmental factor.Mainly have in plant Two kinds of light receptors experience the signal of feux rouges/far-red light and blue light respectively.Light receptor progress in arabidopsis is more protruded, mesh The light receptor being had found in preceding arabidopsis：(1) light receptor for absorbing feux rouges/far-red light (wavelength is 600~750nm) is photosensitive color Plain (phyA, phyB, phyC, phyD, phyE)；(2) cryptochrome of blue light/UV-A (wavelength is 320~500nm) is absorbed (CRY1, CRY2), image assesment (PHOT1, PHOT 2) and LOV/F-box/Kelch domain albumen (ZTL, FKF, LKP2)； (3) UV-B (wavelength is 282~320nm) acceptor UVR8.These light receptors, optical signal is experienced jointly, and cooperateed with photosynthetic pigments Regulating development of plants and energy production.

Plant responding blue light before twoth century early in just being had been reported that, Ahmad and ashmore is utilized in arabidopsis within 1993 T-DNA insertions obtain the insensitive mutant hy4 of a class blue light.Later screening cDNA library be separated at first first blue light by Body-cryptochrome 1 (CRY1).1996, arabidopsis cryptochrome 2 (CRY2) was also separated and obtained.It also found in arabidopsis CRY3 (Kleine etc., 2003 without photomorphogenesis function；Huang etc., 2006).

Subsequent cryptochrome is accredited in other plant and other species such as bacterium, animal even human body.Not jljl Although the homology of inter-species cryptochrome has difference, but its function and arabidopsis are much like.That is its N-terminal sequence and DNA photolyases Very high homology, mostly exercises the function of light receptor.Cryptochrome can be divided into：CPD photodestruciton enzymes, 6-4 photodestruciton enzymes, plant hidden flower Pigment, five subfamilies of animal cryptochrome and CRY-DASH.CPD photodestruciton enzyme, that 6-4 photodestruciton enzyme repairs cyclobutane respectively is phonetic Pyridine dimer, pyrimidine-pyrimidine ketone photoproduct (Sancar A, 2003).Research thinks that Plants and Animals cryptochrome does not have at present There is photodestruciton enzymatic activity.CRY-DASH albumen can be directly bound DNA or RNA, but without the activity of photodestruciton enzyme, can pass through Controlling gene transcription regulates and controls growth course (Hitomi K, 2000；Brudler R,2003；Worthington E N, 2003).But also it was reported that external CRY-DASH albumen, including arabidopsis CRY3, the pyrimidine two in single stranded DNA can be repaired Aggressiveness (Huang Y, 2006；Selby C P,2006；Klar T,2007).

Its function of the structures shape of protein.Cryptochrome has two main domains, and N-terminal is similar to photolyase PHR areas and the extension area CCT of C-terminal different aminoacids length.PHR domains are very high with photolyase similitude, can bind hidden pattern (Cashmore etc., 1999 in chromophore and participation homodimerization reaction in element；Lin and Shalitin, 2003；Lin and Todo, 2005), CCT domains can be the effect area of cryptochrome by the interaction with protein come communicating optical signals.

Cryptochrome is considered as by photolyase evolves (Sancar A, 2003).Photodestruciton enzyme is the big Huang of a class Fibroin family can repair pyrimidine dimer (Todo etc., 1996).Different due to its substrate specificity can be divided into again：Repair ring The CPD photolyases (i.e. photolyase) of butane pyrimidine dimer and the 6-4 photolyases for repairing 6-4 pyrimidine-pyrimidine photoproducts.

The photolyase having now been found that containing two chromophoric groups i.e. catalytic group-riboflavin (FAD) and catches light group-tetrahydrochysene Folic acid MTHF or 8-HDF.Photodestruciton enzyme can be incorporated on DNA pyrimidine dimers, and this combination is independent of light.Absorb a light After son, photolyase starts light reparation reaction.

Although the PHR domains sequence of cryptochrome and photolyase are closely similar in primary structure, by two-part structure domain Constitute：N-terminal α/β domain and C-terminal α helical domains, the two domains are connected by Loop rings.But both higher structure difference It is very big, therefore there is very big difference (Brautigam etc., 2004 in function；Huang etc., 2006), there is photolyase light to repair work( Can, cryptochrome can not be combined the function of also not repairing pyrimidine dimer with DNA.

Cryptochrome is mainly reflected in the following aspects with the interstructural difference of photolyase：(1) ATP binding abilities.Light DNA, and cryptochrome combination ATP can be combined by solving the FAD lands of enzyme, and the former mainly exercises the work(of reparation pyrimidine dimer Can, and the latter ATP is penetrated into the nest of FAD passages, and phosphate group is close to PHR surfaces, and such conformation is conducive to hidden flower The phosphorylation reaction that pigment blue light is relied on.(2) the electrically charged difference of surface institute.How negatively charged cryptochrome surface is, and photolyase It is more positively charged, thus the latter can combine negatively charged DNA (Huang etc., 2006), the difference of surface potential is explained just Why not cryptochrome possesses the activity of photodestruciton enzyme DNA plerosis.The surface of cryptochrome negative electrical charge is further supported so One hypothesis, the cryptochrome CCT domains Hui YuPHR domains of phosphorylation are separated after illumination, cause the change of conformation, specific site it is sudden and violent Dew so that the protein-protein interaction for relying on blue light is produced, so that communicating optical signals.In addition, PHR areas are except that can catch light Homodimer can also be formed outside signal (energy), this normally functioned to cryptochrome most important (Sang etc., 2005； Yu etc., 2007a).

Cryptochrome has an extension area i.e. CCT domains in C-terminal.In different plant species cryptochrome CCT sequences difference compared with Greatly, but they contain a common motif-DAS motif (DQXVP-Adidic-STAESSS), this motif is also an identification The important mark (Lin and Shalitin, 2003) of cryptochrome.CCT domains are the function effects of plant and animal cryptochrome Area is answered, they can provide the plasticity of structure for signal protein, the structure of change can provide more albumen action sites, or The specificity or compatibility of effect, can recognize different signal member or be recognized by different adjustment albumen, therefore in cellular localization With played in protein-interacting it is very important effect (Ahmad etc., 1998b；Yang etc., 2000；Wang etc., 2001；Lin And Todo, 2005).

The phosphorylation and degradation pathway that plant cryptochrome blue light is relied on are described in detail below：

(1) Phosphorylation events that arabidopsis cryptochrome blue light is relied on

Arabidopsis cryptochrome CRY1 and CRY2 can be phosphorylated, and it mainly has following feature：

1) the special Phosphorylation events of blue light.By arabidopsis etiolated seedling from dark be transferred to blue light (>15μmol m-2s-1) Under can detect the phosphorylation of cryptochrome in a short time, and phosphorylation activity can't detect in etiolated seedling.Cryptochrome The special phosphorylation of blue light, increases therewith with the increase phosphorylation degree of blue light strength and light application time.It is transferred to feux rouges Or phosphorylation activity is can't detect in the etiolated seedling under far-red light, returned again to after the processing of etiolated seedling blue light under feux rouges or far-red light Understand dephosphorylation (Shalitin etc., 2002,2003 quickly；Yu etc., 2007b).

2) Phosphorylation events occur in cryptochrome C-terminal.Experiment in vitro is shown, from the arabidopsis of expression in escherichia coli Phosphorylation in vitro detection is carried out after CRY1N ends and C-terminal albumen are purified, as a result shows that C-terminal has an obvious phosphorylation signal, and N End does not have；Phosphorylation can also be reduced (Ahmad etc., 1998b) after total length CRY1 C-terminal mutation.In vivo studies shows, transgenosis table Cause arabidopsis continuation photoresponse up to GUS-CCT fusion proteins, and GUS-CCT is also by the transgene expression of lasting phosphorylation GUS-CCT fusion proteins cause arabidopsis continuation photoresponse, and GUS-CCT be also by lasting phosphorylation (Ahmad etc., 1998a；Yu etc., 2007b).Transgenosis is overexpressed GFP-CRY2 fusion proteins has the phosphoric acid for relying on blue light in arabidopsis body Change phenomenon, it is consistent with endogenous CRY2.Being overexpressed CRY2-GFP arabidopsis then has continuation photoresponse, while the albumen is also gathered around There are the Phosphorylation events of continuation.Transgenosis CRY2-GR albumen is positioned in kytoplasm in the case of no dexamethasone, CRY2-GR is not phosphorylated, and the CRY2 of overexpression can not recover the phenotype of mutant, has been added after dexamethasone processing, GR's Lead lower CRY2 to navigate in nucleus, can be phosphorylated, and with physiologically active, recover the normal phenotype of mutant (Tessadori F,2007)。

Scholars speculate that the cryptochrome CCT surface negative charges of phosphorylation have broken the interaction with PHR, so as to influence Cryptochrome and interaction (Yu etc., 2007b of signal member；Yang etc., 2000；Liu etc., 2008；Yu etc., 2009b).

3) autonomous phosphorylation.Arabidopsis cryptochrome CRY1 does not have sequence homology with protein kinase, but shows in vitro Autophosphorylation activity is shown.At present there is opposite result in the research for the external autophosphorylations of arabidopsis cryptochrome CRY1, Studies have reported that it is blue light dependence, blue light reinforcement, also studies have reported that it is not dependent on (the Shalitin of blue light D,2003；Bouly J P,2003；Ozgur S,2006).Bypass and whether rely on blue light, at least these experimental results are all demonstrated Arabidopsis CRY1 has autophosphorylation activity in vitro.Whether arabidopsis CRY1 autophosphorylation influences CRY1 phosphoric acid enough Change, or CRY1 relies on the phosphorylation of blue light and also needs to the auxiliary of other kinases and be also not very clear.Currently without on arabidopsis The report of CRY2 autophosphorylations.

(2) key character that arabidopsis CRY2 albumen relies on the degrading activity signal protein of blue light is exactly the fast of albumen Speed metabolism is so that the state that organism is returned to before signal stimulus.The fast degradation class for relying on feux rouges is undergone with phytochrome phyA Seemingly, cryptochrome CRY2 also undergoes the fast degradation for relying on blue light, and is by ubiquitination/26S proteasome pathway (Shalitin etc., 2002；Yu etc., 2007b), and cryptochrome CRY1 does not possess degrading activity (Lin, 1998 for relying on blue light； Ahmad M,1998).The degraded for having studies have shown that cryptochrome CRY1 in soybean depends on blue light, and cryptochrome CRY2 Non-degradable under blue light (Zhang etc., 2008).Arabidopsis CRY2 degraded depends on FAD, when CRY2 is mutated (CRY2D387A) Lose after the ability for combining FAD, the albumen of the mutation no longer undergoes the degradation process of Induced by Blue Light (Liu etc., 2008).CRY2's Degraded needs the blue light illumination of lasting high light intensity, and the degrading activity that CRY2 relies on blue light needs PHR domains and CCT domains simultaneously (Ahmad M,1998).It is consistent with this conclusion, continue the GUS-CCT2 non-degradable under blue light (Yu, 2007) of phosphorylation.CRY2 Degraded it is relevant with E3 ligases COP1 (Wang, 2001), but COP1 is not the unique E3 ligases of CRY2, because CRY2 Degrade in the mutant cop1-4 and cop1-6 that COP1 functions are reduced by partial impairment, and in the mutation of COP1 amorphs (Shalitin D, 2002) is wholly absent in body cop1-5.These results imply that CRY2 degraded is possible to also involve To other E3 ligases.

Arabidopsis cryptochrome CRY2 phosphorylation and degraded are occurred in core, and CRY2 phosphorylation is CRY2 degradeds It is necessary, CRY2 mediate regulation and control of the blue light to hypocotyl growth and photoperiod control blooms be also in core, illustrate light by Body CRY2 completes life cycle after its whole translation in core, from it is protein modified to function again to protein degradation (Yu, 2009)。

From being found cryptochrome CRY1, CRY1, its homologous gene CRY2 in arabidopsis, other species it is homologous The extensive functions of gene C RY are found successively.The Function Identification of arabidopsis cryptochrome mainly passes through genetic method, analysis CRY1 or CRY2 Gene Deletion mutant cry1, cry2, and it is overexpressed transfer-gen plant CRY1, CRY2 phenotype.These Research has shown that arabidopsis cryptochrome CRY1, CRY2 have mainly mediated blue light to promote building up and photoperiod control for light form respectively That makes blooms.Certainly, they have also mediated other blue responses, and cryptochrome is the different angles for how coordinating to play the part of in vivo actually Color, its Regulation Mechanism is required for further research with illustrating.Cryptochrome CRY3T-DNA insertion mutation bodies cry3 is not obvious Phenotypic alternation, its specific physiological function is also not very clear.Current research is main to know that CRY3 has reparation single stranded DNA Biochemical activity, it is possible to take part in the protection of organelle gene group.Except arabidopsis, most of species (from bacterium to the mankind) are all It is found that the presence of cryptochrome.Cryptochrome is closely related with biological clock, and nearest research is also shown that cryptochrome and biology Clock is not only relevant with crop yield, also affects human health, including cancer, sleep disordered and many mistakes physiologically Adjust.Understand the molecular mechanism of cryptochrome and blue light reaction, not only there is great meaning to the preliminary basic process for understanding life Justice, and also with certain practical value in terms of agricultural and medicine.

The function of gene is often closely related with its Subcellular Localization.Arabidopsis cryptochrome CRY1 and CRY2 gene is in quilt There are expression (Ahmad M, 1993 in all cell types and all organelles that detected；Lin,1996；Toth R, 2001).It is related that the comprehensive expression of cryptochrome regulates and controls multi-signal approach to it.Started with cryptochrome endogenesis promoter glimmering The expression of light element enzyme reporter gene, as a result show cryptochrome mRNA expression is hardly influenceed by blue light, but by biological clock Regulation and control, expression quantity is issued to peak in light, is darkling preferably minimized value (Toth R, 2001).Cryptochrome CRY1 albumen Light does not influence level, and CRY2 protein level then reduces (Lin, 1996) under blue light.The change of protein level also with hidden flower The activity that pigment relies on light intensity is related.CRY1 expression can be detected in cytoplasm and nucleus, the expression in its cell The influence (Wu G, 2007) of amount not light.It has been reported that the CRY1 mediation blue lights in core promote the depolarising of film, suppress hypocotyl Growth, the CRY1 mediation blue lights in kytoplasm promote the growth (Wu G, 2007) opened with root of cotyledon.It is different with CRY1, Cryptochrome CRY2 mainly completes process after its whole transcription in core, including rely on the phosphorylation of blue light and degraded (Yu, 2007).The CRY2 of nuclear location has mainly mediated the induction to blooming and the suppression (Yu, 2007) to hypocotyl growth.Also there is report Road claims the transcription that the CRY2 of nuclear location take part in gene and the chromatinic concentration of interphase in cell division (Tessadori F, 2007； Charron J B,2009).As can be seen here, its function of orientating as of gene provides clue.

The photomorphogenesis (being called de-etiolation) of plant includes several morphological changes：Hypocotyl growth suppresses, cotyledon expands Open and Development of Chloroplasts.The growth of Xanthophyll cycle hypocotyl, stimulates the expansion of cotyledon, promotes plastid to the transformation of chloroplaset (Nemhauser J,2002).Arabidopsis light receptor cryptochrome and phytochrome have mediated de-etiolation process, depend primarily on The regulation and control of gene transcription level and post-transcriptional level are promoted with photomorphogenesis (Jiao Y, 2007).To weakening de-etiolation The gene mutation body progress genetic analysis of response, many genes of discovery (such as COP1, SPA1, HY5/HYH, HFR1, PP7, HRB1, SUB1, OBP3, SHB1, BIT1, ATAB2 etc.) it take part in the de-etiolation response that cryptochrome regulates and controls.Except COP1, SPA1, HY5/HYH, are that the function of how participating in cryptochrome is also not very clear for these genes.

Xanthophyll cycle hypocotyl growth is to study one of sign of de-etiolation.Cryptochrome CRY1 discovery is just being derived to indigo plant The analysis (Koornneef M, 1980) for the mutant hy4 that hypocotyl growth disinthibites under light.CRY1 is overexpressed plant presentation pair , compared with wild type, there is short hypocotyl (Lin, 1995) under lasting blue light in the high susceptibility of blue light.CRY2 is also assisted in Regulation of the blue light to hypocotyl growth, but its effect is weaker relative to CRY1, and mainly mediation low intensity blue light (<10μ Mm-2s-1) to the suppression of hypocotyl growth, it is possible to which fast degradation is relevant under high intensity blue light with CRY2.Double deletion mutations Body cry1cry2 shows the hypocotyl longer than cryptochrome list deletion mutant cry1, cry2, explanation under lasting blue light The two cryptochromes suppress there is functional redundancy (Mockler T C, 1999) in hypocotyl growth in mediation blue light.By right Wild type, double deletion mutant cry1cry2, CRY1 are overexpressed the analysis of plant, it is found that CRY1 mainly mediates 390-480nm Photoresponse (Ahmad M, 2002) in UV-A and blue light range.

Blue light is mediated to suppress the cell mechanism of hypocotyl growth for cryptochrome, Spalding and his colleague have been done greatly Quantity research, propose cryptochrome have activated ion channel, cause plasma membrane to depolarize, thus suppress cell elongation (Spalding E, 1988；Cho M H,1996).They are it is further proposed that the CRY1 of nuclear location has mediated the mistake of hypocotyl growth and membrane depolarization Journey.Rely on core CRY1 plasma membrane depolarising in the several seconds after illumination just to occur, imply that its reaction mechanism will be transcribed significantly faster than Regulation and control or protein degradation regulation and control.In addition, the blue light that deletion mutant cry1, cry2, phyA and phot1 show decrease is lured Plasma membrane depolarising response (the Parks B M, 1999 led；Folta K M,2001).But phot1 is not occurred under blue light The hypocotyl of elongation.The initial period that Phot1 is in photoresponse in influence of the blue light to cell elongation is mediated is possible to, and CRY1 function is to keep blue light to the suppression of hypocotyl growth (Parks B M, 2001；Folta K M,2003).

Regulation and control of the light to hypocotyl growth are main or by way of gene regulation.Numerous studies show, cryptochrome CRY1 can suppress the degraded of the transcription factor of COP1 mediations, such as HY5/HYH, HFR1 (Osterlund M T, 2000；Duek P D,2004).This class transcription factor is degraded in the dark, sees after light, and light receptor (cryptochrome or phytochrome) suppresses COP1 Activity, cause the accumulation of transcription factor, so as to change the expression of metabolic enzymes or signal protein, such as auxin, rape Plain steroids, gibberellin, catalyze and synthesize with the metabolic enzyme of degradation of cell wall etc., can explain light to hypocotyl to a certain extent The influence of growth.

With Xanthophyll cycle hypocotyl growth on the contrary, luminous energy promotes wealthy of cotyledon, promote the elongation of cotyledon cell.Cryptochrome CRY1 and CRY2 have mediated the influence that blue light grows to cotyledon jointly, unlike, the CRY1 of kytoplasm positioning mediates this response, And the CRY2 in core take part in this photoresponse (Wu G, 2007), the same response of CRY mediations of diverse location makes people obscure.It is hidden Anthocyanidin CRY1 has mediated influence of the high low intensive blue light to wealthy of cotyledon, and CRY2 then mainly mediates the sound of low intensity blue light Should, it is similar (Lin, 1998) with suppressing hypocotyl growth situation.CRY1 or CRY2 transgenic seedlings are overexpressed in lasting blue light Under show the cotyledon bigger than wild type, illustrate that the concentration of intracellular cryptochrome have impact on the wealthy Zhang Chengdu of cotyledon.Hidden pattern Element mediation blue light suppresses hypocotyl growth, but mediation blue light promotes wealthy of cotyledon, illustrates to deposit between the different function of cryptochrome There are different functions in the cryptochrome of different regulatory mechanisms, or Different Organs or tissue.

Except the influence to hypocotyl and cotyledon, cryptochrome can also mediate blue light to adjust the development of plastid, chloroplaset Generation, relies primarily on the regulation and control to gene expression dose, including to the coding karyogene of plastid albumen and luring for plastid transcript profile Lead (Jiao Y, 2007；Fuglevand G,1996；Thum K E, 2001), such as protein D 1 of PSII reaction centers, D2 codings Gene psbA, psbD are regulated and controled by karyogene SIG5, and SIG5 expression is increased under blue light by CRY1 mediation.Therefore, it is hidden Anthocyanidin transcribes the expression of the karyogene needed by regulating and controlling plastid, so as to adjust the development of chloroplaset.

Cryptochrome has also regulated and controled the photoperiod except promoting Arabidopsis thaliana Seedlings photomorphogenesis in the growth course of plant What is controlled blooms.Cry2 mutant flowering time under long-day conditions is much later than wild type, but is bloomed under short-day Time is unaffected, illustrates that cryptochrome CRY2 take part in the regulation and control (Guo H, 1998) of the approach of blooming controlled the photoperiod. In addition, studies have found that the allele for the Late Paleoeene fha that cry2 is exactly screened previously, with reference to these results of study： Cry is bloomed (Guo H, 1998 as co, ft, gi by Photoperiod pathway regulation and control；Koornneef M,1998).By turning Gene arabidopsis and soybean transient expression research have shown that：Cryptochrome GmCRY1a and GmCRY2a in soybean both participate in light form Into process, GmCRY1a effect becomes apparent from；Imply that GmCRY1a plays important to the approach of blooming of soybean Photoperiod Act on (Zhang, 2008).

Cryptochrome CRY2 protein levels show the circadian rhythm for relying on blue light under short-day, and do not have under the long-day Have the phenomenon, illustrate CRY2 protein levels change be possible to explain it participate in photoperiodic signal mechanism (Mockler T, 2003).Analysis to CRY2 allele EDI, more supports this saying.EDI is nearby to intend southern in tropical archipelago under the line The photoperiod insensitive early blossoming quantitative trait locus (Alonso-Blanco C, 1998) screened during mustard Cvi is environmental.QTL EDI has been navigated to CRY2 genes by Identification, so that the point mutation for finding V367M is to cause EDI independent of photoperiod early blossoming The reason for, intracellular CRY2V367M albumen is more more stable than CRY2.The stability of CRY2V367M albumen with not by the photoperiod Regulation is possible to explain its early blossoming phenotype independent of the photoperiod.

Cryptochrome CRY1 is possible to also influence arabidopsis flowering time.Under some experiment conditions, cry1 mutant is presented Go out the phenotype of late flower, but also have under some experiment conditions, cry1 mutant flowering time does not have difference with wild type (Bruggemann E,1996；Blazquez M A,2003).Be likely to be that the slight difference of experiment condition causes it is this not Unanimously.Because the double deletion mutants of cry1cry2, which are bloomed, under blue light is later than single mutant and wild type, illustrate CRY1 and CRY2 work( Can redundancy, energy Accelerate bloom (Liu, 2008).In addition, cry2 mutant inhibits the phenotype of phyB mutant early blossoming, imply CRY2 and phyB play opposite effect (Mockler T C, 2002) in mediation feux rouges suppresses to bloom.

Biological clock is the internal yardstick of organism vital movement, and it enables the change in organism adaptively ball border, Keep day-night cycle and the circulation of the photoperiod of 1 year (Harmer S L, 2009) of about 24 hours.Biological clock has regulated and controled plant Growing for thing, promotes the adaptability of plant.One of biological clock is influenceed by environment simultaneously, just, optical signal Biological clock, the physiologically active that can promote organism are synchronous with surrounding environment day-night cycle (Sancar A, 2000).Scientists Find that cryptochrome is played an important role in biological clock successively in arabidopsis, drosophila and mouse, cryptochrome is considered as The internal members of one main light receptor or biological clock promote transmission (the Emery P, 1998 of optical signal；Somers D E, 1998).Arabidopsis cryptochrome mutant weakens biological clock to the regulation and control of gene downstream (Somers D E, 1998).Intend The biological clock cycle of lengthening is presented in southern mustard cry1 mutant under blue light, and cry2 mutant is only presented slightly under low intensity blue light The biocycle of shortening, cry1cry2 double-mutants show the biocycle of lengthening under blue light.Except changing for biocycle Become, cry1cry2 has no effect on the rhythm and pace of moving things, it is not the inherent member (Devlin of biological clock oscillator to imply arabidopsis cryptochrome P F,2000)。

Influence of the light to biological clock how is mediated also to be not very clear for arabidopsis cryptochrome.It has been reported that hidden pattern Element adjusts influence (Yu J W, 2008) of the light to clock possibly through suppressing COP1 activity so as to influence ELF3 and GI. In addition, the interaction CRY1-phyA, CRY2-phyB, CRY1-ZTL and phyB-ZTL between the light receptor reported be conducive to it is whole Closing light signal joint effect biological clock (Jarillo J A, 2001).Light receptor mediation light is also needed to the Regulation Mechanism of biological clock Further research.

Shadow rings the development of guard cell and the opening of stomata.Light is one more complicated to the influence that guard cell develops Process, include the starting of asymmetric cell division, the increment of precursor, the differentiation of Stomacal guard cell, the formation of stomata. Research shows that blue light, feux rouges and far-red light can stimulate the development (Kang C Y, 2009) of guard cell.Genetic analysis show Cryptochrome has mediated blue light to stimulate the development of stomata, and its regulatory pathway is different from known LRR receptor kinases, MAPK and indulged The related regulated and control network of many transcription factors (Kang C Y, 2009).

Except the influence developed to stomata, cryptochrome can also mediate the opening of light regulation stomata.It is understood that light can Promote the opening of stomata, it is allowed to which gas exchanges carry out photosynthesis or transpiration (Zeiger E, 1977).And image assesment is to adjust The main light receptor of stomatal opening is controlled, mediation blue light adjusts the depolarising of guard cell's film potential, causes flowing and the stomata of water Opening (Schroeder J I, 2001).The nearest double deletion mutants of some results of study report cry1cry2 add plant The drought tolerant of thing, is to be attributed to the opening (Mao J, 2005) that blue light promotes stomata.The missing of cryptochrome reduces blue light The opening of air holes, and it is overexpressed the opening that CRY1 adds stomata.The stomata remained in the double deletion mutants of image assesment is opened Phenomenon is put, is eliminated in cryptochrome and image assesment four lack mutant, also, it is prominent in the double missings of image assesment to be overexpressed CRY1 Response of the stomatal opening to blue light can be recovered in variant, imply that cryptochrome take part in the letter of Induced by Blue Light stomatal opening really Number approach (Mao J, 2005).With de-etiolation response, influence of the cryptochrome to development and the opening of stomata is also relied on COP1, but correlation, the phase of cryptochrome/COP1 and MAPK approach between cryptochrome/COP1, image assesment regulatory pathway Mutual relation is currently not very clear.

Early in 1998, Ahmad seminar found that the double deletion mutants of cryptochrome cry1cry2 lack and relies on blue light Phototropism, and CRY1 is overexpressed and then strengthens phototropism response (Ahmad M, 1998).But then studies have found that image assesment rather than Cryptochrome has mainly mediated photostrophism (Christie J M, 1998).The double deletion mutants of cryptochrome cry1cry2, The double deletion mutants of phytochrome phyAphyB reduce phototropism, but the important parameter of phototropism does not have apparent change (Tsuchida-Mayama T,2010).Thus researchers think that image assesment has mediated influence of the blue light to phototropism, promote The opening of stomata, the motion of chloroplaset.Cryptochrome and phytochrome are possible to participate in or have assisted some regulation and control of image assesment (Whippo C W,2003).Recently it has been reported that cryptochrome CRY1, CRY2 and phytochrome phyA forward directions adjust RPT2 albumen The formation for the auxin gradient that accumulation, image assesment are relied on, promotes phototropism growth, while also negative regulation relies on GA phototropism suppression Approach processed (Tsuchida-Mayama T, 2010).

Except phototropism, light has an effect on the geotropism (Hangarter R P, 1997) of plant.Feux rouges can influence arabidopsis Hypocotyl Bendroquinon in growth medium, is mainly mediated by phytochrome (Robson P R, 1996).However, blue light is to geotropic shadow Sound is often covered by phototropism, therefore this kind of experimental analysis needs to carry out under the background that image assesment is mutated.By cryptochrome CRY1 or CRY2 is overexpressed to be lacked in mutant to cry1cry2phot1phot2 tetra-, occurs that random hypocotyl is curved under blue light Song, and do not have under dark.This similar to phytochrome mediation feux rouges it is demonstrated experimentally that suppress geotropism, cryptochrome is mediated Blue light suppresses (Ohgishi M, 2004) to geotropic.The geotropism of light receptor mediation is possible to mainly adjust by light receptor Save hormone metabolism, transhipment, the expression of signal transduction gene realization.For example, cryptochrome mediation blue light suppresses PGP19/ABSB9 The expression of gene.Auxin transhipment of one ABC type of PGP19/ABSB9 gene codes, transhipment take part in auxin Transport and Bendroquinon in growth medium.Genetic analysis show that PGP19 is upper (Nagashima A, 2008) in cryptochrome.Thus, Cryptochrome mediates suppression of the blue light on PGP19 gene expressions to explain blue light to geotropic influence.Cryptochrome is to ground The response of property also involves COP1 signal pathways, and cop1 mutant shows continuation photomorphogenesis and apogeotropism phenotype (Cao D,2000；Yang,2000).

Light can adjust the development of root, include extension, the generation of lateral root, development of plastid and the tropism growth (Feldman of root L J,1984).It is reported that blue light stimulates the extension of root, and cryptochrome CRY1 mediated this response (Canamero R C, 2006).CRY2 is opposite with CRY1 function in the growth regulating to root.In addition, there are some researches show kytoplasm and core CRY1 have can It can respectively facilitate or suppress the extension (Wu G, 2007) of root.Except the influence extended to root cells, blue light can also influence root leaf green The development of body, is mainly mediated by CRY1 (Usami T, 2004).

Migrant such as birds and insects are believed to experience magnetic field of the earth, thus, it is possible to distinguish the direction migrated. The magnetic field induction of animal depends on light, and research at present thinks that photoinduction light receptor produces the proton pair of magnetic responsiveness, is transferred to god Through system, animal is set to experience the direction (Ritz T, 2000) in magnetic field.The light receptor is considered as cryptochrome in drosophila, and The photochemical mechanism of cryptochrome mediation magnetic field induction is independent of tryptophan triplet electron transmission in albumen and FAD photo-reductions Reaction (Gegear R J, 2010).Scientists also studied response of the plant to magnetic field, it is found that magnetic field can promote arabidopsis Cryptochrome CRY1 mediation blue lights suppress the growth of hypocotyl, the accumulation for promoting anthocyanidin and rely on the CRY2 of blue light degraded, And the reaction is dependent on FAD photo-reduction photoactivation mechanism (Solov'yov I A, 2007).Also studies have reported that hypocotyl Growth, the accumulation of anthocyanidin, the expression of related gene are not exposed to the influence (Harris S R, 2009) of magnetic field intensity.Magnetic field The influence of blue response is mediated to also need to further verification to cryptochrome.

Research finds that under flu mutant backgrounds arabidopsis cry1 mutant suppresses to rely on blue light, ROS programmed cell Property dead (PCD) (Danon A, 2006).Flu mutant is in photosynthetic pigment and phytochrome chromophore chlorophyll and blue-green algae courage (Meskauskiene R, 2001) found in the research of plain biosynthesis pathway.After flu is lacked, the mutation grown under light Body, which is transferred under dark, can accumulate more protochlorophyllides, when this mutant is placed back under light, protochlorophyll The oxygen that luminous energy can be transferred to ground state by sour fat molecule promotes oxygen to excitation state.As ROS important sources, oxygen promotes PCD, draws Experience light has been played to dark again to the death (Op den Camp R G, 2003) of the mutant flu under light.Flu mutant PCD Phenotype only relies upon blue light rather than feux rouges, and thus scientists contemplate the regulation and control that cryptochrome is possible to take part in PCD.CRY1 Mutation can save the PCD phenotypes of flu mutant, imply that CRY1 has mediated the PCD processes for relying on blue light, it may be possible to by luring The expression of stress response gene is led to amplify PCD processes (Danon A, 2006).

The function of blue light is relied on except more than, cryptochrome also possesses the activity of blue light non-dependent, for example mediate feux rouges and Far-red light is responded.The biological clock cycle of lengthening is presented in cry2 mutant seedlings under feux rouges, and hypocotyl occur under far-red light prolongs Stretch defect (Mas P, 2000).The cotyledon that reduction is also presented in the double deletion mutants of cry1cry2 under other illumination conditions opens Degree, CRY2 Cvi ecotypes allele C RY2V367M presented under far-red light reinforcement cotyledon open (Botto J F, 2003).It is generally acknowledged that cryptochrome does not absorb the light of long wavelength, but the chromophore FAD that cryptochrome is included possesses different Redox state, it is possible to absorb light (the Henbest K B, 2008 more than 600nm；Ozturk N etc., 2008).Therefore, Influence of other optical wavelength to cryptochrome 26S Proteasome Structure and Function can not be excluded.In addition, the interaction between light receptor is also explained Response of the light receptor in the case where not sharing the same light.

Arabidopsis cryptochrome CRY experienced a series of biochemical reactions, including electron transmission, phosphorylation, ubiquitination, Change protein conformation transmission blue light signals.For the pathway of cryptochrome optical signal, current research mainly proposes two The mode of kind：A kind of mode is cryptochrome by being expressed with transcription factor CIB interaction controlling genes；Another is hidden pattern Element by with SPA1/COP1 composite bulk phase interaction regulatory protein stability.Both the above signal transduction path all relies on indigo plant The protein-interacting controlling gene expression that light is relied on, so as to influence growth and development of plants.

Signal protein in arabidopsis cryptochrome pathway includes：

(1) transcription factor CIB

Determined by the yeast two-hybrid screening under blue light to the special cryptochrome interaction albumen of first blue light within 2008 Justice is CIB1 (CRY-interacting bHLH1).In yeast, CIB1 and CRY2 interaction are that blue light wavelength is special, phase The intensity of interaction depends on blue light strength, and dependent on chromophore FAD.In the Arabidopsis thaliana Seedlings cell under blue light CIB1-CRY2 complexs can be detected, and be can't detect under feux rouges.CIB1 exercises transcription in transient transcription factorial experiment The function of the factor depends on cryptochrome and blue light.Arabidopsis CIB1 can combine G-box (CACGTG) in vitro, in vivo can G-box and E-box (CANNTG) transcription is enough influenceed, illustrates that CIB1 combines DNA activity in vitro and vivo transcription regulation is lived Property has differences.A kind of possible explanation is exactly CIB1 and other same family genes (CIB3,4,5 etc.) formation heterologous two in vivo Aggressiveness, so as to change its compatibility to different DNA.It fact proved, at least four CIB1 related genes can be with CIB1 Or CRY2 interactions, including CIB1, CIB3, CIB4, CIB5, and some of them can form heterodimer with CIB1, jointly Influence the expression of CRY2 controlling genes.Consistent with this argument, single deletion mutant cib1 is not any under experimental conditions Phenotype, the wild type to be later than and double deletion mutant cbi1cib5 are bloomed, illustrates there is functional redundancy between CIB albumen.Will CIB1 is overexpressed can cause early blossoming to wild type background, and not have phenotype under cry1 cry2 mutant backgrounds, illustrate CIB1 Cryptochrome is depended on to the promotion bloomed.Detection to CIB1 promoters, finds it in all organelles and cell type It is active, illustrate CIB1 in addition to being bloomed in vascular bundle cell with CRY2 interaction regulations, CIB1 albumen also has can The expression of other photoresponse genes can be regulated and controled with CRY interactions in other cells.Certainly, existing detection is implied at present CIB1 is possible to be not involved in regulations of the CRY to de-etiolation, because what CIB albumen list missing or many missings were all responded without de-etiolation Novel presentation (Liu and lin, result is not delivered).

(2) ubiquitination E3 ligases COP1

Ubiquitination ligase COP1 plays key player (Deng X in Plant Light morphological grad image and light receptor function W,1992).Afunction mutant cop1 shows continuation photomorphogenesis phenotype, occur under dark short hypocotyl, Cotyledon, the expression of increased anthocyanidin and the false demonstration of photoinduction gene opened.It is overexpressed GUS-CCT1 and GUS-CCT2 Cop1 phenotypes are showed, DNA chip is analyzed, and the gene of change is expressed in cop1 has 80% in GUS-CCT1 and GUS-CCT2 Expression be also affected, similar phenotype implys that relation functionally, make scientists started to cryptochrome and The discussion of COP1 relations.Find that CCT1 and CCT2 can interact with COP1 in yeast two-hybrid system, it is thin in arabidopsis CRY2 and COP1 is identified in born of the same parents by co-immunoprecipitation to interact, GFP-CCT1 is with COP1 common locations in nucleus nucleome (Wang H,2001).It is a key point of photomorphogenesis regulation and control to the regulation of protein stability, there are some researches show COP1 is situated between The degraded that numerous optical signal opalescences are relied on, including HY5/HYH, LAF1, HFR1, CRY2, phyA, CO, GI (Duek P are led D,2004；Liu,2008；Shalitin D,2002；Hardtke C S,2000；Jang S,2008).It is subsequently assumed that photoactivation Cryptochrome be possible to directly or indirectly interact with COP1, suppress its E3 connection enzymatic activitys, change COP1 substrate proteins White stability, development (Yang, 2000 of final regulation plant；Wang H,2001).In addition, the phase of cryptochrome and COP1 Interaction is not dependent on (Liu, 2008 of blue light；Yang,2000；Wang H, 2001), this just proposes a query, CRY2 It is the influence for how mediating light to COP1 activityIt is a kind of may be exactly after illumination CRY change itself biochemical activity or COP1 Caryoplasm is distributed, and alternatively possible is exactly protein-interactings of the CRY with COP1 interactions by way of light is relied on, and carrys out joint effect COP1 activity.

(3) coiled-coil/WD repeat Protein Ss PAs

SPA1 is the special signal mediating proteins of phytochrome phyA, suppresses building up for light form in arabidopsis.It is included COP1 WD duplicate domains are highly similar in WD duplicate domains, sequence.SPA1 can be with COP1 interactions, and its coiled-coil domain is It is necessary with COP1 interactions.SPA1 and COP1 interaction promotes ubiquitinations of the COP1 to HY5, HFR1, has mediated phyA signals Conduction (Duek P D, 2004；Saijo Y,2003).SPA1 and COP1 interaction is photaesthesia, and light can suppress theirs Interaction, suppresses COP1 E3 ubiquitinations enzymatic activity (Saijo Y, 2003,2008).But, just how to suppress actually SPA1 and COP1 interaction, the activity for how about suppressing COP1 is not clear at present.

Nearest research shows that SPA1 can interact with CRY1 and CRY2, and is to rely on blue light and chromophore FAD , action intensity increases with the increase of blue light light intensity.Except SPA1, arabidopsis also have three SPA1 related genes SPA2, SPA3、SPA4.SPA4 under blue light with CRY1 and CRY2 interactions.Functional redundancy is there may exist between SPAs genes, it is common to participate in The blue light signals approach of cryptochrome regulation and control.Researchers further demonstrate SPAs genes in cryptochrome from science of heredity angle Regulate and control the role in blue light signals approach.Under lasting blue light, mutant cry1 shows long hypocotyl, and under spa1spa4 Plumular axis is shorter than wild type, and spa1spa4cry1 hypocotyl is slightly longer than wild type but considerably shorter than cry1, illustrates that SPAs take part in The hypocotyl growth of cryptochrome regulation and control suppresses, and positioned at the upper of CRY1.SPA1 has also assisted in the photoperiod control of CRY2 dependences The approach of blooming of system.Cry2, which is bloomed, under long-day is later than wild type, and spa1 is without obvious flowering phenotype, double deletion mutants Cry2spa1 flowering times are consistent with spa1, illustrate that SPA1 is located at CRY2's in the approach of blooming that CRY2 regulates and controls photoperiod control Downstream.CRYs has also mediated blue light to adjust the expression (Sellaro R, 2009) of SPAs gene transcription levels.

Blue response approach is described in detail below

(1) CRY2-CIB1 approach

Arabidopsis cryptochrome CRY2 and bHLH transcription factors CIB1 experienced the interaction for relying on blue light.CIB1 can be combined E-box in FT gene promoters, CRY2 adjust FT expression by the interaction with CIB1 in transcriptional level, promote the photoperiod What is controlled blooms.FT encodes a moveable transcription factor, and apical meristem Accelerate bloom gene is moved to from leaf Expression.Homologous gene interactions of the CRY2 also with CIB1, experiment in vitro prove CIB1 and other homologous genes (CIB3, CIB4, CIB5 heterodimer) is formed, the E-box of FT promoters is attached to.CIB albumen take part in CRY2 signal transduction way jointly Footpath, promotes to rely on the formation of the bud of photoperiod.

There are some problems to need further inquire into for CRY2-CIBs regulatory pathways.For example：Whether CRY2 influences CIB1 With reference to DNA affinityCryptochrome is how to mediate blue light to influence CIB1 transcriptional activityIn addition, CIB albumen is not having Ubiquitination -26S proteasome degradation pathways are undergone under conditions of blue light, blue light is the stability for how promoting CIB albumen How CIB albumen special accumulation under blue light and signs of degradation cryptochrome CRY2 blue light under are explainedExcept promoting light week What the phase controlled blooms, and cryptochrome has also regulated and controled many other responses, and whether CIB has intervened these response approachWhether also deposit In the transcription factor with CRY1 interactions so that cryptochrome CRY1 transmits light letter from the expression of transcriptional level direct regulation and control gene Number

(2) CRY-SPA1/COP1 approach except by the interaction with transcription factor from the expression of transcriptional level regulatory gene, Cryptochrome also passes through the expression of post-transcriptional mechanism indirect adjustments and controls gene.Cryptochrome has mediated blue light to live E3 ligases COP1 Property suppression, such as CRY1 mediation blue light inhibit COP1 rely on transcription factor HY5 (Long Hypocotyl5), HYH (HY5 Homologue), the degraded of HFR1 (Long Hypocotyl in Far-Red1) albumen, these transcription factors are rung in de-etiolation The metabolic enzyme of the expression of regulatory gene in answering, such as auxin, Brassinosteroids, gibberellin, and synthesis and degradation of cell wall Class.CRY2 has mediated blue light to suppress the degraded of transcription factor CO (Constans) albumen that COP1 is relied on, and CO is rush under the long-day Enter the important member bloomed, it can promote FT expression on transcriptional level.It has recently been demonstrated that cryptochrome is logical Cross the interaction communicating optical signals with protein complexes SPA1/COP1, CRY and COP1 interaction is independent of light, but CRY can be with The interaction that the Protein S PA1 experience light of COP1 interactions is relied on.CRY and SPA1 interaction is that blue light is special, and SPA1 exists It is located at CRY downstream in heredity.The interaction that CRY and SPA1 relies on blue light explains CRY is how to mediate light pair to a certain extent COP1 function inhibitios.Surprisingly there are some differences in structure similar CRY1 and CRY2 and SPA1/COP1 interaction. CRY1-SPA1 interaction inhibits SPA1-COP1 interaction (Sellaro R, 2009 in yeast and arabidopsis；Lian H L, 2011；Liu B, 2011), CRY1 has mediated suppression of the blue light to COP1 activity in the way of rival, and elder generation is explained just Preceding discovery：SPA1-COP1 interactions receive the suppression (Saijo Y, 2003) of light.With CRY1 on the contrary, dependent on blue light CRY2-SPA1 interaction has no effect on SPA1-COP1 interaction, and CRY2-SPA1 interaction adds in yeast Strong CRY2-COP1 interaction, promotes the formation (Zuo Z, 2011) of CRY2-COP1 complexs in plant. CRY2-SPA1 interaction promotes CRY2-COP1 interaction to be detected by yeast three-hybrid, CRY2-SPA1 The formation of CRY2-COP1 complexs is promoted only to be observed in the transfer-gen plant that SPA1 is overexpressed.Cryptochrome CRY1 and CRY2 and the SPA1/COP1 differences interacted are possible to interact therewith site related.CRY1 C-terminal CCT domains and SPA1 C-terminal CC-WD domains interaction, and CRY2 N-terminal PHR domains and SPA1 N-terminal kinase domain interact.SPA4 also with CRY1 and There is the interaction for relying on blue light in CRY2.

Soybean is used as a kind of preferable photoperiod research material very long history, but given birth to the molecule of its photoperiodical reaction Thing mechanism is still known little about it so far.Set about studying the molecular biology problem of its photoperiodical reaction, Ke Yicong from cryptochrome The characteristics of upstream of signal transduction starts to understand soybean photoperiodical reaction, so not only improves the soybean photoperiod research of developing as early as possible Frontier, also can for deeper into progress research invaluable experience be provided, taken a firm foundation, theoretical anticipated with very important Justice.Research before shows that soybean cryptochrome GmCRY1 is acted on substantially in soybean photoperiodical reaction, and with the hidden flower of arabidopsis Pigment CRY2 features are similar, and the more similar CRY1 of GmCRY2.GmCRY1 and CRY2 similarity is mainly shown as：1. present Obvious circadian rhythm；2. flowering of plant can be promoted.Except that GmCRY1 is overexpressed in CRY1 deletion mutants, it is short Accelerate bloom under day, and CRY2 Accelerate blooms under long day.GmCRY2 and CRY1 similarities are, both without obvious circadian rhythm It can not be obviously promoted and bloom, but can be degraded under GmCRY2 blue lights and CRY1 can not (Zhang etc., 2008).

It is overexpressed in soybean or RNAi knocks out whether GmCRY genes can change sensitivity of the soybean blossoming to the photoperiod Property, it is not yet reported that before this research.In addition, whether the GmCRY in soybean carries out blue light by being interacted with GmCIB Signal transmission is also unclear.We have found in soybean and have cloned 9 GmCIBs genes homologous with arabidopsis CIB1 at present, The GmCIB1 and GmCRY2 special interactions under blue light by yeast two-hybrid assay preliminary identification.This research will pass through Interaction between means research GmCRYs and the GmCIBs albumen such as yeast two-hybrid, BiFC and Co-IP；Further obtain The genetic transformation soybean strain that GmCRY is knocked out with GmCIB1 gene overexpressions or RNAi, with reference to the phenotypic analysis of transgenic progeny GmCRY in the downstream effects target gene that (ChIP) result finds GmCIB1, analysis soybean is tested with chromosome co-immunoprecipitation to pass The GmCIB signal paths of blue light signals, and GmCRY and effect of the GmCIB1 genes in soybean Senescence manipulation are passed, is soybean The further further investigation of photoperiod and germplasm innovation lay the first stone.

Invention summary

In an aspect, the present invention relates to a kind of gene of separation, it has following nucleotide sequence：

(1)SEQ ID NO:Sequence or its complementary series shown in 1 or 3；

(2) under stringent hybridization condition with SEQ ID NO:The sequence of 1 or 3 hybridization；

(3) with SEQ ID NO:1 or 3 have the sequence of at least 85%, 90%, 95% or 99% homogeneity, and it regulates and controls leaf Sub- aging and bloom；Or

(4) by SEQ ID NO:Sequence shown in 1 or 3 passes through the missing of wherein one or more nucleotides, displacement, insertion Or add and derivative resulting sequence.

In another aspect, the present invention relates to a kind of DNA molecular of separation, it includes above-mentioned gene and its variant, The DNA molecular has the biological function of GmCIB1 genes or GmCRY2 genes.

In another aspect, the present invention relates to a kind of recombinant vector, it is comprising above-mentioned gene or such as DNA molecular.

In another aspect, the present invention relates to a kind of protein of separation, it has following amino acid sequence：

(1)SEQ ID NO:Amino acid sequence shown in 2 or 4；

(2) by the sequence of above-mentioned gene code；Or

(3) displacement comprising one or several amino acid residues and/or missing and/or the SEQ ID NO of addition:2 or 4 institutes The amino acid sequence shown, the protein has regulation and control plant leaf aging and the function of blooming.

In another aspect, the present invention relates to a kind of host cell, it is carried comprising above-mentioned gene, DNA molecular, restructuring Body or protein.

In another aspect, the present invention relates to a kind of genetically modified plants, it includes above-mentioned gene, DNA molecular, restructuring Carrier or protein.

In one embodiment, described genetically modified plants are monocotyledon or dicotyledon, preferably intend south Mustard or soybean.

In another aspect, planted the present invention relates to described gene, DNA molecular, recombinant vector or protein in regulation and control Thing leaf senescence and the application in blooming.

In one embodiment, described plant is monocotyledon or dicotyledon, preferably arabidopsis or big Beans.

In another aspect, the method for genetically modified plants is cultivated the present invention relates to a kind of, including above-mentioned gene is led Enter in purpose plant to obtain genetically modified plants, so as to regulate and control the leaf senescence of the genetically modified plants and bloom.

In one embodiment, the method for cultivating genetically modified plants further comprises the kind for obtaining the genetically modified plants Son.

In one embodiment, the plant is monocotyledon or dicotyledon, preferably arabidopsis or soybean.

In another aspect, the present invention relates to it is a kind of regulate and control plant leaf aging method, including by above-mentioned gene Or carrier is introduced into the plant so that it is expressed in plant, so as to regulate and control the leaf senescence of plant.

In another aspect, the present invention relates to it is a kind of regulate and control flowering of plant method, including by above-mentioned gene or load Body is introduced into the plant so that it is expressed in plant, so as to regulate and control blooming for plant.

In another aspect, the present invention relates to a kind of method for producing genetically modified plants, the genetically modified plants include Said gene, DNA molecular or the recombinant vector of introducing, or the above-mentioned protein of its recombination expression, this method include obtaining institute State the seed of genetically modified plants, and by planting the seed to obtain new genetically modified plants.

Brief Description Of Drawings

Fig. 1 shows the chromosome mapping of soybean CIB genes.

Fig. 2 shows the homology between soybean CIB gene family members.

Fig. 3 shows arabidopsis and the analysis of soybean CIB conserved positions, and arrow (top) represents the ammonia guarded in each domain Base acid.

Fig. 4 shows the gene structure pattern of soybean CIB genes.

Fig. 5 shows Subcellular Localizations of the soybean CIB in protoplasts of Arabidopsis thaliana broken by ultrasonic.

Fig. 6 shows expression pattern of the soybean CIB gene families in different tissues organ.

Fig. 7 shows expression pattern of the soybean CIB gene families in different development stage.

Fig. 8 a-8i respectively illustrate the photoperiod expression pattern of soybean CIB1-9 genes, and wherein Fig. 8 a are CIB3 genes Photoperiod expression pattern, Fig. 8 b are the photoperiod expression pattern of CIB2 genes, and Fig. 8 c express for the photoperiod of soybean CIB1 genes Pattern, Fig. 8 d are the photoperiod expression pattern of soybean CIB4 genes, and Fig. 8 e are the photoperiod expression pattern of soybean CIB5 genes, figure 8f is the photoperiod expression pattern of soybean CIB6 genes, and Fig. 8 g are the photoperiod expression pattern of soybean CIB7 genes, and Fig. 8 h are big The photoperiod expression pattern of beans CIB8 genes, and the photoperiod expression pattern that Fig. 8 i are soybean CIB9 genes, wherein short-day (SD；8 hours illumination/16 hour dark, a and b) or long-day (LD；16 hours illumination/8 hour dark, d) c and processing 48 are small When, continuous illumination (LL is gone to respectively；A and c) or continuous darkness (DD；B and d) processing 48 hours.Standard error (n as shown in the figure =3).

Fig. 9 a-e respectively illustrate the expression pattern of GmCIB1-9 in the soybean varieties of different latitude source, and wherein Fig. 9 a is not GmCIB2＆3 expression pattern in the soybean varieties of same latitude source, Fig. 9 b are GmCIB1＆4 in the soybean varieties of different latitude source Expression pattern, the expression pattern that Fig. 9 c are GmCIB5＆6 in the soybean varieties of different latitude source, Fig. 9 d are that different latitude source is big GmCIB7＆8 expression pattern in beans kind, and the expression pattern that Fig. 9 e are GmCIB9 in the soybean varieties of different latitude source.

Figure 10 shows that soybean GmCIB1 is overexpressed the phenotype of transformation of Arabidopsis thaliana plant.

Figure 11 shows that soybean GmCIB4/5/6/8 is overexpressed the phenotype of transformation of Arabidopsis thaliana plant.

Figure 12 shows that GmCRY1 is overexpressed plant related phenotype of aging under continuous shine, wherein (A-C)：Wild type and In GmCRY1-OX transfer-gen plants the single leaf or cotyledon of different growing stage according to aging aspects can be divided into three classes (green, do not have There is aging；Yellow, slight aging；It is withered, complete aging), the ratio shared by per class is such number and the total ratio of plant It is worth (n >=10).(D-H):The picture of the single leaf of different ageing phase plant or cotyledon.(I):Wild type and two GmCRY1-OX turn The immunoblotting assay of GFP-GmCRY1 and GmCRY1 expressions in gene plant.Total protein extract is used for 10%SDS- PAGE is analyzed, and GmCRY1 antibody is used for immuning hybridization.WT, wild type KN18；GmCRY1-OX, GFP-GmCRY1 are overexpressed out of office In raw type KN18.

Figure 13 shows that GmCRY2 is overexpressed plant related phenotype of aging under continuous shine, wherein (A-C)：Wild type and The single leaf or cotyledon of different growing stage can be divided into three classes (such as Figure 12 according to aging aspects in GmCRY2-OX transfer-gen plants It is shown), the ratio shared by per class is such number and the ratio (n >=10) of plant sum.(D-F,H):Different ageing phases The phenotype of plant list leaf or cotyledon.(G):GFP-GmCRY2 expressions in wild type and two GmCRY2-OX transfer-gen plants Immunoblotting assay.Total protein extract is analyzed for 10%SDS-PAGE, and GmCRY2 antibody is used for immuning hybridization.NS, The non-specific band of GmCRY2 antibody is used to indicate applied sample amount control；GmCRY2-OX, GFP-GmCRY2 are overexpressed in wild type KN18 In.

Figure 14 shows GmCRY2-RNAi related phenotypes of aging under continuous shine, wherein (A-C)：Wild type and The single leaf or cotyledon of different growing stage can be divided into three classes (as schemed according to aging aspects in GmCRY2-RNAi transfer-gen plants Shown in 12), the ratio shared by per class is such number and the ratio (n >=10) of plant sum.(D-H):Different ageing phases The phenotype of plant list leaf or cotyledon.(I):GmCRY2 expressions in wild type and two GmCRY2-RNAi transfer-gen plants Immunoblotting assay.Total protein extract is analyzed for 10%SDS-PAGE, and GmCRY2 antibody is used for immuning hybridization.NS, The non-specific band of GmCRY2 antibody is used to indicate applied sample amount control；GmCRY2-RNAi, GmCRY2 RNA disturb plant.

Figure 15 shows that GmCIB1 is overexpressed plant related phenotype of aging under continuous shine, wherein (A-C)：Wild type and The single leaf or cotyledon of different growing stage can be divided into three classes (such as Figure 12 according to aging aspects in GmCIB1-OX transfer-gen plants It is shown), the ratio shared by per class is such number and the ratio (n >=10) of plant sum.(D-H):Different ageing phases are planted The phenotype of the single leaf of strain or cotyledon.(I):YFP-GmCIB1 expressions in wild type and two GmCIB1-OX transfer-gen plants Immunoblotting assay.Total protein extract is analyzed for 10%SDS-PAGE, and GFP antibody is used for immuning hybridization.NS, GFP antibody Non-specific band is used to indicate applied sample amount control；GmCIB1-OX, GFP-GmCIB1 are overexpressed in wild type KN18.

Figure 16 shows the table of wild type and transfer-gen plant Determination of Chlorophyll content, photosynthetic rate and aging related genes Up to situation, wherein (A)：Chlorophyll content (white post) and leaf are green in the big single leaf of surrounding in wild type and each transgenic line Plain a:The measure (black post) of b ratios.FW：Fresh weight.Standard error is as shown in the figure (n=10).(B)：Light in leaf growth process Synthesize variation tendency.Selection is planted in the second compound leaf under continuous illumination as measure object, with LI-6400 instruments to after planting The plant of 31 days to 43 days is measured, and determines once within every two days.PSR：Photosynthetic rate.(C)：Wild type and each transgenic line In four aging related genes (GmAPGL5, GmWRKY53, GmSAGL12 and GmAGL12) rna expression situation, selected materials For the single leaf that will turn yellow under continuous illumination.Standard error is as shown in the figure (n=10).(D)：In wild type and each transgenic line Rna expression situation under the GmPaO blue lights processing of chlorophyll degradation related gene.Material process is：Grown under dark 12 days Seedling, is transferred under blue light (~22 μm of ol m-2s-1) and handles 6 hours, respectively under dark, blue light illumination 1 hour, 3 hours and 6 Hour materials (B1, B3 and B6).Standard error is as shown in the figure (n=3).

Figure 17 shows opening in GmCRY1-OX, GmCRY2-OX, GmCRY2-RNAi and GmCIB1-OX transfer-gen plant Flower phenotype, wherein (A)：Flowering time is determined；(B)：Number of blade when blooming.Plant is planted under continuous illumination (LL), works as open country When the raw compound leaf of type the 7th occurs, all plant are included into each transfer-gen plant and are transferred under the conditions of short-day (SD) (8 hours Illumination/16 hour dark).Standard error is as shown in the figure (n >=15).

Figure 18 shows GmFTs and GmTFLs the mRNA expressions in different genotype, wherein (A)：WT and GmCRY1- GmFTs and GmTFLs mRNA expressions in OX transfer-gen plants；(B)：In WT and GmCRY2-OX transfer-gen plants GmFTs and GmTFLsmRNA expressions；(C)：GmFTs and GmTFLs mRNA expressions in WT and GmCRY2-RNAi transfer-gen plants； (D)：GmFTs and GmTFLs mRNA expressions in WT and GmCIB11-OX transfer-gen plants；The material of measure be Figure 17 in retouch The plant stated takes the 1st seven compound leaf after continuous illumination goes to short-day growth 3 days.Standard error is as shown in the figure (n=3).

Figure 19 shows Yeast two hybrid assay GmCRY1, the interaction between GmCRY2 and AtCRY1 and GmCIBs, its In (A)：The analysis of histidine auxotrophy does not interact between showing GmCRY1 and GmCIBs.(B)：Histidine auxotrophy point Analysis shows the interaction for having blue light to rely between GmCRY2 and GmCIB1.(C)：The analysis of histidine auxotrophy shows AtGmCRY1 There is the interaction that blue light is relied on respectively between GmCIB1, GmCIB4, GmCIB8.Yeast cells containing albumen shown in coding exists (~22 μm of ol m-2s-1) growth is respectively used to histidine auxotrophy analysis in (A-C) for three days under dark or blue light.(+)：Ferment Mother cell, which is grown in, to be lacked in tryptophan and leucine, but the culture medium containing histidine.(-)：Yeast cell growth is lacking In tryptophan, leucine, and the culture medium of histidine.(D)：Betagalactosidase activity detects to analyze blue light (~30 μm of ol ) or dark lower interaction between GmCRY2 and GmCIBs m-2s-1.The yeast cells of albumen shown in incubated overnight expression is simultaneously pressed 1/5x dilutes, and is transferred under blue light and cultivates after dilution dark culturing to OD600=0.3-0.4.Standard error (n as shown in the figure =3).

Figure 20 shows the interaction that GmCRY2 is relied on GmCIB1 blue lights, wherein (A)：Betagalactosidase activity shows Show feux rouges (R30 ,~30 μm of ol m-2s-1), 2 hours GmCRY2 of blue light (B30 ,~30 μm of ol m-2s-1) or dark processing and Interaction between GmCIB1.(B)：Yeast cells feux rouges (R30 ,~30 μm of ol m-2s-1), the blue light of albumen shown in expression Betagalactosidase activity when (B30 ,~30 μm of ol m-2s-1) or dark different disposal time.(C)：Different blue light strengths Under the processing of (30~70 μm of ol m-2s-1) and light application time, beta galactosidase when GmCRY2 and GmCIB1 interact is lived Property.(A-C) albumen expressed by yeast cells is as shown in the figure.Standard error is as shown in the figure (n=3).(D)：BiFC experiments show Interaction in GmCRY2 and GmCIB1 bodies.The protoplast of the mesophyll cell of the Arabidopsis thaliana Seedlings of surrounding is grown under long-day It is detached for the plasmid of albumen shown in cotransformation expression figure.Blue light (~22 μ after dark culturing 12-14 hours after cotransformation Mol m-2s-1) processing 30 minutes or be still positioned in dark be used as control.A is arranged:YFP fluorescence；B is arranged:Autofluorescence；C is arranged:It is bright ；D is arranged:A, b, c are superimposed；Scale is 20 μm of (E)：Under blue light (~22 μm of ol m-2s-1) and dark processing, microscope is regarded The ratio of cell containing YFP fluorescence in 100 cells of Yezhong cotransformation expression nYFP-GmCIB1 and cCFP-GmCRY2 plasmids Value.(F)：CoIP experiments show that the GmCRY2-GmCIB1 of blue light dependence complex can be formed in tobacco body.Using containing The Agrobacterium injection tobacco leaf of albumen plasmid shown in coding.WT and injected plant it is dark in place three days after, be transferred to indigo plant Under light (~22 μm of ol m-2s-1) or stay in the dark.MYC antibody is used for the immuning hybridization of total protein and MYC- jel products, The protein film is peelled off Flag antibody immuning hybridization again is used after antibody.

Figure 21 shows the interaction that GmCRY2N terminal domains are relied on GmCIB1 blue lights, wherein (A)：GmCYR2 and GmCIB1 protein structure schematic diagrames.The domain of GmCYR2 and GmCIB1 interactions is labeled out (pink colour shade).(B)： Auxotrophy experiment indicates the interaction that blue light is relied between GmCRY2N and GmCIB1.The name of domain is using (A) figure institute Show.The yeast cells of albumen shown in expression is grown on the culture medium in fig. 19 a, is respectively placed in dark or blue light (~25 μ Mol m-2s-1) under.(C)：Betagalactosidase activity when GmCRY2N or GmCRY2C domains are acted on GmCIB1.Expression The yeast cells of shown albumen is placed under dark or blue light (25 μm of ol m-2s-1) (left side), or carries out different blue light strengths (B30 to B70,30~70 μm ol m-2s-1) and light application time processing (right side).

Figure 22 shows the interaction that GmCRY1PHR domains are relied on GmCIB1 blue lights, wherein (A)：GmCYR1 and GmCIB1 protein structure schematic diagrames.The domain of GmCYR1 PHR domains (GmCRY1N485) and GmCIB1 interaction is used Pink colour shade is marked.(B)：Auxotrophy experiment shows each domain of GmCRY1 and GmCIB1 interphase interaction situations. The name of domain is using shown in (A) figure.The yeast cells of albumen grows described in Fig. 3 .8A on culture medium shown in expression, point It is not placed under dark or blue light (~25 μm of ol m-2s-1).(C)：When GmCRY1N or GmCRY1C domains are acted on GmCIB1 Betagalactosidase activity.The yeast cells of albumen shown in expression is placed under dark or blue light (25 μm of ol m-2s-1) (left side), Or carry out the processing (right side) of different blue light strengths (B30 to B70,30~70 μm ol m-2s-1) and light application time.(D)：BiFC Experiment shows GmCRY1 or GmCRY2 different structure territory and interaction situations of the GmCIB1 in protoplast.Shown in expression The plasmid of albumen passes through Fig. 3 .9 (D) methods described cotransformation protoplast.After blue light (22 μm of ol m-2s-1) is handled 30 minutes In fluorescence microscopy Microscopic observation.Scale is 10 μm.

Figure 23 shows the interaction that GmCIB1N terminal domains are relied on GmCRY2 blue lights, wherein (A)：Auxotrophy Experiment shows GmCIB1N (GmCIB1N217) or GmCIB1C (GmCIB1C218-420) phase interaction between GmCRY1 or GmCRY2 Use situation.The yeast cells of albumen shown in expression is grown on the culture medium in fig. 19 a, be respectively placed in dark or blue light (~ 25μmol m-2s-1).(B)：BiFC experiments show GmCIB1N and GmCRY2 interaction situations.(C-D)：GmCIB1 difference knots Betagalactosidase activity when structure domain is with GmCRY1 or GmCRY2 effects.The yeast cells of albumen shown in expression be placed in it is dark or Under blue light (25 μm of ol m-2s-1) (C), or carry out different blue light strengths (B30 to B70,30~70 μm of ol m-2s-1) and light According to the processing (D) of time.

Figure 24 shows GmCIB1 and interactions of the GmCRY1/GmCRY2 in soybean body, wherein (A, B)：Co-IP is tried Test in display soybean body the formation WT (KN18) of the special GmCRY2-GmCIB1 or GmCRY1-GmCIB1 complexs of blue light and 35S::YFPGmCIB1 transfer-gen plants are planted respectively to be grown three weeks under the conditions of short-day.It is small that seedling is transferred to processing 18 under dark When after carry out blue light (B, 22 μm of ol m-2s-1) processing, processing time be (D, 0 minute；B30,30 minutes；B60,60 minutes B120,120 minutes).YFP antibody is used for the immuning hybridization of total protein (Input) and GFP- jel products (FP-IP), by the egg Tunica albuginea, which is peelled off, uses GmCRY2 (A) or GmCRY1 (B) antibody immuning hybridization again after antibody.

Figure 25 shows the interaction that the external blue lights of GmCIB1 and GmCRY1/GmCRY2 are relied on, wherein (A)：In vitro Pull-down experiments show the interaction of GmCRY2 and GmCIB1 blue lights specifically.Gel containing Flag antibody and table respectively Insect cell lysate up to GmCIB1-flag and GmCRY2 albumen is mixed.Mixture is in blue light (B, 22 μm of ol M-2s-1) and under dark handle, processing time is as shown in the figure.With reference to albumen rinsing after elute with Flag (GmCIB1- Flag) antibody carries out immuning hybridization, peels off after the antibody with GmCRY2 immuning hybridizations again.(B)：External pull-down experiments table The interaction of bright GmCRY1 and GmCIB1 blue lights specifically.Test method is as described such as figure (A), with reference to albumen rinsing after under elution To carry out immuning hybridization with Flag (GmCIB3-Flag) antibody, peel off after the antibody with GmCRY1 immuning hybridizations again.

Figure 26 shows the interactions of GmCIB1-DNA in vitro.RBSS result of the tests show that GmCIB1 is protein bound DNA sequence dna and the conserved sequence for being listed in bottom.Bottom sequence is the protein bound DNA sequence dnas of GmCIB1, after bottom is compares CANNTG.

Figure 27 a-e show the mutual of GmCIB1 and GmFT3, GmFT4, GmTFL1, GmTFL3 and GmWRKY53 chromosome Effect, wherein upper drawing shows selected gene (Figure 27 a-GmFT3, Figure 27 b-GmFT4, Figure 27 c-GmTFL1, Figure 27 d- GmTFL3, Figure 27 e-GmWRKY53) promoter region (arrow), 5 ' UTR (black line), extron (grey square frame), introne (black Square frame), 3 ' UTR (black line).Black circles represent the position where E-box (CANNTG).Region of DNA domain shown in black line is set Primer can expand to or amplification less than region.Figure below shows ChIP-qPCR results.Select wild type (WT) or YFP- GmCIB1 is overexpressed the seedling of plant (GmCIB1-OX) as the material of separation chromosome segment (~500bp), plant strain growth 21 Dark lower placement 18 hours, are transferred under blue light (Blue, 22 μm of ol m-2s-1) and handle (left side) or continue to be placed on dark after it (Dark) under (right side), immunoprecipitation is carried out with GFP antibody, obtained DNA precipitates the primer for qPCR.%Input:1% The amount of beginning chromosome segment is used for Input.Standard error is as shown in the figure (n=3).

Figure 28 shows the interaction of the GmCRY2a and GmCIB1 in response to blue light in yeast cells.(A) beta galactose Glycosides enzyme (β-gal) analysis shows are in blue light (30 μm of ol m^-2s^-1) or the yeast cells of dark treatment in GmCRY2a and GmCIB1 and The interaction of GmCIB1 homologues.(B) β-gal analysis shows are in feux rouges (R30,30 μm of ol m^-2s^-1), blue light (B30,30 μ mol m^-2s^-1) or dark (D) handle the interaction of GmCRY2a and GmCIB1 in the yeast cells of 2 hours.Show that expression is each Plant bait and the yeast cells of prey.The independent average value repeated of display three and standard deviation (A, B).(C) β-gal analysis shows Within the shown period in response to it is different can flow rates blue light (D, secretly；B30,30μmol m^-2s^-1；B50,50μmol m^-2s^-1； B70,70μmol m^-2s^-1) GmCRY2a and GmCIB1 interaction.β-gal the activity respectively handled is in irradiation with the time It is linear to improve, and represent point and the linear regression curves fitting of β-gal activity.(D) it is different can flow rates linear regression curves Slope is as shown in (C).Show that three independent average (± SD) repeated (are carried out by SPSS programs in figure Jonckheere-Terpstra trend analyses, P=0.003, n=3), show measuring depending on luminous intensity for binding kineticses. (E) signal of GmCRY2a and GmCIB1 domain (khaki shade) needed for display GmCRY2a-GmCIB1 interactions Figure.

Figure 29 shows the interaction of the GmCRY2a and GmCIB1 in response to blue light in external and plant cell.(A) The GmCRY2a-GmCIB1 that pull-down analysis shows blue light dependence interacts in vitro.Be coupled anti-Flag antibody (α- Flag the insect of sepharose 4B) and expression 6His-GmCIB1-Flag (GmCIB1) and 6His-GmCRY2a (GmCRY2a) is thin The lysate mixing of born of the same parents.Mixture carries out blue light (B, 22 μm of ol m^-2s^-1) or dark treatment shown in time.With reference to protein Elute after washing, and analyzed by the Western blotting of anti-Flag antibody (α-Flag), slitting and anti-with anti-GmCRY2a Body (α-GmCRY2a) is detected again.(B) BiFC analysis shows the plasmid with coding nYFP-GmCIB1 and cCFP-GmCRY2a The GmCRY2a-GmCIB1 interactions of blue light dependence in the protoplasts of Arabidopsis thaliana broken by ultrasonic of cotransfection.Long day (LD, 16h light/8h Incubated in the plasmid cotransformation of protein, dark shown in the mesophyll protoplast coding of the 4 week old plants grown under the conditions of secretly) 12 hours and it is subsequently transferred to blue light (22 μm of ol m^-2s^-1) in 30 minutes, afterwards carry out confocal microscopy analysis.A columns, YFP is glimmering Light；B columns, autofluorescence；C columns, bright field；D columns, the merging rods on a, b and c column, 10 μm.(C) to display BiFC fluorescence signals The number of protoplast is counted.Each sample includes at least 50 protoplasts.Show average value and standard deviation (n=3). P=0.00026 (Student ' s t inspections).(D) in vitro Co-IP analysis shows tobacco (Nicotiana Benthamiana the formation of the blue light dependence of GmCRY2a-GmCIB1 complexs in).As shown, tender leaf is to carry coding The Agrobacterium infiltration of GmCIB1-Flag (GmCIB1) or GmCRY2a-Myc (GmCRY2a) plasmid, is kept for 3 days in the dark, And it is subsequently exposed to blue light (B, 22 μm of ol m^-2s^-1) 1 hour or be maintained in dark (D).Protein extract has with coupling The agarose of anti-Myc antibody is incubated 60 minutes at 4 DEG C together.Collect pearl and wash 3 times, then elution IP products.Total protein The Western blotting of matter extract (Input) and IP products is suitable using anti-Myc antibody (α-Myc) and anti-Flag antibody (α-Flag) Carry out to sequence.(E) in Co-IP analysis shows soybean the blue light dependence of GmCRY2a-GmCIB1 complexs formation.Wild type (WT) soybean KN18 and overexpression 35S::The soybean transgene system (being 2) (GmCIB1-OX-2) of YFP-GmCIB1 transgenosis is in SD Lower growth 2 weeks.Plant be transferred in 18 hours and exposed to blue light (B, 22 μm of ol m^-2s^-1) shown in time (D, 0min； B30,30min；B60,60min；B120,120min).The egg that the agarose for having anti-YFP antibody (α-YFP) using coupling is carried out The Western blotting of white matter extract (Input) and IP products is detected by anti-YFP antibody (α-YFP), slitting and passed through Anti- GmCRY2a antibody (α-GmCRY2a) is detected again.

Figure 30 .GmCIB1 are the DNA- associated proteins interacted with E box (CANNTG).(A) via random incorporation position The comparison for the DNA sequence dna that point selection (RBSS) analysis is selected by GmCIB1 protein.More than 90% selected by GmCIB1 Sequence include E box elements (CANNTG), such as (http by being calculated in sequence of threads:// Weblogo.berkeley.edu/) (B) competes the phase interaction for the E-box DNA that EMSA analysis shows GmCIB1 and DIG- is marked With.GmCIB1-DNA interacts to be competed with unlabelled wild type E box (Ewt) or mutant E box (Em4), in such as (C) It is shown.Black wedge shape represents all dosages (12.5X, 25X, 50X, molar excess) of competitor.(C) wild type E-box DNA (Ewt) and mutant E-box sequences (Em) competitor DNA sequence dna.(D) use the competition EMSA's of Ewt or other competitors Quantitative analysis.Unlabelled competitive oligonucleotide person exist (+UOC) under GmCIB1- bonding probes signal divided by be not present The signal of unlabelled competitive oligonucleotide person (- UOC) GmCIB1- bonding probes, and it is expressed as RBU (with respect to bonding unit).

The activating transcription factor that Figure 31 .GmCIB1 are adjusted by GmCRY2 in response to blue light.(A) E-box drivings it is double- The structure of Luc reporter genes and restructuring E-box DNA sequence dna.(B) image of luciferase activity is shown.(C) relative report Double-luc analyses of gene activity.(D) GmCRY2a is for DNA- binding activity of the GmCIB1 and E-box DNA in response to blue light Inhibition effect GmCIB1-DNA binding analysis.

Figure 32 .GmCRY2a and GmCIB1 adjust leaf aging.(A-C) Western blotting show GmCRY2a-ox plants, The expression of GFP-GmCRY2a fusion proteins or endogenous GmCRY2a albumen in GmCIB1-RNAi plants or wild-type plant (WT), Or in GmCIB1-ox plants YFP-GmCIB1 fusion proteins expression.Examine two independent strains of each genotype.Always Protein extract is analyzed in 10%SDS-PAGE for exempting from for being detected by α-GmCRY2a (A, B) or α-GFP antibody (C) Epidemic disease trace.The non-specific band (NS) recognized by antibody is used separately as loading control.(D-F) image of typical cotyledon shows The different degrees of aging of strain shown in shown growth phase.WAS, sowing Later Zhou Dynasty, one of the Five Dynasties number.(G-I) cotyledon and single leaflet are according to it It is divided into three groups of (greens, no aging in the aging degree of shown stage of development；Yellow, slight aging；Dry, complete aging).Leaf Senescence index is calculated as the percentage of each group of the total number of sheets (n >=10) relative to single plant.(J-O) corresponding gene type (J- L chlorophyll content (chlorophyll a+b) and GmCRY2a-OX-1 (M)), GmCRY2a-RNAi-1 (N), GmCIB1-OX-2 (O) The chlorophyll a of leaf:B ratios (M-O) and WT comparison.Shown in collecting in the stage of development in continuous white light growing plants two The biased sample of individual single leaflet and the leaflet of the first two three is measured for the two.Show average value and standard deviation (n=3).

Figure 33 genetically engineered soybeans show leaf senescent phenotypes.(A) genetically engineered soybean (CRY2ox) for being overexpressed GmCRY2a shows The leaf senescent phenotypes of delay are shown.Plant grows 8.5 weeks in continuous illumination.(B) expression GmCRY2a-RNAi transgenosis is big Beans (CRY2RNAi) show the leaf senescent phenotypes of acceleration.Plant grows 7.5 weeks in continuous illumination.(C) it is overexpressed GmCIB1 Genetically engineered soybean (CIB1ox) show the leaf senescent phenotypes of acceleration.Plant grows 7.5 weeks in continuous illumination.

Figure 34 .GmCIB1 combination GmWRKY53b chromatin with promote its mRNA express.(A) quantitative RT-PCR shows even GmWRKY53b rna level in the leaf with shown phenotype grown in continuous light.Relative expression units (REU) pass through Standardization of the GmWRKY53b signals relative to the signal of ACTIN11 internal contrasts is measured, and shows standard deviation (n= 3).2T2,2T4,2T6 or 2T8:Second three leaflet collected from 2WAS, 4WAS, 6WAS or 8WAS (the sowing Later Zhou Dynasty, one of the Five Dynasties) plant. GmWRKY53b differential expression is examined by Student t and analyzed between WT and transgenosis system:For 2WAS's or 6WAS GmCRY2a-ox and 6WAS GmCRY2a-RNAi or GmCIB1-ox, respectively p=0.007,0.046,0.006 or 0.005. (B) chart describes the promoter (arrow) and 5 ' UTR (white box) of the prediction of GmWRKY53b genes.Black circles represent E Box (CANNTG) position.The different zones of 2880bp WRKY53b genomic DNAs are expanded by PCR.Short underscore represents single The center of individual overlapping PCR products, asterisk represents the transcription initiation site (TSS) of presumption, numerical monitor TSS upstreams (+) or under Swim the position (bp) of (-).(C) using shown primer sets to GmCIB1-OX-2 the and WT plants collection from different developmental phases The ChIP-qPCR analyses that sample is carried out.Plant is in continuous emerging middle growth.U2, U3 or U4 represent 2WAS, 3WAS or 4WAS stage Single leaflet.ChIP samples are by anti-YFP Antibody preparations and carry out qPCR analyses.ChIP-qPCR result by relative to The IP signal normalizations of corresponding input signal and quantify, it is shown that standard deviation (n=3).

Figure 35 .GmCIB1 combine the E-box DNA sequence dnas being located in GmWRKY53b chromatin.EMSA shows GmCIB1 eggs In vain with the compatibility for each DNA probe for corresponding respectively to chromatinic c, f, k, o or q regions of GmWRKY53b, as illustrated in figure 34b. The DNA sequence dna of each probe is in bottom.

Figure 36 blue lights suppress GmCIB1 and the chromatinic combinations of GmWRKY53b.(A) GmCIB1 in response to blue light with The comparison of the compatibility of the chromatinic each regions (a-r, Figure 34 B) of GmWRKY53b.Grown in short photoperiod day (8hL/16hD) 3 week old plants be transferred in dark 18 hours, then keep in the dark or exposed to blue light (22 μm of ol m^-2s^-1).Collect First three leaflet is analyzed for ChIP.DBU (different bonding units) is calculated by following formula:[the IP/ dark places of (GmCIB1/WT) Manage the input of (GmCIB1/WT) of sample]/[input of (GmCIB1/WT) of the IP/ blue lights processing sample of (GmCIB1/WT)], Show standard deviation (n=3).GmCIB1 passes through Student t from the different compatibilities in the chromatinic single regions of GmWRKY53b Inspection is analyzed, for " a ", " f " or " k " region, respectively p=0.8,0.002 or 0.007.(B) working model is depicted Suppress for the blue light of the GmCRY2a- mediations of the GmCIB1- dependences activation of leaf aging.PHR and CCE:Photolyase homologous region Domain and the extension of CRY C- ends.

Describe in detail

Following definition and method are provided to preferably define the present invention and instruct this area skill in an embodiment of the present invention Art personnel.Unless otherwise noted, otherwise term will be understood according to the conventional use of implication of various equivalent modifications.

As used herein, term " restructuring " refer to generally do not found in nature and thus by people intervene produce Raw DNA and/or protein and/or organism form.Such people, which intervenes, can produce recombinant DNA molecules and/or recombinant plant. As used herein, " recombinant DNA molecules " be comprising can not together naturally occurring DNA molecular combination and be what people intervened As a result DNA molecular, such as by the DNA molecular constituted of at least two heterologous DNA moleculars each other, and/or be artificial DNA molecular that is synthesis and including polynucleotide sequence derived from the polynucleotide sequence from nature is typically found in, And/or the side of the transgenosis comprising the genomic DNA for being artificially integrated into host cell and the genome for the host cell being connected Wing DNA DNA molecular.The example of recombinant DNA molecules is described herein because of transgenosis to arabidopsis, corn or paddy gene Insertion in group and the DNA molecular produced, it can ultimately result in recombinant DNA and/or protein molecule and be expressed in the organism.

As used herein, term " transgenosis " refers to the polynucleotides point for being artificially integrated into the genome of host cell Son.Such transgenosis can be heterologous for host cell.Term " genetically modified plants " refers to comprising such transgenosis Plant.

As used herein, term " DNA ", " DNA molecular ", " nucleic acid molecules " refer to the double-strand of genome or synthesis source The polymer or nucleic acid molecule of DNA molecular, i.e. deoxyribonucleotide bases, read from 5 ' ends (upstream) to 3 ' ends (downstream) Read.

As used herein, term " DNA sequence dna ", " nucleotide sequence " and " polynucleotide sequence " refers to generally from 5 ' The sequence of the nucleotides for the DNA molecular that (upstream) end is shown to 3 ' (downstream) ends.

As used herein, term " separation " refers to molecule at least in part with usual in its autologous or native state In be coupled other molecules separation.In one embodiment, term " gene of separation " or " DNA molecular of separation " are Refer to such DNA molecular, its at least in part with autologous or native state usual flank be connected the nucleic acid of the DNA molecular Separation.Thus, for example being blended in the DNA of regulation and control that they are not coupled generally or coded sequence as the result of recombinant technique Molecule is considered herein as separation.Even if this quasi-molecule divides when the chromosome for being integrated into host cell or with other DNA It is also considered as separation that son is present in when in nucleic acid solution together.

As described herein, term " stringent condition " is typically referred to by Sambrook et al., and 1989 and by Haymes et al. ：Nucleic acid hybridization,A practical approach,IRO Press,Washington,DC (1985) condition described in.Suitable stringent condition (such as about 45 DEG C of 6.0x sodium chloride/citric acid of DNA hybridization The 2.0x SSC washings of sodium (SSC), then 50 DEG C) it is known to those skilled in the art, or can be in Current Found in Protocols in Molecular Biology, John Wiley＆Sons, N.Y., 1989,6.3.1-6.3.6.Example Such as, the salinity in washing step can be from 50 DEG C of about 2.0x SSC low stringency condition to 50 DEG C of about 0.2x SSC High stringency conditions.In addition, the temperature in washing step can increase to about from the low stringency condition of room temperature (about 22 DEG C) 65 DEG C of high stringency conditions.Temperature and salt can change, or one kind in temperature or salinity can keep constant, and another One variable changes.For example, medium stringency condition can be about 2.0x SSC salinity and about 65 DEG C of temperature, High stringency conditions can be about 0.2x SSC salinity and about 65 DEG C of temperature.In one aspect of the invention, this hair Bright gene has SEQ ID NO:1 or SEQ ID NO:Nucleotide sequence shown in 3.In another aspect of the present invention, this hair Bright gene and SEQ ID NO:1 or SEQ ID NO:Nucleotide sequence shown in 3 has 80%, 81%, 82%, 83%, 84%, 85%th, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.In the further aspect of the present invention, gene and SEQ ID NO of the invention:1 or SEQ ID NO:Nucleotide sequence shown in 3 has 95%, 96%, 97%, 98%, 99% and 100% sequence identity.

The present invention gene also include GmCIB1 genes by one or more of nucleotides occur missing, displacement, The mutation of insertion or addition and derivative obtained variant sequence thereof.Gene mutation refer to that genomic DNA molecule occurs it is unexpected, can The variation phenomenon of heredity.From molecular level, gene mutation refers to that base-pair composition occurs in structure or arranges suitable for gene The change of sequence.The generation of gene mutation can be it is spontaneous can also induce, the mode of induced mutations includes physical mutagenesis (such as gamma-rays, x-ray, ultraviolet and neutron current), mutagenesis (such as alkylating agent, base analogue and antibiotic) and life Thing mutagenesis (such as some viruses and bacterium).Furthermore, it is possible to make DNA molecular occur in specified location using recombinant DNA technology Specific change, so as to realize directed mutagenesis.Those skilled in the art can use any in these known method of mutagenesis Plant includes the variant that missing, displacement, insertion or the GmCIB1 genes for the mutation added occur for one or more nucleotides to obtain Sequence.

Arabidopsis (A.thaliana), also known as Arabidopsis thaliana, arabidopsis thaliana, Arabian cron.This slight flowering plant is One of model organism in plant science, including science of heredity and development of plants research.Its institute's role in botany Just seemingly small white mouse in medical science such as drosophila in science of heredity.The advantage of arabidopsis is that small (1 teacup can be planted plant Several), often for time short (from germinateing to blooming be no more than 6 weeks), knot many (every plant can produce many grain seeds), live Power is strong (can make artificial culture with ordinary culture medium).The genome of arabidopsis is currently known minimum in Plant Genome. The overall length of each haplochromosome group (n=5) only has 70,000,000 base-pairs, i.e. only the 1/80 of chromosome of wheat group leader, this Just making clone, it has correlation gene to be comparatively easier.Its whole gene group is in the international mouseearcress genes of 2000 Nian You Group cooperative alliances joint is completed, and is also the Plant Genome of first sequencing analysis.Arabidopsis is self-pollination plant, and gene is high Homozygosis is spent, it is very high with chemical factors processing mutation rate, it is readily available the deficiency of various metabolic functions.For example with containing herbicide Culture medium is screened, and the mutation rate for typically obtaining anti-herbicide is 1/100000.Due to there is these above-mentioned advantages, so arabidopsis It is the good material for carrying out genetics research.

In addition to building up flower development ABC models classical in research in phytomorph, nearly ten years, plant scientists profit Arabidopsis modular system is used, similar research has been carried out with the development of organ to plant different tissues.It is prominent by a large amount of arabidopsis The analysis of variant, development of the scientists to plant roots, stem, leaf, flower, embryo and seed, to disease resistance of plant and resistance machine Reason, and signal transduction process etc. caused by the relevant hormone of various vital movements, light and envirment factor has been carried out deep Research, extreme enrichment understanding of the mankind for plant vital activity inherent mechanism.

Plant in the present invention includes but is not limited to monocotyledon and dicotyledon, including crop plants (such as jade Rice), Btassica (for example, wild cabbage, Chinese cabbage, leaf mustard), especially can be used as seed oil source Brassica species, alfalfa (Medicago sativa), paddy rice (Oryza sativa), naked barley (Secale cereale), sorghum (Sorghum Bicolor, Sorghum vulgare), grain is (for example, pearl millet (Pennisetum glaucum), broomcorn millet (Panicum Miliaceum), millet (Setaria italica), dragon's paw grain (Eleusine coracana)), sunflower (Helianthus Annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), Tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanut (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot Esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), mandarin tree (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), crocodile Pears (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew nut (Anacardium Occidentale), macadamia (Macadamia integrifolia), apricot (Prunus amygdalus), beet (Beta vulgaris), sugarcane (Saccharum spp.), oat, barley), vegetables (such as tomato (Lycopersicon Esculentum), lettuce (such as Lactuca sativa), green soya bean (Phaseolus vulgaris), Kidney bean (Phaseolus Limensis), the member such as cucumber (C.sativus) of pea (Lathyrus spp.) and Cucumis, muskmelon (C.cantalupensis) and muskmelon (C.melo)), ornamental plant (such as azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), lotus (Hibiscus rosasanensis), rose (Rosa spp.), tulip (Tulipa spp.), daffodil (Narcissus spp.), morning glory (Petunia hybrida), carnation (Dianthus Caryophyllus), poinsettia (Euphorbia pulcherrima) and chrysanthemum).In specific embodiment, the present invention Plant be crop plants (for example, corn, alfalfa, sunflower, Btassica, soybean, cotton, peanut, sorghum, wheat, Grain, tobacco etc.).In some embodiments, it is preferred being arabidopsis or soybean.

The present invention is cloned into 9 CIB1 homologous genes from soybean (Glycine max), analyzes its gene structure special Point, the spatial and temporal expression profile of different tissues organ and developmental stage mRNA, under the conditions of the different photoperiods with different latitude originate MRNA expression patterns in soybean varieties, and gene overexpression is subjected to Primary Study in arabidopsis to its function, to grind Study carefully the effect that GmCIB genes are played in soybean growth process, Photoperiod, be that the functional study of GmCIB genes is carried For certain theoretical reference.

Genome structure analysis display, GmCIB family genes member all extrons containing different numbers and length and interior UTR containing son, and different length.Complicated genome structure may can also be produced and modified after different genetic transcriptions, simultaneously Along with different gene functions.

Subcellular Localization is found out, although GmCIB is transcription factor but Subcellular Localization is not merely to be located at cell In core, GmCIB6 and GmCIB7 albumen has expression in nucleus and cytoplasm, implys that they may be in cytoplasm It can function.

Each member of spatial and temporal expression analysis display of GmCIBs different tissues organs is different in the expression of different tissues organ, table Expression patterns are also more complicated.See that most gene expression quantity in the organ of flowering period is higher on the whole, thus it is speculated that it is possible this Family gene plays a role in soybean blossoming regulation and control.Research at present shows that arabidopsis CIB1 major functions are that regulation and control are planted Thing is bloomed, and can be speculated for more than and be provided theoretical foundation.The gene of some in GmCIBs also has higher in the histoorgan of single leaf phase Expression, such as GmCIB1/2/3/9 expression quantity in the single leaf just opened is very high, and these possible genes are except there is regulation and control of blooming Effect, may also be participated in other regulatory pathways.

Soybean CIB gene different development stage expression pattern comprehensive analysis is seen, in addition to GmCIB2, other gene aerial parts Expression quantity it is all relatively low, most gene expression quantity higher period is concentrated mainly on nutrient growth later stage, reproductive growth rank at initial stage Section is florescence, and this shows that these genes may participate in the regulation and control of blooming of soybean.But also some genes are at the beginning of reproductive growth Also there is very high expression quantity in stage phase such as single leaf period, and GmCIB1 is very high in single leaf phase and the expression quantity come into bloom, and Develop interstage expression quantity but very low.The possible gene plays important in the primary stage of nutrient growth and reproductive growth Regulating and controlling effect, is served both functions to developmental regulation.And GmCIB6 overall expression is all very low, possible gene pairs development Effect very little in regulation and control, or the gene expressed by certain signal induction.

Both expression patterns are complex in photoperiod and biological clock analysis display, GmCIB family members.On the whole Most gene is regulated and controled jointly by photoperiod and biological clock, and simply two kinds of regulatory mechanisms are occupied an leading position difference.Make a general survey of all Member, GmCIB4 has obvious biological clock rhythm in the expression of long-day conditions, and under short-day, biological clock rhythm substantially by To Photoperiod antagonism, when be transferred to continuous illumination be the overall expression of gene heighten and under the conditions of continuous darkness gene one Directly maintain very low expression.Imply that regulatory mechanism of the gene under the conditions of LD and SD is variant, this may also be by It is strict short-day plant in soybean, there may be different regulatory mechanisms under conditions of two kinds of illumination conditions are totally different.Although Photoperiod and biological clock regulate and control the expression of gene jointly, and the further analysis to each member finds that most gene (is removed Outside GmCIB1 and GmCIB9) under the conditions of SD and LD, there is obvious or not apparent biological clock rhythm, in the expression of one day all There are peak value and valley to occur, be transferred to biological clock rhythm after LL and DD and cut down, and Photoperiod is occupied an leading position, under the conditions of LL The overall expression of gene is improved, and gene maintains this relatively low expression always under the conditions of DD.GmCIB2 makes an exception, in LL Under the conditions of the gene expression be substantially suppressed, and expression quantity is of a relatively high under the conditions of DD, illustrates GmCIB2 expression light According to suppression.

GmCIBs expression patterns are shown in the soybean varieties of different latitude source, are not found between the expression pattern and latitude of gene Clear and definite correlation.Global analysis shows, in the kind in different latitude source, and the expression of gene is all under SD or LD environment There is the increase gradually increased trend with light application time, during increase, some genes exist in the expression of some kind genes Moment at high noon (N) reaches gradually to reduce after peak value, and some can continue to increase until reaching peak value before dark arriving (E). The pattern gradually reduced is presented in the expression of seldom a part of gene.

The GmCIBs being cloned into is built and is overexpressed plant expression vector arabidopsis thaliana transformation, obtained transfer-gen plant is entered Row phenotypic analysis.Under the conditions of long day and short day, the transgenic arabidopsis for being overexpressed GmCIB1/4/5/6/8 shows prematurity Phenotype, it is similar with arabidopsis CIB1 function.Downstream target gene AtFT is expressed in the transfer-gen plant for being overexpressed GmCIB1 Level detection display, AtFT expression is apparently higher than wild type, and turning for AtFT can be improved by illustrating external source GmCIB1 expression Record level is so as to promote plant prematurity.Although functions of the GmCIB1 in arabidopsis is similar to CIB1, soybean is typical case Short-day plant, and arabidopsis is long-day plant, and both Regulation Mechanisms are possible to different, so soybean heredity need to be carried out Function of the GmCIB genes in soybean is further studied in conversion.

In addition, inventor by yeast two-hybrid assay preliminary identification GmCIB1 and GmCRY2 special phases under blue light Interaction.This research is by by mutual between means research GmCRYs and the GmCIB1 albumen such as the double miscellaneous, BiFC and Co-IP of yeast Effect；The Genetic Transformation of Soybean strain that GmCRY is knocked out with GmCIB1 gene overexpressions or RNAi is further obtained, with reference to transgenosis The phenotypic analysis and ChIP of offspring tests to find GmCIB1 downstream effects target gene, is transmitted so as to analyze GmCRY in soybean The GmCIB signal paths of blue light signals, are that soybean photoperiod and the further investigation of leaf senile regulation and control and germplasm innovation lay base Plinth.

Obtain GmCRY1-OX using agriculture bacillus mediated cotyledonary node method for transformation, GmCRY2-OX, GmCRY2-RNAi and GmCIB1-OX genetically engineered soybeans, carry out phenotypic analysis to transfer-gen plant respectively；Utilize yeast two-hybrid, BiFC and immune Co-precipitation etc. means have studied GmCRYs and GmCIB1 by interaction；Analyze what GmCIB1 and DNA interacted in vitro Site, and pass through ChIP Preliminary Experiment Screening and Identifications GmCIB1 downstream effects target gene.Mainly obtain drawing a conclusion：

GmCRY1-OX, GmCRY2-OX, GmCRY2-RNAi and GmCIB1-OX genetically engineered soybean phenotypic analysis result shows, Under continuous illumination, being overexpressed GmCRY2 causes plant leaf aging to postpone, and in GmCRY1-OX, GmCRY2-RNAi and There is the phenotype that blade shifts to an earlier date aging in GmCIB1-OX genetically engineered soybeans, and initial guess GmCRY1 and GmCIB1 is adjusted in leaf senile Play a part of just regulating and controlling during control, and GmCRY2 may suppress the aging of blade.

Be overexpressed GmCRY1 and GmCRY2 and knock out genetically engineered soybean and show Blooming, and be overexpressed GmCRY2, GmCIB1 then causes soybean evening to be bloomed, and implys that GmCRY1 Accelerate blooms, and GmCRY2 and GmCIB1 negative regulation soybean blossomings.Ferment Female double cross and BiFC experiments show, GmCRY2 and GmCIB1 have the interaction of blue light specifically, and this interaction with The enhancing of light intensity and the increase of light application time and strengthen.GmCRY1 and GmCRY2 PHR domains participate in and GmCIB1 blue lights according to Bad interaction.In vivo (GmCIB1-OX genetically engineered soybeans) and external (insect protein expression system) Pull-down test into One step demonstrates the interaction that blue light is relied between GmCRY1/GmCRY2 and GmCIB1.

GmCIB1 shows that GmCIB1 is combined with E-box with interaction result external DNA, ChIP experiment displays, GmCIB1 is combined with the E-box of arabidopsis FT homologous gene GmFT3 and GmFT4 chromosomal regions, also can be with aging related genes E-box in WRYK53 homologous gene GmWRYK53 promoter region Matrix attachment regions is combined, and this combination does not have strict Blue light specificity, thus it is speculated that GmCIB1 may be bloomed in soybean body by negative regulation GmFT expression inhibiting, promotes aging related Gene such as GmWRYK53 transcription and then promotion leaf senile.

The cloning and expression pattern analysis of embodiment 1, soybean CIB family genes

There is the special phase of blue light with cryptochrome CRY2 to first by yeast two-hybrid screening in arabidopsis within 2008 The albumen of interaction, is named as CIB1 (CRY-interacting bHLH1).CIB1 is a member in bHLH families. In yeast cells, CIB1 and CRY2 interaction are that blue light wavelength is special, and the intensity interacted is with blue light strength Enhancing and light application time increase and strengthen.Its N-terminal domain of CIB1 structure function studies have shown that is able to carry out and albumen The function of interaction；And the C-terminal domain containing bHLH motifs participates in the interaction with DNA, so as to be made by cis Regulate and control purpose target gene with element.In arabidopsis CIB1 by and E-box (CANNTG) combinations of FT promoter regions improve FT transcriptional level, so as to start flowering of plant.

This research is cloned into 9 CIB1 homologous genes from soybean (Glycine max), analyzes its gene structure special Point, the spatial and temporal expression profile of different tissues organ and developmental stage mRNA, under the conditions of the different photoperiods with different latitude originate MRNA expression patterns in soybean varieties, and gene overexpression is subjected to Primary Study in arabidopsis to its function, to grind Study carefully the effect that GmCIB genes are played in soybean growth process, Photoperiod, be that the functional study of GmCIB genes is carried For certain theoretical reference.

1. materials and methods

1.1 material

1.1.1 vegetable material

(1) soybean material：

Soybean cultivates agriculture 18 (KN18) and 10 other the cultivated soybean kinds, plants in greenhouse, condition of culture is：Long-day (16h illumination/8h is dark) or short-day (8h illumination/16h is dark), cultivation temperature is 25-28 DEG C.Developmental stage and histoorgan Sample：Soybean is cultivated into agriculture 18 (KN18) to plant under the conditions of short day, cultivation temperature is 25-28 DEG C.

Overground part seedling is taken before single leaf opens；Single leaf phase of opening takes root, hypocotyl, epicotyl, cotyledon, single leaf and stem Point；Root, stem, Dan Ye, the first compound leaf, the second compound leaf, the 3rd compound leaf and flower are taken in florescence (the 4th compound leaf phase)；The 7d after spending, 14d and 21d take the Fruit pod and seed of different development stage；Answered respectively in single leaf phase, the first compound leaf phase, the second compound leaf phase, the 3rd Ye Qi, the 4th compound leaf phase (florescence) take aerial part.Illumination starts rear 30min samplings.

Photoperiod sample：Soybean cultivates agriculture 18 (KN18) and planted respectively under the conditions of long day and short day, when single leaf just deploys At interval of 2h samplings, 24 time points (48h) are respectively taken, seedling is then transferred to continuous illumination (LL) and continuous darkness respectively (DD) under the conditions of, sampled at interval of 2h, respectively take 24 time points (48h).

Different dimensions source soybean varieties sample：From 11 the cultivated soybean kinds, i.e. Heihe 27 (HH27), agriculture 14 of pacifying (SN14) agriculture 18 (KN18), long agriculture 13 (CN13), iron rich 31 (TF31), Ji beans 12 (JD12), middle yellow 13 (ZH13), middle beans, are cultivated Early 2 (GZ2) in 31 (ZD31), Zhejiang spring 3 (ZC3), good fortune beans 1 (FD1) and osmanthus, are planted, single leaf is opened completely under the conditions of long day and short day Sampled when opening, starting 30min (M), light application time intermediate point (N), illumination in illumination respectively terminates preceding 30min (E) samplings.

It is standby with storing in -80 DEG C after liquid nitrogen frozen immediately after all samples sampling.

(2) arabidopsis material：

Arabidopsis wild type Col-0, growth temperature is 21~22 DEG C, light application time be long day (16h illumination/8h dark) and Short-day (8h illumination/16h is dark).

1.1.2 bacterial strain and plasmid

Agrobacterium tumefaciens strain GV3101::PMP90RK is preserved by this laboratory, and coli strain DH5 α are purchased from full formula King Company.Gateway carrier Ts pGWc comes from China Agricultural University associate professor Chen Qijun laboratory.Plant expression vector This laboratory of pLeela, pENSG-YFP-GW is provided.

1.1.3 enzyme and kit

PrimerSTAR amplification enzymes, SYBR Green I Kit are purchased from TaKaRa companies, Gateway LR Clonase Enzyme Mix are purchased from Invitrogen companies, and glue reclaim and the small extraction reagent kit of plasmid are purchased from Axygen companies, and plasmid is small to be carried Middle amount kit is purchased from Tiangeng company.

1.2 experimental method

1.2.1 bioinformatic analysis

(1) determination of candidate gene

According to the CDS (code sequence) and amino acid sequence of arabidopsis CIB1 genes, in http:// Local Blast are carried out in www.phytozome.net websites, the homologous gene in soybean is filtered out.

(2) design of primers is expanded with PCR

According to the CDS sequences of gene, gene cloning primer is designed using Primer Premier 5.0.Real- Time primers are designed using the softwares of Beacon Designer 7.0.Primer synthesis is completed by Shanghai Sheng Gong biotech firms.

(3) chromosome mapping of gene

According to soybean gene networking station Phytozome (http://www.phytozome.net) provide information determine base Because of position on chromosome, drawn according to the length ratio of gene and chromosome with Adode Illustrator CS3.

(4) genomic organization

By the Soybean genomic DNA sequence obtained by prediction with corresponding cDNA gene orders in GSDS (http:// Gsds.cbi.pku.edu.cn) it is compared in website, determines position and the length of gene UTR areas, introne and extron Degree.Carry out drawing gene structure ideograph using Adode Illustrator CS3 sequence lengths ratio.

(5) analysis of homologous fragment

Using the softwares of biological software Clustalx 2.1, DNAStar software kits, the softwares of GeneDoc 2.7, MEGA 4 is soft Part bag, and http://weblogo.berkeley.edu websites are analyzed by the homologous fragment of predicted gene.

The primer and sequence of the clone gene of table 1

Table 2 is used for qRT-PCR primer sequence

(1) RNA extraction

1) liquid nitrogen is added in mortar, rapidly fully grinds sample, about 30mg samples are contained with RNase-Free centrifuge tubes (at 100 μ l scales).

2) 350 μ l RA1Buffer and 3.5 μ l β-ME are added, abundant vortex oscillation makes it mix completely.

3) 8000g centrifuges 1min at room temperature, and supernatant is transferred in the Filter column of pink colour, room temperature 11000g centrifugation 1min, Liquid is transferred to new 1.5ml centrifuge tubes, discards Filter column.

4) ethanol of 350 μ l 70% is added, vortex oscillation 25s is fully mixed.

5) liquid is transferred in the adsorption column of blueness, 8000g centrifuges 30s, RNA is adsorbed on pillar.

6) desalination, adds 350 μ l MDB Buffer, 11000g centrifugation 1min, discards waste liquid.

7) DNA is removed, 95 μ l DNase Reaction Mixture is added to adsorption column, is stored at room temperature 15min.

8) clean, add 200 μ l RA2Buffer, 11000g centrifugation 2min, the collecting pipe more renewed.Add 600 μ l RA3Buffer, 11000g centrifuge 1min, discard waste liquid, add 250 μ l Buffer, 11000g centrifugation 2min, abandon waste liquid.

9) the RNase-Free centrifuge tubes more renewed, 60 μ l RNase-Free H2O, 11000g are added to adsorbed film center 1min is centrifuged, is repeated once, to increase eluting rate.This step is operated on ice.Obtained RNA is put in -80 DEG C of refrigerator preservations.

Table 3 is used for ChIP-PCR primer sequence

Table 4 is used for ChIP-PCR primer sequence

(2) the reverse transcription synthesis of the chains of cDNA first

2) following reagent is sequentially added into nuclease-free PCR pipes on ice：

Total RNA 6μl(0.1ng-5μg)

Oligo(dT)18primer 1μl

nuclease-free 5μl

It is placed in 65 DEG C of reaction 5min in PCR instrument.

2) and then following reagent is added：

Gently mix, 45 DEG C of reaction 60min in PCR instrument.

2) in PCR instrument, 70 DEG C of 5min, terminating reaction.

(3) real-time fluorescence quantitative PCR

Real-time fluorescence quantitative PCR is carried out using ABI StepOne, and fluorescence signal is detected using SYBR Green I.

1) reaction system is 15 μ l：

2) response procedures are：

Two-step method：

Stage1：95 DEG C of 10s of thermal starting；

Stage2：PCR reacts 95 DEG C of 5s, 60 DEG C of 1min, 40Cycles；

Stage3：Melt curve analysis analyze 95 DEG C of 15s, 60 DEG C of 1min, 95 DEG C of 15s.

Three-step approach：

Stage1：95 DEG C of 10s of thermal starting；

Stage2：PCR reacts 95 DEG C of 5s, 58 DEG C of 30s, 60 DEG C of 1min, 40Cycles；

If two-step method is undesirable, optimization is adjusted to annealing temperature using three-step approach.

3) the calculating calculation formula of gene relative expression quantity is：Relative expression quantity (RQ)=2^-△△CT

1.2.3 the clone of gene and vector construction

(1) amplification of purpose fragment：The template of the experiment is the mixing that soybean cultivates each tissue site cDNA of agriculture 18 (KN18) Thing.

PCR reaction systems are as follows：

Response procedures：98 DEG C of denaturation 10s, 57 DEG C of annealing 5s, 72 DEG C of extension 2min 30s, 35 circulations, 25 DEG C of insulations.

(2) PCR primer is reclaimed and product connection

Ago-Gel QIAquick Gel Extraction Kit (being purchased from Axygen companies) reclaims PCR primer, reclaims fragment and linearisation PGWC carriers are attached.Before use, the carrier by Eam1105I digestions and producing two ends has 5 ' T linearized vector. PGWC carriers connection after the PCR primer of recovery and digestion, detection is positive to clone and send sequencing.

The endonuclease reaction system of pGWC carriers is as follows：

React mixed liquor and incubate 4-6h in 37 DEG C.Agarose gel electrophoresis detects the digestion effect of 1 μ l endonuclease reaction liquid, greatly Body estimates carrier segments concentration, and connection is directly used as without purifying endonuclease reaction liquid.

DNA reclaims fragment and pGWC-T connection：

Connection (about 12~16h) is used for transformation experiment to 16 DEG C of reaction solution afterwards overnight.

(3) conversion of bacillus coli DH 5 alpha

A) competent cell of 50 μ l freeze thawing on ice is taken in super-clean bench, 5 μ l connection products is added, gently mixes, ice bath Put 30min.

B) 42 DEG C of water-bath heat shock 45s, are quickly transferred to and place 2min on ice.

C) the LB cultures for adding 500 μ l nonreactives are based in centrifuge tube, 37 DEG C, 200rpm cultures 1h.

D) bacterium solution is added into even spread with the LB flat boards containing corresponding antibiotic (chloramphenicol chl), treating that liquid blows completely After dry, flat board, 37 DEG C of overnight incubations are inverted.

(4) identification of positive colony

With the monoclonal on toothpick picking flat board, in the LB plate streakings containing chloramphenicol antibiotics, then toothpick is existed Be gently agitated in pipe containing PCR reactant mixtures it is several under, carry out bacterium colony PCR and identify.Can with the multiple monoclonals of picking, and Mark is carried out on flat board, to have increased access to the probability of positive colony.Flat board is placed in 37 DEG C of incubated overnights afterwards.

PCR system：

PCR programs：Response procedures：98 DEG C of 6min, 94 DEG C of 30s, 57 DEG C of 30s, 72 DEG C of 2min, 35 circulations,

PCR primer, the line on correspondence flat board, is sent bacterium solution to be sequenced, obtained through electrophoresis detection, the clone of purposeful fragment It is connected with the positive colony of positive gene.

(5) plasmid extraction shakes bacterium solution for correct monoclonal, expansion is sequenced, and plasmid is extracted with kit, and step is as follows：

A) bacterium solution of 2ml overnight incubations is taken, 12000g centrifugation 1min abandon most supernatant.

B) add 250 μ l Buffer S1 (containing RNase) pipettor piping and druming suspended bacterials to precipitate, must ensure It is even.

C) 250 μ l Buffer S2 are added, leniently spins upside down and is well mixed for several times, thalline is fully cracked, until Form bright solution.This step is cleaved no more than 5min to prevent DNA.

D) 350 μ l Buffer S3 are added, it is gentle and be sufficiently mixed for several times, 12000g centrifugations 10min.

E) draw supernatant and be transferred to preparation pipe (being placed in 2ml centrifuge tubes), 12000g centrifugation 1min abandon filtrate.

F) pipe will be prepared to put back in 2ml centrifuge tubes, plus 500 μ l Buffer W1,12000g centrifugation 1min, abandon filtrate.

G) pipe will be prepared and put back to centrifuge tube, plus 700 μ l Buffer W2,12000g centrifugation 1min, filtrate is abandoned；Repeat one It is secondary.

H) pipe will be prepared to put back in 2ml centrifuge tubes, 12000g centrifugations 1min.

I) pipe will be prepared to move into clean 1.5ml centrifuge tubes, in absorption periosteum center plus 40 μ l

DdH2O, is stored at room temperature 1min.12000g centrifuges 1min, elutes DNA.

(6) structure of expression vector

The expression vector used in this experiment contains Gateway recombination systems, and plasmids of the pGWC with target gene is made For entry vector (Entery vector), the structure of destination gene expression carrier can be completed by LR reactions.

LR reaction systems：

25 DEG C of reactions are stayed overnight.Reaction solution converts bacillus coli DH 5 alpha, screening positive clone.

(7) preparation and conversion of Agrobacterium competence

1) preparation of Agrobacterium competence

Picking Agrobacterium GV3101::PMP90RK and EHA105 single bacterium colonies are respectively placed in LB liquid of the 5ml containing corresponding antibiotic In body culture medium, GV3101::PMP90RK resistances are：100 μ g/ml rifampins (Rif), 50 μ g/ml kanamycins (Kan), 50 μ G/ml gentamicins (Gen)；EHA105 resistances are：100 μ g/ml rifampins (Rif).28 DEG C of overnight incubations；Take incubated overnight bacterium Liquid

500 μ l are inoculated in fluid nutrient mediums of the 50ml LB containing corresponding antibiotic, and 28 DEG C of cultures to OD600 are about 0.5； 30min is placed on ice；4 DEG C, agrobatcerium cell is resuspended with the 10mM CaCl2 of 15ml precoolings in 5,000rpm centrifugation 10min, 4 DEG C, 5,000rpm centrifuges 10min；Precipitation is resuspended with the 10mM CaCl2 of 2ml precoolings, 100 μ l/ pipes are dispensed on ice, liquid nitrogen flash freezer, -80 DEG C preserve.

2) Agrobacterium-mediated Transformation

Take 100 μ l competent cells to thaw on ice, add after the mixing of 1 μ g DNAs, 30min, liquid nitrogen flash freezer are placed on ice It is immediately placed in 37 DEG C of water-bath 5min after 3-5min, adds 1ml nonreactive LB fluid nutrient mediums, 28 DEG C, bacterium after 160rpm recoveries 3-5h Liquid is uniformly applied on the solid medium containing corresponding antibiotic.28 DEG C are inverted 2~3d of culture, choose positive gram of single bacterium PCR identifications It is grand.

1.2.4 protoplast transformation and transient expression observe Subcellular Localization

(1) it is small to carry middle amount kit and extract DNA (extracting method carries middle amount kit plasmid extraction side with reference to Tiangeng is small Method)

(2) prepared by protoplasts of Arabidopsis thaliana broken by ultrasonic

A) the young tender Arabidopsis leaf of non-bolting is taken, blade is cut to 0.5mm-1mm sizes, blade tip and petiole is discarded.

B) broken leaf is soaked in 10ml enzymolysis liquids, light culture 3-4h, until protoplast will be completely dissociated.

C) microscopy examines hydrolysis result.

D) 200 mesh stainless steel screen filtration protoplasts allow liquid slowly to be slided along tube wall, liquid relief into new centrifuge tube The pipette tips that device is used should shear tip, it is to avoid protoplast ruptures in suction process.

E) low speed 100g centrifuges 1min, abandons supernatant, collects protoplast；

F) protoplast is resuspended with the W5 fluid nutrient mediums of isometric precooling, 100g centrifugation 1min, supernatant discarding (repeats one It is secondary).

G) protoplast is resuspended in the W5 liquid of isometric precooling, and 30min is placed on ice.

H) 100g centrifuges 1min, collects protoplast, and protoplast is resuspended in 1/10 volume MMg liquid.

(3) PEG converts protoplast

A) 10 μ l DNAs (10-20 μ g), the 100 above-mentioned protoplasts of μ l, gently springing are sequentially added in 2ml centrifuge tubes Tube wall, 110 μ l PEG-4000 solution are added after fully mixing.Mixture is stored at room temperature 30min.

B) 440 μ l W5 fluid nutrient mediums are added, are fully mixed.

C) low speed 100g centrifugations 1min, supernatant discarding, collect protoplast.

D) 500 μ l W5 liquid are added, 100g centrifugation 1min collect protoplast, are repeated once.

E) 1ml W5 fluid nutrient mediums are added in Tissue Culture Plate after protoplast is resuspended, 25 DEG C of lucifuge culture 18- 20h。

F) 100g centrifuges 1min, then only leaves 100 μ l liquid suspension protoplasts, remaining supernatant fluid is discarded, used Laser confocal microscope imaging is preserved.

1.2.5 arabidopsis genetic transformation and Phenotypic Observation

(1) preparation of Agrobacterium

1) by the Agrobacterium (GV3101 containing purposeful carrier::PMP90RK) it is inoculated in LB (containing corresponding antibiotic) liquid Culture medium；28 DEG C, 180-210rpm incubated overnights；

2) draw 500 μ l bacterium solutions to be coated on the LB solid mediums containing corresponding antibiotic, 28 DEG C of overnight incubations.

3) 30ml YEB fluid nutrient mediums sweep and bacterial plaque are resuspended, and re-suspension liquid, which is used, contains 5% sucrose and 0.01%Silwet- L 77 aqueous solution is diluted to 120ml, for transformation of Arabidopsis thaliana.

(2) conversion of arabidopsis

1) the arabidopsis kind grown under long-day, cuts first inflorescence after bolting；Treat within one week or so that more buds expose When can be used for convert；

2) Agrobacterium-mediated Transformation immersion flower bulb flower bud 10-20s.Transformed plant is covered into lucifuge, moisturizing with black plastic bag, Polybag is raised after 16-24h；

3) plant strain growth situation is regarded, occurs after next group bud (after two weeks), second of conversion can be carried out.Look after and turn meticulously Plant after change, sowing is used for the screening for converting positive plant.

(3) screening and Preliminary Identification transformation of Arabidopsis thaliana offspring

The seed received after conversion is T1 generations, is uniformly seeded in soil, cotyledon opens completely after 7-10d, can now spray Spill 1:Second of spray after 1000 BASTA, 5-7d.All wilting is dead for one week or so non transformed plants, and plant now is Transgenosis T2 is for plant, while Preliminary Identification can be carried out for transfer-gen plant to T1 by extracting DNA, PCR detection.Individual plant T2 is harvested for seed.

(4) statistics of arabidopsis flowering time and blade

Seed is placed in containing in the culture dish for soaking filter paper, and then 4 DEG C of dark processing 3-4d are sowed on soil, are stayed within three weeks or so It is lower to grow the consistent and preferable seedling of growing way.Grown under conditions of control and seed to be detected are placed in equally, each strain is true Possess 10-40 individual plant.Count flowering time when from planting to blooming and the average value and standard deviation of the lotus throne number of blade.

2. result and analysis

The clone of 2.1 soybean CIB genes

According to arabidopsis CIB1 sequences, Blast analyses are carried out at soybean gene networking station, soybean CIB gene families are found In have 12 members's (table 5), wherein GmCIB1, GmCIB2, GmCIB3, GmCIB11 and arabidopsis CIB1 homology are higher, Speculate that they there may be similar function with CIB1.And homologous gene of other members in arabidopsis belongs to bHLH families. 9 GmCIB genes are cloned into altogether at present.

The chromosome mapping of 2.2 soybean CIB genes

According to website http:The soybean gene group relevant information that //www.phytozome.net is provided draws 12 GmCIB Gene station-keeping mode figure (Fig. 1) on chromosome.12 gene distributions on 11 chromosomes of soybean, except GmCIB2 and GmCIB8 co-localizations are on No. 4 chromosomes, and physical distance is close.Other 10 genes are distributed in different chromosomes On.In terms of the distributing position of each gene on chromosome, this 12 genes be mostly distributed in chromosome it is long-armed on, only GmCIB5, GmCIB9, GmCIB10 are located on the short arm of a chromosome.

2.3 soybean CIB genetic homologies and conserved positions analysis

2.3.1 homology analysis

CIB1 and CIB5 regulation and control are bloomed in arabidopsis.The homology analysis of gene finds (Fig. 2), soybean CIB genes with Arabidopsis CIB1, CIB5 homology are mostly more than 30%.GmCIB1, GmCIB2, GmCIB3, GmCIB11 and arabidopsis CIB1 homology is higher, respectively 41.6%, 42.1%, 39.2% and 39.4%, and their homologys with CIB5 are slightly lower In the homology with CIB1, homology is 30% or so.GmCIB9 and GmCIB10 and CIB1 homology 30% or so, and Homology with CIB5 is higher, and respectively 51.2% and 50.5%.

The height of homology is uneven between soybean CIB genes, and such as GmCIB1 and GmCIB2 homologys are up to 91%, and this The homology of two genes and other genes is significantly lower than this numerical value.GmCIB1 except with GmCIB11 homology it is higher in addition to, with The homology of other genes is relatively low.Further analysis finds to be divided into homology between gene two-by-two in 12 soybean CIB genes Very high 6 pairs of combinations, and this two gene and other intergenic tetraploid rices are low.This be probably because soybean is ancient tetraploid, In long-term evolution selection course, two genes of separate sources, which are retained, forms 2 copies so that many genes with Multicopy mode is present.

5 soybean of table, 12 CIB gene family members

Note：A sequence informations come from

Phytozome(http://www.phytozome.net/cgi-bin/gbrowse/soybean/) b is local Blastp compares arabidopsis thaliana protein database.

2.3.2 conserved positions are analyzed

Arabidopsis CIB1 is typical bHLH family genes, containing conservative bHLH domains, to the guarantor of soybean CIB families Keep structure domain analysis and find (Fig. 3), all genes all contain bHLH domains, and contain and arabidopsis identical conservative Site, the typical tryptophan Try (R) in Basic basic regions [except GmCIB8 is serine Ser (S)], the bright ammonia of Helix helical regions Proline Pro (P) in sour Leu (L), Loop Lu puff rings.Functionally see, arabidopsis bHLH domains are participated in DNA's Interaction, is combined to regulate and control the expression of downstream targets target gene by the cis-acting elements with promoter region.The CIB family of soybean So conservative bHLH domains are contained in race, if also assist in and DNA cis-acting elements combination, it is not clear at present.

Further analysis is carried out to the homology of conserved sequence to find, GmCIB7 and GmCIB8 evolution outermost branch, GmCIB1/2/3/11, GmCIB4/6/12 and GmCIB9/10 gather for a class respectively.Although the conserved domain of gene is conservative Site is almost consistent, but it is probably the different selection pressure during long-term evolution that the homology of other amino acid is very low And the function differentiation caused.

The gene structure of 2.4 soybean CIB genes

Arabidopsis CIB1 encodes the display of plot structure, gene can be functionally divided into N-terminal and C-terminal, N-terminal contains one section of core Positioning signal (NLS) participates in the interaction entered between nuclear location and albumen of gene, and the bHLH domains of C-terminal can be with DNA combines (functional element), regulates and controls the expression of downstream target gene.Soybean CIB gene code plot structures are similar with CIB1, bHLH knots Structure domain also is located at the C-terminal of gene.

Genome structure analysis display, the conservative of soybean CIB genomes is than relatively low, because each genome structure Composition is complicated.Fig. 4 shows that each gene contains different number of extron and introne, and 5 ' all containing different length UTR.Other genes do not have in addition to GmCIB1/2/3/11 contains 3 ' UTR, this just with this four genes closer phase in evolution It coincide.From the number of extron and introne, GmCIB1/4/5/6 contains 7 extrons and 6 intrones, wherein The length difference of each corresponding extrons of GmCIB4/5/6 and introne is not very big.GmCI1/8/11 is outer containing 6 Aobvious son and 5 intrones, and GmCIB8 the 5th length of intron is most long in all genes.It is outer aobvious contained by GmCIB12 Subnumber mesh is up to 12, and introne number is up to 9 contained by GmCIB9.

The Subcellular Localization of 2.5 soybean CIB genes

In order to primarily determine that the positioning of soybean CIB genes in the cell, soybean CIB genes and yellow fluorescence protein are constructed (YFP) expression vector of fusion, protoplast transformation is carried out using arabidopsis mesophyll cell, to the GmCIB albumen cloned Subcellular Localization is analyzed.As a result display (Fig. 5) has table except GmCIB6 and GmCIB7 albumen in nucleus and cytoplasm Reach, other GmCIB albumen are positioned in nucleus.

The expression pattern analysis of 2.6 soybean CIB genes

2.6.1 soybean CIB genes are expressed in the expression pattern of different tissues organ

Using soybean single leaf phase, florescence and fruiting period 20 different tissues organs in soybean CIB gene families The expressions of 9 genes analyzed.As a result display (Fig. 6) soybean CIB genes are different in the expression of different tissues organ. GmCIB1, GmCIB2, GmCIB3 expression pattern are similar, the expression quantity in the histoorgan of single later stage leaf phase and early stage in florescence It is higher, and the expression quantity in early stage single leaf phase and later stage in florescence histoorgan is relatively low.These three genes are in single leaf phase and open Expression quantity is all very high in single leaf at florescence, in addition second compound leaves of the GmCIB1 and GmCIB2 in florescence, GmCIB1 florescence Expression quantity in first compound leaf is also very high.The mRNA expression analysis of other 6 genes shows that they are when blooming the later stage and bearing pods Expression quantity in the histoorgan of phase is higher.Expression quantity of the GmCIB5/6/7/8/9 in floral organ is all of a relatively high, GmCIB7 In the second compound leaf height expression in florescence, GmCIB4 and GmCIB5 the later stage experssion amount that bears pods are very high, and are expressed in mature seed Amount is reduced sharply, and the possible gene is not involved in the after ripening of seed.In contrast, GmCIB6/7/8/9 is bearing pods later stage experssion amount very It is low, and expression quantity increases severely in mature seed, the after ripening of these possible gene seeds is relevant.The scientific appraisal of the inference is also needed Further explore.

The sample of the soybean different tissues organ of table 6

2.6.2 expression pattern of the soybean CIB genes in different development stage

The sample of the soybean different development stage of table 7

Soybean CIB genes are different (Fig. 7) in the expression pattern of different development stage, from the overall expression pattern of all genes See, in soybean CIB genes in addition to GmCIB2, the expression quantity of other gene aerial parts is all relatively low, expression quantity higher period is main Compound leaf period is concentrated on, Individual genes (GmCIB1) also have very high expression quantity in single leaf phase.The expression of individual gene shows Show, GmCIB6 entirety expression is relatively low, and GmCIB8 overall expression is higher.GmCIB1/3/4/7 has just opened in the 3rd compound leaf Expression quantity when opening is very high, and GmCIB1 has expression in single leaf of each developmental stage.Further analysis find GmCIB4 and GmCIB7 developmental stage expression patterns are similar, very high except starting to open up into expression during the 4th compound leaf starts to open in the 3rd compound leaf Outside, the expression in other periods is all very low.GmCIB5 and GmCIB9 the Fruit pod stage of development expression pattern on the contrary, GmCIB5 table It is continuously increased but is reduced sharply in seed maturity expression with Fruit pod development up to amount, and GmCIB9 tables when Fruit pod just germinates Higher level is reached up to amount, its expression quantity constantly lowers with the development of Fruit pod, but in the expression quantity play of the mature seed gene Increase.Such expression pattern may be relevant in the function that developmental stage is exercised with gene.

2.6.3 the photoperiod expression pattern of soybean CIB genes

Sample materials involved by soybean CIB photoperiod expression patterns are as follows：Agriculture 18 (KN18) seedling is cultivated to plant respectively Yu Chang (LD) and (SD) under the conditions of short day, sample are the single leaf just deployed, have just turned on light and have started sampling constantly, have been taken at interval of 2h Sample, respectively takes 24 time points (48h).Seedling is then transferred to continuous illumination (SD-LL/LD-LL) and continuous darkness respectively (SD-DD/LD-DD) under the conditions of, sampled at interval of 2h, respectively take 24 time points (48h).Each group sample is used for the GmCIBs photoperiods Expression analysis, as a result shows that (Fig. 8) GmCIBs photoperiod differential expression is very big.Table under the conditions of GmCIB1 (Fig. 8 a) SD and LD Expression patterns are different, under the conditions of SD, and circadian rhythm change is presented in gene expression, and downward trend is presented in gene expression after turning on light, and enters The expression of gene is also in low expression level when dark, and 4 hours gene expression peak values occur before bright light.It is transferred to continuous illumination Afterwards, the expression circadian rhythm of gene changes, and multiple expression peak values occurs.And when being transferred to continuous darkness, its circadian rhythm entirety mould Formula change is little, but the expression peak value backward delay of gene two hours.Under the conditions of LD, GmCIB1 expression does not have obvious light Circadian rhythm.

There there is no GmCIB2 expression under the conditions of Fig. 8 b show photoperiod expressions of the GmCIB2 under the conditions of SD and LD, LD Obvious circadian rhythm phenomenon.And GmCIB2 expression quantity after turning on light gradually is reduced under the conditions of SD, its table when entering dark Reached up to amount minimum, the expression of subsequent gene gradually increases, and the expression of 4 hours genes reaches highest before turning on light.Turn when from SD Gene expression integral level has declined when entering LL, although under expression peak value does not change at the time of appearance, but its amplitude is obvious Drop.And from SD be transferred to DD when the overall expression of gene greatly improve, the peak value of gene expression is also capped.Can further it push away Survey, GmCIB2 expression is regulated and controled under the conditions of SD by the photoperiod, while also by the inhibitory action of light.

GmCIB1 photoperiod expression pattern is similar under the conditions of SD and LD, and gene expression has the slightly shorter time after turning on light Reduction, (i.e. moment at high noon) gene expression gradually increases after 6 hours, and gene expression in 6 hours reaches highest after light-off, then expression Amount is reduced sharply.

When being transferred to LL respectively, the peak vanishes of gene expression, gene expression high level in maintaining.And work as and be transferred to DD When, the expression quantity of gene is substantially reduced.Above phenomenon illustrates that GmCIB1 mRNA expresses the positive regulation and control of light.

GmCIB4 photoperiod expression pattern analysis shows (Fig. 8 d), and the gene has the obvious daily cycle rhythm and pace of moving things in LD, The expression quantity highest of 6 hours genes after turning off the light, and other time its expression quantity it is relatively low.Expression pattern is several after LL is transferred to Do not change, be transferred to after DD and express peak value postponement 2h appearance.Gene expression reaches highest after 6 hours in bright light under the conditions of SD, It is in then reduction trend.But the expression of gene substantially increases when being transferred to LL, and gene is constantly in relatively low table under the conditions of DD Up to level.Under possible SD, Photoperiod intensity is more than the biological clock regulation and control in plant, and illumination promotes turning for the gene Record level.And under the conditions of LD, the biological clock regulation and control of the gene play a leading role.

Fig. 8 e are shown under the conditions of SD, and the peak value expressed occurs after illumination 4h in GmCIB5 expression quantity.It is transferred to LL conditions The lower gene dosage has different degrees of increase, but expression peak vanishes.And under the conditions of DD, the expression of gene substantially drops It is low.Under the conditions of LD, GmCIB5 expression quantity has individual short time (2h) reduction after bright light, then with the increase of light application time Its expression quantity is continuously increased, and its expression quantity reaches highest after illumination 6h.Speculate that the expression of the gene is mainly regulated and controled by the photoperiod, And the positive regulation and control of light.

GmCIB6 expression pattern (Fig. 8 f) is similar to GmCIB5, and Photoperiod is occupied an leading position., should under SD and LD Obvious circadian rhythm is presented in gene.And this species rhythm is broken under LL and DD, gene expression water under the conditions of LL is showed Smooth body is increased substantially, and gene expression is significantly reduced under the conditions of DD.

GmCIB7 expression analysis shows (Fig. 8 g), and the gene is in SD and LD without obvious biological clock or photoperiod Rule.After LL is transferred to, with the increase of light application time, its expression quantity is constantly raised to, although have an expression to reduce sharply the moment, but then It expresses again quick increase.And under the conditions of DD, the expression of the gene is substantially lowered and maintains very low expression always. Result above shows that the gene is not regulated and controled by biological clock, and illumination may regulate and control its transcriptional level.

GmCIB8 has obvious biological clock rhythm under the conditions of SD and LD it can be seen from Fig. 8 h.Arriving with illumination under SD Carry out expression quantity constantly to raise, the 2h after turning off the light, expression peak value occurs, and subsequent expression quantity is reduced sharply, and the 4h before turning on light, expression quantity is dropped to Low ebb.Under the conditions of LD, 6h gene expression amounts reach highest after illumination, and then expression is gradually reduced, and 6h expression quantity is minimum before turning on light. This species rhythm phenomenon disappears under the conditions of LL and DD, and the overall expression of gene has carrying by a relatively large margin under the conditions of LL Height, and reduced levels are then maintained under the conditions of DD.

GmCIB9 expression of results shows (Fig. 8 i), and the biological clock rhythm of the gene and photoperiod do not advise significantly Rule, the complicated weave in of this two regulating and controlling effect may be concured during gene expression, or the gene table Controlled up to by more complicated regulatory mechanism.

2.6.4 different latitude source soybean varieties in GmCIB gene families expression pattern

Choose in 11 different the cultivated soybean kinds of latitude, the greenhouse that SD and LD is planted in respectively, M, N, E tri- is taken respectively The sample at individual moment, the expression analysis in different cultivars for GmCIBs genes.As a result (Fig. 9) is shown, GmCIBs genes Expression pattern in different cultivars is different with the change of latitude.The kind (40 ° -50 °) being distributed in high latitude, no matter GmCIB1 expression has and first increases the trend (Fig. 9 a) reduced again under SD or LD, i.e. moment (N) expression quantity highest at noon. The expression of gene is on a declining curve under the conditions of low latitudes kind (25 ° -30 °), SD, and expression quantity gradually increases under LD.

Under the conditions of SD, GmCIB2 expression patterns are substantially the same in different cultivars, in addition to CN13, TF31, ZD31, other kinds In expression of the gene in one day present and first increase the trend that reduces afterwards, at noon the moment reach peak value.And under the conditions of LD, it is several Opposite expression pattern is presented, in high latitude kind (HH27, SN14), the moment reaches valley at noon for the expression of gene, Expression quantity highest at the moment the dusk.With the reduction of latitude, the expression of gene is from receiving to continue to increase after illumination, and at candlelight carves Reach highest.It can be seen that under the conditions of LD, GmCIB2 is in KN18, CN13, and lasting reduction is expressed from after turning on light in TF31, and in other product Plant the trend for nearly all presenting and continuing to increase.

GmCIB1 genes show (Fig. 9 b) in the expression pattern analysis of different cultivars, with the increase of latitude, its expression quantity Peak value constantly move forward, in the cultivar of -50 ° of distributions of 38 ° of latitude, at candlelight under the conditions of SD and LD carves GmCIB1 transcription Horizontal highest.In ZD31 and ZC3, under SD and LD the expression pattern of gene just on the contrary, under short-day gene expression present first Increase, at noon the moment reach peak value, subsequent expression quantity is gradually reduced, and the long-day present first reduce in increased trend. At two compared with low latitudes kind FD1, GZ2, gene expression not light cycle influences, expression pattern is almost identical, with illumination Its expression quantity of the increase of time constantly rises, at noon moment expression quantity highest.

The expression for the cultivar that GmCIB4 genes are distributed in high latitude is can be seen that from Fig. 9 b, not by the shadow of photoperiod Ring, sustainable growth trend after illumination is presented in its expression in HH27, SN14, KN18, half an hour reaches expression peak value before turning off the light. With the reduction of latitude, gene expression peak value is constantly moved forward, at noon moment (N) arrival, and subsequent expression quantity is gradually reduced.And By equatorial kind (FD1 and GZ2), its expression first has individual downward trend under the conditions of LD, and (N) reaches low ebb the moment at noon, Then expression gradually increases.And under the conditions of SD, low latitudes (ZC3, FD1 and GZ2) kind expression pattern is similar to high latitude kind, Its expression quantity continues to increase.

Fig. 9 c are shown, under the conditions of LD, expression of the GmCIB5 genes in low latitudes (25 ° -31 °) cultivar (outside GZ2), Continue to increase in illumination period, highest is reached when that will enter dark.This trend and its expression pattern under the conditions of SD It is similar.Expression of the GmCIB5 genes (being distributed in middle high latitude) under LD environment, which is presented, in other kinds first increases the mould reduced afterwards Formula.Under the conditions of SD and LD, the expression peak value of GmCIB6 genes is mostly before light-off in each kind, but the trend increased is not Together, the expression sustainable growth of high latitude kind GmCIB6 genes from after turning on light, with the reduction of latitude, the transcriptional level of the gene Gradually reduced with the increase of light application time, reach after low ebb and gradually increase.

GmCIB7 and GmCIB8 genes show (Fig. 9 d) in the expression pattern analysis of each kind, the table of two genes under the conditions of LD Expression patterns are roughly the same, gradually increase with the increase expression quantity of light application time, and (N) reaches peak value the moment at noon.SD conditions Lower GmCIB7 genes also show above-mentioned expression pattern in the kind that high latitude and low latitudes are distributed, and GmCIB8 genes are then showed Go out the trend that expression continues to increase, (E) expression quantity highest before dark is entered.

GmCIB9 gene expression analysis shows that the gene expression peak value constantly moves forward with the reduction of latitude, and SD bars Translational speed under part is faster than LD conditions.Under the conditions of SD, GmCIB9 genes increase again in the kind that high latitude is distributed by first reducing Plus expression pattern, enter it is dark before reach peak value.As its expression of successively decreasing of latitude is in first increasing the trend that reduces afterwards, Moment at high noon, (N) reached peak.And the lasting reduction of (FD1 and GZ2) its expression in the kind of relatively low Latitude Distribution.LD conditions Under, the moment reaches peak value to GmCIB9 genes at noon in low latitudes kind (FD1 and GZ2), with the increasing of kind Latitude Distribution Plus its expression is continuously increased, expression quantity reaches highest before light-off.

2.7 phenotypic analysis of the soybean CIB gene overexpressions in arabidopsis

Soybean GmCIBs is building up on 35S plants over-express vector (pLeela) respectively, difference arabidopsis thaliana transformation is simultaneously right Its function is analyzed.5 gene arabidopsis of GmCIB1/4/5/6/8, which have been obtained, through Basta screenings is overexpressed transfer-gen plant. The transfer-gen plant screened is planted under the conditions of SD and LD respectively, analysis phenotype is further looked at.GmCIB1, which is overexpressed, to be intended Southern mustard shows early blossoming phenotype (Figure 10) in SD and LD.Flowering time and number of blade statistics display, under the conditions of SD and LD, turn Gene plant flowering time is substantially earlier than wild type, and the number of blade is also less than wild type.GmCIB1 to being overexpressed (LD) in plant Show that GmCIB1 gene overexpressions in transfer-gen plant, AtFT expression quantity is bright with AtFT mRNA expression testing results It is aobvious to be higher than wild type.Imply that the overexpression of GmCIB1 genes promotes AtFT expression, so that plant Blooming. Functions of the GmCIB3 in arabidopsis is also same with arabidopsis CIB1 function phases.But whether it also has similar function in soybean, Specific below it will discuss.Equally, tetra- genes of GmCIB4/5/6/8 are overexpressed in arabidopsis respectively, under the conditions of SD and LD Arabidopsis Blooming (Figure 10) can be promoted, flowering time is also significantly reduced earlier than wild type, lotus throne number of sheets mesh, illustrate this four Individual gene also functions to the effect for regulation and control of blooming in arabidopsis.But their functions in soybean, it is not clear at present.

3. discuss

The clone of 3.1 soybean CIB gene families

This experiment determines 12 members in CIB gene families by the homologous comparison of bioinformatics in soybean, most Preceding 9 GmCIBs genes have first been predicted, with the continuous announcement of soybean genomic sequence, then GmCIB10/ have been predicted again 11/12 3 genes.Preceding 9 GmCIBs genes have been cloned in this experiment at first based on the reason for time and converting material.Clone institute It is the mixture that soybean cultivates agriculture 18 (KN18) Main Tissues organ cDNA with template, avoiding problems gene in different tissues The gene cloning that differential expression is caused is difficult.The code area of 9 GmCIBs genes of clone and the sequence of prediction are coincide, and respectively will It is gene constructed to arrive on the carriers such as the Subcellular Localization expression vector that plant over-express vector, YFP are merged, and yeast two-hybrid, Studied to the inside and outside function to these genes.

The gene structure of 3.2 soybean CIB gene families

Soybean CIB gene families member is carried out by bioinformatics tools and some biological softwares relatively raw in detail Thing bioinformatics analysis.Chromosome mapping, which is shown, removes GmCIB2 and GmCIB8 co-localizations on same chromosome (chr.4), other 10 gene distributions are on different chromosome.Although physical distances of the GmCIB2 and GmCIB8 on same chromosome is close, But both homology is not high, 27.3% is only reached, thus it is speculated that in being probably long-term evolution, the homologous gene that selection pressure is caused Function differentiation.

Between 12 GmCIB members, homology very high 6 pairs of assortments of genes between two genes are formed, homology highest reaches 93.0% (GmCIB9 and GmCIB10), minimum also reaches 80.1% (GmCIB7 and GmCIB8).From chromosome distribution See, two higher genes of homology are respectively positioned on coloured differently body, this is probably due to soybean is ancient tetraploid, the two of separate sources Individual gene is retained by long-term evolution atomization, so as to form 2 copies, these be located on coloured differently body and Homologous higher paired gene exactly supports this judgement.

GmCIB homologous genes functionally whether there is redundancy, not have been reported that also at present.Consider from evolution angle, repeat Gene is influenceed to different directions to develop by long-term evolution selection pressure, such as：The forfeiture (pseudogene) of function, function (different genes copy function is thin for reduction (expression reduction, miopragia), gain-of-function (differentiating new function) and function refinement Change and regulate and control same process).Spatial and temporal expression profile progress analysis to soybean CIB gene family members will be helpful to understand at it The function exercised in soybean.

Arabidopsis CIB1 is typical bHLH albumen, contains typical bHLH conserved domains.Soybean CIB gene families are protected Keeping property domain analysis shows, each member is containing the higher bHLH domains of conservative.Arabidopsis CIB1 studies have shown that BHLH motifs can promote FT mRNA expressions, so as to open by being combined with the E-box (CANNTG) in FT gene promoters area Animals and plants bloom (Liu etc., 2008).GmCIB families possess so conservative domain, and whether its function is similar with CIB1, This is also the problem of we am interested.The N-terminal of arabidopsis CIB1 genes and C-terminal function it is different, N-terminal contains one section of nuclear location Signal, the interaction for entering core and protein-protein with gene is relevant.The C-terminal of gene contains bHLH domains and participated in and DNA Combination.The code area structure prediction of GmCIB family genes is similar with arabidopsis CIB1, and bHLH conserved domains also are located at gene C-terminal.The conserved domain homology analysis of GmCIB family members is found, each intergenic domain homology and gene Slightly difference between coded amino acid total length homology, this is probably caused by other amino acid are different in addition to conserved amino acid.This can Can be also that plant is the different function differentiation for selecting homologous gene formed by pressure of adaptation during long-term evolution.

The expression pattern of 3.3 soybean CIB gene families

Phenotype of the 3.4 soybean CIB gene families in arabidopsis

4. brief summary

12 arabidopsis CIB1 homologous gene is predicted in soybean, 9 GmCIBs are cloned into altogether, by being predicted The bioinformatic analysis of gene, to the gene that is cloned into different tissues organ and the mRNA of developmental stage spatial and temporal expression mould Formula, biological clock and/or photoperiod and the mRNA expression patterns in different dimensions originate soybean varieties are analyzed, subcellular fraction Positioning, and gene overexpression arabidopsis is subjected to initial analysis to gene function.Obtained Main Conclusions is as follows：

Soybean CIB gene family members have 12, respectively (GmCIB2 and GmCIB8 on 11 different chromosomes It is distributed in same chromosome).Homology analysis shows that family member is divided into 6 pairs, and the intergenic homology of each pair is very high, but Homology between other members is relatively low, and paired gene is respectively positioned on coloured differently body, may be ancient tetraploid with soybean And long-term evolution selection is relevant.

Soybean CIB genes have conservative bHLH domains.The genome structure of each member is more complicated, all containing difference The extron and introne of number and length, and the not UTR of length.

Soybean CIB and YFP fusion proteins show its Subcellular Localization research own through protoplast transformation transient expression Gene is all positioned in nucleus, and has Individual genes (GmCIB6 and GmCIB7) also to have expression in cytoplasm, illustrates these The function of gene is exercised and is not limited solely to nucleus, may also assist in the partial function regulation and control in cytoplasm.

The expression of soybean CIB gene different tissues organs shows that most of member is higher in the tissue expression in florescence.No Show with developmental stage expression pattern, most gene when nutrient growth is transitioned into reproductive growth (i.e. florescence) expression quantity by Edge up height, thus it is speculated that the family member may be related to regulation and control of blooming.And Individual genes such as GmCIB1 is in single leaf phase and comes into bloom Expression quantity it is all very high, thus it is speculated that the gene may play important regulation and control in the primary stage of nutrient growth and reproductive growth and make With being served both functions to developmental regulation.Photoperiod and biological clock expression pattern analysis show that GmCIB genes are by both machines The dual regulation and control of system.See on the whole, positive regulation and control of the most gene (in addition to GmCIB4) by the photoperiod.Different latitude source soybean GmCIBs expression patterns are shown in kind, and GmCIBs is with latitude change without obvious correlation.

The GmCIBs being cloned into is overexpressed into arabidopsis, obtained overexpression GmCIB1/4/5/6/8 transgenosis intends south Mustard shows prematurity phenotype under the conditions of long day and short day, shows these functions and arabidopsis CIB1 in arabidopsis Function it is similar.

The functional study that embodiment 2, GmCRY1＆2 and GmCIB1 regulation and control are bloomed with aging

This research will pass through the phase between the means research GmCRYs and GmCIB1 albumen such as the double miscellaneous, BiFC and Co-IP of yeast Interaction；The Genetic Transformation of Soybean strain that GmCRY and GmCIB1 gene overexpressions or RNAi are knocked out further is obtained, with reference to turning base GmCIB1 downstream effects target gene is found because of phenotypic analysis and the ChIP experiment of offspring, is passed so as to analyze GmCRY in soybean The GmCIB signal paths of blue light signals are passed, are that soybean photoperiod and the further investigation of leaf senile regulation and control and germplasm innovation are laid Basis.

1. materials and methods

1.1 material

1.1.1 vegetable material

Soybean varieties is cultivate agriculture 18, and tobacco bred is Nicotiana Benthamiana.

1.1.2 bacterial strain and plasmid

Agrobacterium and coli strain be the same as Example 1, yeast strain Mav203 preserve for this laboratory.Insect cell elder brother Worm cell sf9 applies a public laboratory from Tsing-Hua University.

Yeast two-hybrid vector pDEST22, pDEST32；Plant expression vector pGWB11, pGWB17；BiFC carriers pCCFP-X、pNYFP-X；Insect expression vector pFASTBacHTA is preserved by this laboratory.

1.2 method

1.2.1 Genetic Transformation of Soybean and Phenotypic Observation

(1) Genetic Transformation of Soybean：The over-express vector containing target gene and RNAi carrier of structure are utilized into optimization Agriculture bacillus mediated cotyledonary node method, is transferred to soybean varieties and cultivates in agriculture 18 (KN18), obtains homozygosis and can stablize the transgenosis of heredity Plant, the function of Primary Study gene.

(2) identification of transfer-gen plant and the acquisition of homozygous line：Three are mainly taken for the transfer-gen plant of stable heredity Individual method is identified：One is Analysis of Resistance, and above expression vector is all marked with herbicide screening, can removed with certain density Careless agent sprays preliminary screening to possible transfer-gen plant；Second, identified using molecular biology method, using RT-PCR and Western-blot proves that the target gene being incorporated into genome is expressed really；3rd, analyzed using genetic method, weeding Whether agent resistant maker gene isolates with target gene, and whether target gene meets Mendel's rule in descendant inheritting, and The homozygous transgenic plant of the stable heredity of screening.

(3) phenotypic analysis of transfer-gen plant：The overexpression and RNA interference transfer-gen plant of the stable heredity of analysis are with compareing Phenotypic difference between plant.

1) flowering time and number of blade statistics：Different transfer-gen plants and wild type were planted in long-day and short day respectively According to number of blade when in greenhouse, counting flowering time and blooming.

2) statistics of leaf senile difference：Based under continuous illumination some transfer-gen plant early ageing phenomenons it is obvious, Different transfer-gen plants and wild type are planted under the conditions of continuous illumination, statistics cotyledon and single leaf start to turn yellow, turn yellow and The time come off.

3) measure of Chlorophyll and chlorophyll a, b content：The plant in continuous illumination is planted, when the single leaf of early ageing plant When starting to turn yellow, sample is fully ground after taking 5 different single leaves, liquid nitrogen frozen respectively per strain.500 μ l are added per sample to extract Liquid [50mM lithium phosphates pH7.2；1mM；120mM；5% (v/v) glycerine；1mM PMSF；0.2%] mix, take 100 μ l to add respectively 80% acetone, after fully mixing, is placed in -20 DEG C of avoid light place 1h, 12800g centrifugation 3min.Spectrophotometric determination 663nm and The absorbance value of 646nm wavelength.Chlorophyll concentration is calculated using below equation：

Chlorophyll content (mg/L)=20.2A646+8.02A663；

Chlorophyll-a Content (mg/L)=13.19A663-2.57A646；

Content of chlorophyll b (mg/L)=22.1A646-5.269A663.

4) measure of photosynthetic rate：The difference of continuous illumination plantation is determined in different time sections using photosynthetic instrument LI-6400 The photosynthetic rate of plant, single leaf of the tested plant of selection, the second compound leaf and the 4th compound leaf are used as measure object.

5) aging related genes and regulation and control related gene expression situation analysis of blooming are selected under continuous illumination, early ageing plant As materials time point when single leaf just changes, different transfer-gen plants and wild type are drawn materials, senile correlation is used as Because (GmAPGL5, GmWRKY53, GmSAGL12, GmAGL12) expresses the template determined.After continuous illumination switchs to short-day three days The template for taking the 7th compound leaf to be determined as regulation and control related gene (GmFTs and GmTFLs) expression of blooming.(RNA extractions, Real-time PCR be the same as Examples 2)

1.2.2 yeast two-hybrid

Yeast two-hybrid assay is carried out according to Clontech handbooks.GmCRYs and GmCIBs is building up to yeast respectively double miscellaneous Hand over carrier pDEST32 (Bait) and pDEST22 (Prey), corotation yeast strain Mav203.With the experiment point of histidine auxotrophy The interaction of albumen between analysis GmCRYs and GmCIBs, drops in three by yeast clone and lacks growth (- His-Trp-Leu) on plates, one group puts Put under dark, one group, with 25 μm of olem-2s-1 blue light illuminations, grows 2 to 3 days in 28 DEG C of incubators.To interactions between protein Quantitative analysis is using quantitative beta galactosidase experiment, using CPRG as chromogenic substrate.Picking yeast monoclonal lacks SD trainings in two Lucifuge incubated overnight in base (- Trp-Leu) is supported, OD600 to 1.8 is treated, it is about 0.2 that OD600 is diluted to SD, then lucifuge culture 1- 2h, is then divided into two groups, and one group is placed under 22 μm of olem-2s-1 blue lights, and one group is placed in identical culture environment with masking foil parcel Under, different time points collect yeast detection betagalactosidase activity in 0-150min.Whole process keeps yeast growth dense OD600 is spent between 0.4-0.8.

1.2.3 BiFC is tested

GmCRY1/GmCRY2 and GmCIB1 are building up on expression vector pCCFP-X, pNYFP-X respectively, pass through plasm Body converts (method be the same as Example 1), Confoal micro- Microscopic observation GmCRY1/GmCRY2 and GmCIB1 interaction situation.

1.2.4 Western blotting (western blot)

(1) Western blotting operating process

1) protein extraction：Arabidopsis thaliana Seedlings are collected in 1.5ml centrifuge tubes, 20-30 μ L 4 sample buffers is added, grinds After mill is abundant, 95 DEG C are boiled sample 5min, put cooled on ice, and 15min is centrifuged in maximum speed.

2) SDS-PAGE protein isolates.

3) by protein delivery to NC films, 1h is closed with 5% skimmed milk power (being dissolved in PBST solution).

4) primary antibody normal temperature is added to be incubated 1h.

5) film is washed with PBST solution three times, each 6min.

6) add secondary antibody and be incubated 1h

7) film is washed with PBST solution three times, each 6min.

8) chromogenic substrate is added to be developed.

9) film is hybridized：Film is washed with film washing liquid three times, each 15min is closed with milk again, add second of antibody.

(2) solution is prepared

1) gel liquid storage：Acrylamide 29.2g, Bisacrylamide 0.8g, plus ddH2O are settled to 100mL, filter Paper filtering is after 4 DEG C of preservations.

2) 1.5M Tris-HCl buffer solutions, pH8.8

36.3g Tris-base are weighed in 70mL ddH2O, are settled to HCl tune pH value to 8.8, plus ddH2O 100mL, autoclaving, room temperature preservation.

3) 0.5M Tris-HCl buffer solutions, pH6.8

6g Tris-base are weighed in 70mL ddH2O, 100mL is settled to HCl tune pH value to 6.8, plus ddH2O, it is high Pressure sterilizing, room temperature preservation.

4) 10%SDS

10g SDS are weighed, 100ml, autoclaving, room temperature preservation are settled to ddH2O.

5) 10%AP

0.1g Ammonium Persulfate are weighed, 1mL is settled to ddH2O, in -20 DEG C of preservations.

6) 10% separation gel (5mL)

7) 4% concentration glue (2mL)

8) 10x protein electrophoresises liquid (1000mL)

30g Tris, 144g Glycine, 10g SDS, plus ddH2O are settled to 1000mL.

9) 10x transferring films liquid (1000mL)

30g Tris, 144g Glycine, plus ddH2O are settled to 1000mL.

10) 1x transferring films liquid (1000mL)

The transferring film liquid of 100ml 10,700ml ddH2O, 200mL methanol.

11)10x PBS(1000mL)

80g NaCl,2g KH2PO4,2g KCl,2₄1.42g Na2HPO.7H O。

12)1x PBST

100mL 10PBS, 1mL Tween20, plus ddH2O are settled to 1000mL.

13) film washing liquid (1000mL)

15g Glycine, adjust pH value to be 2.5 with HCl, plus ddH2O is settled to 1000mL.

14) sample buffer

25.2g Glycerol, 0.02g Bromophenol Blue, 4g SDS, 20ml 1M Tris-HCl pH6.8, 3.1g DTT, plus ddH2O are settled to 50mL, and packing is after -20 DEG C of preservations.

1.2.5 the vivoexpression of GmCRY1＆2 and GmCIB1 albumen and purifying

The His-GmCRY1 of restructuring, His-GmCRY2, His-GmCIB1-Flag albumen are by sf9 insect cell cell System's expression.GmCRY1, GmCRY2 and GmCIB1-Flag coding region sequence are connected respectively to by EcoRI and XhoI digestions PFASTBacHTA carriers, by recombinating Bacmid DNA mediated transformation sf9 insect cells.Build and conversion process reference Invitrogen Bac to Bac Baculovirus Expression System。

Protein purification：Virus infection cell (3000g, 10min) is collected by centrifugation, be resuspended in XB solution (500mM NaCl, 50mM Tris-HCl pH 7.8,0.5%Triton X100,4.8mM β-ME, 1mM PMSF add fresh), ice bath 30min, ultrasonication (SONICS＆MATERIALS.INC, Model VC505) is no longer sticky up to cell solution, then hypervelocity Centrifuge (45000g, 60min, 4 DEG C), collect supernatant and the filtering membrane filtration with 0.22 μm.With Ni-NTA (Invitrogen) nickel Post adsorbs destination protein, dialyses and concentrates (Amicon Concentrator 30KD cut off).

1.2.6 the interaction of external albumen and albumen

External GmCRY1＆GmCRY2 and GmCIB1 interaction：

1) GmCRY1, GmCRY2 and GmCIB1-Flag of insect cell expression cell are cracked.By GmCRY1 and GmCRY2 cell pyrolysis liquid is mixed with GmCIB1-Flag cell pyrolysis liquid respectively.

2) the cell high speed centrifugation after cracking goes precipitation, and filters supernatant with 0.22 μm of filter.

3) lysate concentration is determined with BCA Protein Assay System (Pierce).

4) XB buffer solutions are used by cell lysis buffer to 1 μ g albumen/μ L.

5) 1mL GmCRY1, GmCIB1-Flag lysate mixed liquors and GmCRY2, GmCIB1-Flag lysate are taken respectively Mixed liquor and 20 μ L protein-A/G agarose are incubated in 1h, removal lysate and the non-spies of protein-A/G agarose The albumen that the opposite sex is combined.

6) by the protein-A/G agarose solution for being connected to Flag antibody be added separately to GmCRY1-GmCIB1 and In GmCRY2-GmCIB1 mixed pyrolysis liquid, it is placed on ice, different time is handled under dark or blue light (22 μm of olem-2s-1) (10min,30min,60min,120min)。

7) 1000g, 3min, 4 DEG C, collect Agarose, with WB2 buffer solutions (500mM NaCl, the 50mM Tris- of ice bath HCl pH 7.8,0.1%Triton X100,1mM PMSF) wash 4 times.

8) Agarose beads are resuspended in the sample buffers of 30 μ L 2,95 DEG C are boiled 10min.Take 10 μ l SDS- PAGE is separated, and detects target protein with Western Blot.0.2% Input is taken as control.With GmCRY1 and GmCRY2 Antibody detect GmCRY1 and GmCRY2 respectively, with Flag antibody test GmCIB1.

1.2.7 the interaction of vivo protein and albumen

(1) detection of soybean interaction of in vivo proteins

Soybean cultivates agriculture 18 and 35S::YFPGmCIB1 is overexpressed transfer-gen plant and is planted in respectively under short-day three weeks or so. Plant is transferred under dark condition blue light after 18h (B, 22 μm of ol m-2s-1) processing, and processing time is D, 0min；B30, 30min；B60,60min；B120,120min.Different processing times are drawn materials respectively, and sample is cut at slice MG132 lucifuges 3-4h is managed, liquid nitrogen frozen is tested for co-immunoprecipitation.Total protein extract (Input) and coupling anti-GFP antigen gel posts Product (GFP-IP) under dragging carries out immuning hybridization, subsequent ECL films stripping buffer using anti-YFP antibody Anti-YFP antibody is washed off, is immunized and combined again with anti-GmCRY2 or anti-GmCRY1 antibody.

(2) tobacco transient expression detection protein-protein interaction

1) Agrobacterium of the picking containing purposeful expression vector (pGWB11-GmCIB1, pGWB17-GmCRY2) is in 3-5mL LB Culture medium (+50mg/L Kana+50mg/L Rif), 250rpm, 28 DEG C of shaken cultivations are stayed overnight (16h).OD600=1-2

2) with 1；500—1:1000 are forwarded to fresh LB (+10mM MES+20uM AS+50mg/L Kana+ 50mg/L Rif), 250rpm, 28 DEG C of shaken cultivations are stayed overnight (16h).OD600=1

3) 4000rpm centrifuges 10min, removes supernatant

4) thalline is resuspended with infiltration buffer (10mM MES, 150uM AS, 10mM MgCl2) (can first use The infiltration buffer of bacterium solution half volume are resuspended).Measure re-suspension liquid OD600 numerical value.

5) infiltration buffer adjust pGWB11-GmCIB1 and pGWB17-GmCRY2 re-suspension liquids to OD600= 1.5, P19 re-suspension liquids are to OD600=1.

6) each bacterium solution (being remixed before also can first separating standing, injection) is mixed in equal volume, is being stored at room temperature re-suspension liquid 3 hours More than (but the not too long time, it is proposed that do not exceed 6 hours).

7) tobacco prepares：Tobacco can be taken out from culturing room within one day before the injection, be placed on laboratory upstairs, because The tobacco leaf water content of culturing room is high, and bacterium solution is difficult that infiltration injection is entered.The blade of selected injection wants full extension, close Preferably, too tender bad injection, too old vigor is not strong for the blade on top.

8) an aperture is pricked with syringe needle in vacuum side of blade, has spent the syringe alignment osculum of syringe needle, another hand hand Refer to and gently block syringe and aperture across blade (dynamics is needed oneself to know from experience, and has weighed very much bacterium solution and has not pushed away not come out, and too light bacterium solution is pushed away Do not enter, experience is used with forefinger side is relatively good), piston is promoted, bacterium solution is injected into blade, has expired whole leaf until water stain Piece (if blade is larger, pricks 2-4 aperture).It is labelled on petiole.

9) tobacco injected is it is noted that moisturizing, greenhouse moves to dark placement two days later after placing one day, and portion of material is blue Light (B, 22 μm of ol m-2s-1) handles 2h, and the material that dark and blue light is handled is drawn materials respectively, rapidly with liquid nitrogen frozen, for exempting from Epidemic disease is co-precipitated.

1.2.8 DNA-binding is tested

(1) preparation of material：

1)64bp dsDNA:64bp ssDNA containing 16bp random nucleic acid sequences in the middle of composition sequence.

Random 5’-TGGAGAAGAGGAGAGTGGGCNNNNNNNNNN

Binding-F NNNNNNCTCTTTTGCATTCTTCTTCGATTCCGGG-3’

Random 5’-CCCGGAATCGAAGAAGAATGCAAAAGAGNNN

Binding-R NNNNNNNNNNNNNGCCCACTCTCCTCTTCTCCA-3’

RBingF 5’-TGGAGAAGAGGAGAGTGGGC-3’

RBingR 5’-CCCGGAATCGAAGAAGAATGCAAAAGAG-3’

2)Protein:Insect cell expression His-GmCIB1

3) 5x DNA combination buffers：

(2) test procedure：

1) dsDNA synthesis:50ul PCR reaction system moderate mixing RBSS-F and RBSS-R (200pmole).PCR Following react is carried out in instrument：100℃2min；45 DEG C are slowly cooled to, 10min is kept；72℃10min.

2) insect cell expression His-GmCIB1, adsorbs destination protein using Ni-NTA (Invitrogen) nickel post, obtains It is coupled His-GmCIB1beads, SDS PAGE detection protein contents.

3) DNA is combined：Added in 0.5ml centrifuge tubes

5ul DNA random fragments (step 1 or 6)

5ul His-GmCIB1beads (10ug albumen)

2.5ul 5x DNA combination buffers

React at room temperature 10min.(note：The beads of destination protein is not contained as negative control)

4) 500ul 1x DNA combination buffers rinse beads, 2000rpm, 4 DEG C of centrifugations, abandon supernatant.Repetition is washed 5 times.

5) DNA that elution beads is combined:Add the 50mMTris-HCl (pH that 10ul contains 10mM reduced glutathiones 8.0) buffer solution elution beads, gently mixes 10min, 2000rpm, 4 DEG C of centrifugations, supernatant is transferred in a new pipe.

6) using the DNA fragmentation that affords as template, performing PCR amplification is entered by primer of F/R.

Response procedures are：94℃30s,40℃1min,72℃20s；94 DEG C of 30s, 60 DEG C of 10s, 72 DEG C of 10s, 30 circulations.

7) 2.5% Ago-Gel detection PCR primer.

8) repeat step 3 to 7, five times, until the amount of PCR primer is enough, i.e., 20 circulations are with regard to that can detect PCR productions Thing.

(there is conditions of streaking in third round PCR primer, should now reduce period and shorten extension of time).Third round PCR Program：94℃30s,40℃1min,72℃20s；94 DEG C of 30s, 60 DEG C of 10s, 72 DEG C of 6s, 22 circulations；Fourth round PCR programs： 94℃30s,40℃1min,72℃20s；94 DEG C of 30s, 60 DEG C of 10s, 72 DEG C of 5s, 20 circulations；Five to seven wheel PCR programs：94 ℃30s,40℃1min,72℃20s；94 DEG C of 30s, 60 DEG C of 10s, 72 DEG C of 3s, 18 circulations.

9) last wheel PCR primer is connected on cloning vector (TA cloning kit), connection product conversion large intestine bar Bacterium DH5 α.

10) screened through blue hickie, picking hickie carries out sequencing analysis.

1.2.9 ChIP is tested

(1) it is crosslinked：

1) soybean wild type and plant in 35S::YFPGmCIB1 is overexpressed transfer-gen plant and is planted in respectively under short-day Three weeks or so.Dark place is placed after 18h, blue light (B, 22 μm of ol m-2s-1) processing 2h, is taken respectively under 4g blue lights and dark condition Spire.

2) spire taken is cut into slice to be put in 50ml triangular flasks, is separately added into 37ml crosslinked fluids, room temperature in vacuo pump Interior crosslinking 10-15min.(the transparent shape of blade after crosslinking)

3) 2.5ml 2M glycine (final concentration of 100mM), room temperature vacuum pumping pump 5min are added in the triangular flask of crosslinking To terminate cross-linking reaction.

(2) separation of chromosome with it is ultrasonically treated：

4) sample after being crosslinked is washed three times with sterile, is blotted as far as possible and is used liquid nitrogen frozen rapidly after blade surface.

5) it is fully ground sample, it is ensured that sample is in freezing state in process of lapping.

6) sample after grinding is fitted into 50ml centrifuge tubes, and the nucleus extraction liquid of 25ml precoolings is resuspended.

7) quick oscillation sample, until fully mixing, during which sample holding is placed on ice.

8) re-suspension liquid after four layers of filtered through gauze, 11000g, 4 DEG C of centrifugation 20min.This is it will be appreciated that bottom of the tube has closely Linen particle.

9) supernatant is abandoned, greyish white coloured particles (i.e. nucleus) are resuspended in the karyorhexis liquid of 2ml precoolings.(each sample takes out 5 μ l and used Compared in the fragment after ultrasound).

10) obtained sample is divided into four parts, 500 μ l in every centrifuge tube.It is ultrasonically treated that DNA is cut into 500bp or so Fragment.Ultrasonically treated program is：Ultrasound intensity is 25%, and 30s is stopped per ultrasound 15s, altogether ultrasound 5 times, and sample is kept on ice Place.

11) sample after ultrasound, 13800g, 4 DEG C of centrifugation 10min.

12) supernatant (totally four pipe) collection of same sample is bonded to together.5 μ l samples corresponding with step 9 are taken out respectively Product carry out electrophoresis, 1% Ago-Gel, 100-120V, electrophoresis 30min.Ultraviolet lower observation, if the sample after ultrasound is in 200- There is the band of fuzzy hangover sample in 1000bp, and is relatively concentrated in 500bp or so, illustrates that the ultrasonic effect of DNA sample is relatively good.Instead It, ultrasonic effect is not good, further to optimize ultrasound condition.

(3) immunoprecipitation：

13) 100 μ l ultrasonically treated chromosome samples lysate is taken to dilute ten times into 1ml.

14) the salmon sperm DNA/protein A of 50 μ l pre-equilibrations are separately added into the sample after diluting Agarose beads, 4 DEG C gently upset be incubated 1h, go in sample with salmon sperm DNA/protein A agarose The DNA fragmentation of beads non-specific bindings.

15) 3,800g, 4 DEG C of centrifugation 2min, discard agarose beads.

16) the appropriate antibody (anti-GFP) of 5 μ l is added in supernatant, 4 DEG C gently overturn overnight incubation.

17) the salmon sperm DNA/protein A of 80 μ l pre-equilibrations are added in the reaction solution in (16) Agarose beads, 4 DEG C are continued to be incubated 2h.

18) 3,800g, 4 DEG C of centrifugation 2min, supernatant discarding collects the agarose beads for combining chromosome.

19) Agarose beads cleaning, is separately added into below 1ml buffer solutions, 4 DEG C gently upset mix 5min, 3, 800g, 4 DEG C of centrifugation 2min, abandons supernatant.The order of buffer solution is used in cleaning：Less salt rinsing liquid is rinsed once, high salt rinsing liquid Once, LiCl rinsing liquids are rinsed once for rinsing, and TE buffer solutions are rinsed twice.

20) elution buffer that 250 μ l are newly prepared is added in the centrifuge tube containing agarose beads, ambient temperature with gentle 15min is overturn, immune combination complex is eluted from agarose beads.

21) 3,800g, 4 DEG C of centrifugation 2min, supernatant is transferred in a new centrifuge tube.

22) elution buffer that 250 μ l newly the prepare centrifuge tube containing agarose beads into (21) step is added In, ambient temperature with gentle upset 30min, elution is immune again combines complex, 3,800g, 4 DEG C of centrifugation 2min.

23) by 21) and 22) eluent mixes totally 500 μ l twice in step.At the same time, the chromosome after 50 μ l ultrasounds Fragment (coming from step 12) adds 450 μ l elution buffers and is used as input (positive control).

(4) crosslinking and protein digestibility are removed：

24) cross-linking reaction is gone:20 μ l 5M NaCl, 65 DEG C of overnight incubations are added in each sample into step 23.

25) digestion of protein：Sequentially added in each sample：10μl 0.5M EDTA,20μl 1M Tris-HCl (pH6.5), 1 μ l proteinaseK (20mg ml-1), 45 DEG C of incubation 1.5h.

(5) DNA is precipitated：

26) phenol/chloroform/isoamyl alcohol of isometric (550 μ l) is added, gently upset is mixed.

27) 13,800g, 4 DEG C of centrifugation 15min, supernatant is transferred in a new 2ml centrifuge tubes.

28) sequentially add：The absolute ethyl alcohol of 2.5 times of volumes, the 3M sodium acetates (pH5.2) of 1/10 volume, 4 μ l glycogens (20mg ml-1), -80 DEG C of precipitation DNA are stayed overnight.

29) 13,800g, 4 DEG C of centrifugation 15min.

30) supernatant, the ethanol of 500 μ l 70% rinsing precipitation are abandoned.13,800g, 4 DEG C of centrifugation 10min.

31) supernatant, drying at room temperature precipitation are abandoned.

32) 50 μ l TE buffer solutions DNA, -80 DEG C save backup.

(6)Real-time PCR：

33) real-time fluorescence quantitative PCR is carried out using Roch480, utilizes LightCycler 480SYBR Green I Master detects fluorescence signal

34) performing PCR detection is entered using gene specific primer, the involved gene of this experiment is:GmFT3、GmFT4、 GmTFL1、GmTFL3、GmWRKY53。

35) reaction system is 15 μ l：

Response procedures are：

Stage1：95 DEG C of 5min of thermal starting (20 DEG C/s).

Stage2：PCR 95 DEG C of 5s (20 DEG C/s) of reaction, 55 DEG C of 10s (20 DEG C/s), 72 DEG C of 30s (20 DEG C/s), 45Cycles.The calculating of gene relative expression quantity is calculated using %Input methods

(7) solution is prepared：

1) cross-linking buffer：0.4M sucrose, 10mM Tris-HCl (pH8.0), 1mM PMSF, 1mM EDTA, 1% Formaldehyde, matching while using

2) nucleus dissociation buffer solution：0.25M sucrose, 15mM PIPES (pH6.8), 5mM MgCl2,60mM KCl, 15mM NaCl, 1mM CaCl2,0.9%TritonX-100,2mgml -1pepstain A, 2mgml -1aprotinin, 4 DEG C of guarantors Deposit.

3) Nuclei lysis buffer：50mM HEPES (pH7.5), 150mM NaCl, 1mM EDTA, 1mM PMSF, 1% SDS, 0.1%Na deoxycholate, 1%Triton X-100,1mg ml -1pepstain A, 1mg ml -1aprotinin

4) elution buffer：0.5%SDS, 0.1M NaHCO3

5) less salt wash buffer：150mM NaCl, 20mM Tris-HCl pH 8,0.2%SDS, 0.5%Triton X-100,2mM EDTA, 4 DEG C of preservations.

6) high salt wash buffer：500mM NaCl, 20mM Tris-HCl pH 8,0.2%SDS, 0.5%Triton X-100,2mM EDTA.4 DEG C are preserved.

7) LiCl wash buffers：0.25M LiCl, 1%sodiumdeoxycholate (NaTDC), 10mM Tris-HCl pH8,1%NP-40,1mM EDTA.4 DEG C are preserved.

8) TE buffer solutions：8,4 DEG C of preservations of 1mM EDTA, 10mMTris-HCl pH.

9)PMSF：200mM, -20 DEG C of preservations are configured to methanol.

10)Pepstatin A：1mg ml -1, -20 DEG C of preservations are configured to methanol.

11)Aprotinin：1mg ml -1, -20 DEG C of preservations are configured to water.

12) the salmon sperm DNA/protein A agarose beads of pre-equilibration:

The method that protein A agarose beads are balanced with lysis buffer is as follows：Depending on the number of sample, difference 50 μ l salmon sperm DNA/protein A agarose beads are taken in 1.5ml centrifuge tubes, 3,800g, 4 DEG C of centrifugations 2min, abandons supernatant.50 μ l lysis buffers are added, 4 DEG C gently overturn mixing 2min, 3,800g, 4 DEG C of centrifugation 2min, abandon supernatant, Repeat this step once.Protein agarose A are resuspended in 50 μ l lysis buffers.

13)EDTA：50mM

2. result and analysis

The phenotype of 2.1 GmCRYs and GmCIB1 overexpressions or RNAi Soybean transgenic plants

GmCRY1-OX, GmCRY2-OX, GmCRY2-RNAi and the GmCIB1-OX obtained by Genetic Transformation of Soybean turns Gene plant, wild type (WT) and each transfer-gen plant are planted under continuous illumination, respectively with corresponding antibody to transgenosis Plant carries out western detections, as a result show be overexpressed GmCRY1 in plant GmCRY1-OX, GmCRY2-OX and GmCIB1-OX, GmCRY2 and GmCIB1 are expressed (Figure 12 I, 13G, 15G) respectively, and endogenous GmCRY2 in GmCRY2-RNAi transformed plants Significantly reduce (Figure 14 I).Leaf senile phenotypic analysis shows, GmCRY1-OX, GmCRY2-RNAi and GmCIB1-OX transgenosis Plant, plants two weeks or so, and cotyledon comes off substantially earlier than wild type, gradually turns yellow with the growth list leaf of plant, and turn yellow Time also morning and wild type, these three transgenic lines show the phenotype of early ageing.And GmCRY2-OX transfer-gen plants are either Cotyledon, which comes off, or single leaf turns yellow all is later than wild type, illustrates that the aging of the transfer-gen plant is delayed by.Above phenotype implys that GmCRY1 and GmCIB1 may promote plant senescence, and GmCRY2 suppresses the process of leaf senile.

Further the relevant parameter of analysis and characterization leaf senile is shown, Chlorophyll content and chlorophyll a:B measurement results Show (Figure 16 A), GmCRY1-OX, GmCRY2-RNAi and GmCIB1-OX transfer-gen plant Determination of Chlorophyll content is significantly lower than open country Raw type WT, and chlorophyll a:B ratios are higher than WT, because then chlorophyll b is first transformed into chlorophyll a during chlorophyll degradation Degradation pathway is entered back into, therefore in degradation process Determination of Chlorophyll a:B ratios can be raised.The result also implys that three above turns base Because the degradation speed of strain Determination of Chlorophyll is higher than wild type control.And then on the contrary, chlorophyll contains in GmCRY2-OX transfer-gen plants Measure and be higher than WT, and chlorophyll a:B ratios are less than WT, thus it is speculated that the degraded of the transfer-gen plant Determination of Chlorophyll is delayed by.To continuous light The measure of photosynthetic rate is carried out according to the plant of lower growth 31 days, the second compound leaf and the 4th compound leaf are selected respectively as measure object, Determine once within every two days, as a result METHOD FOR CONTINUOUS DETERMINATION two weeks shows (Figure 16 B), the photosynthetic rate of each measured strain is with blade Its photosynthetic rate of the increase of growth time is gradually reduced, from GmCRY1-OX, GmCRY2-RNAi in terms of the trend that photosynthetic rate is reduced It is higher than wild type with photosynthetic rate reduction speed in GmCIB1-OX transfer-gen plants, and it is photosynthetic in GmCRY2-OX transfer-gen plants Rate reduction amplitude is less than WT.

MRNA level in-site to aging related genes determines analysis shows, the transfer-gen plant occurred in senescence phenotype, these The expression of aging related genes substantially rises, and very low (Figure 16 C) in the expression of GmCRY2-OX transfer-gen plants. As homologous gene GmWRYK53 in soybean of arabidopsis typical aging related genes WRKY53, SAG12, APGL5, The expression quantity of GmSAGL12, GmAGL12, GmAPGL5 in GmCRY2-OX transfer-gen plants is far below WT, and in GmCRY1- Expression quantity in OX, GmCRY2-RNAi and GmCIB1-OX transfer-gen plant far above WT, especially GmWRYK53, The expression quantity of tri- genes of GmSAGL12, GmAGL12 is higher than 10 times or so of wild type.Imply that GmCRY1, GmCRY2 and GmCIB1 may participate in the regulation and control of plant senesecence.Play an enzyme of speed limit during PaO gene code chlorophyll degradations (Phephorbide a oxygenase), the expression analysis to the homologous gene GmPaO of the gene in soybean shows (Figure 16 D), The etiolated seedling of the lower growth 12 days of dark is transferred under blue light respectively, and blue light is handled 6 hours, in GmCRY1-OX, GmCRY2-RNAi It is very low with the adusk expression quantity of the gene in GmCIB1-OX transfer-gen plants, with its table of the increase of blue light light application time Gradually increase up to amount and far above in wild type, especially GmCRY2-RNAi transfer-gen plants in blue light illumination 6 hours the base 30 times higher than wild type or so of the expression quantity of cause.And in GmCRY2-OX transfer-gen plants, the gene maintains very low table always Up to level.Speculate that blue light may induce the table of GmPaO in GmCRY1-OX, GmCRY2-RNAi and GmCIB1-OX transfer-gen plant Reach, so as to accelerate the process of leaf senile.And GmPaO expression is suppressed in GmCRY2-OX transfer-gen plants, so its leaf Piece aging is delayed by.

GmCIB1 gene overexpressions have been obtained to the transgenic arabidopsis of Blooming into arabidopsis in embodiment 1, Speculate that GmCIB1 plays an important role to regulation and control of blooming.And the phenotype of late flower is shown in GmCIB1-OX genetically engineered soybeans (Figure 17), implys that GmCIB1 also plays the effect for regulation and control of blooming, but with arabidopsis CIB1 effect on the contrary, in soybean GmCIB1 suppresses to bloom.GmCRY1-OX and GmCRY2-RNAi transgenosis, which is planted, to be shown to the analysis of other transfer-gen plant flowering phenotypes Strain shows the phenotype of early blossoming, and GmCRY2-OX transfer-gen plants then show late flower.Speculate that GmCRY1 and GmCRY2 is being bloomed Opposite effect is played in regulation and control, the former Accelerate bloom and the latter suppresses to bloom.To the GmFTs in WT and transfer-gen plant and GmTFLs mRNA expression is analyzed, and as a result shows that (Figure 18) plants in GmCRY1-OX and GmCRY2-RNAi transgenosis GmFTs expression has different degrees of raising in strain, and GmTFLs expression is very low.And in GmCIB1-OX and GmCRY2-OX GmTFLs mRNA level in-site substantially rises in transgenic line, and GmFTs expression quantity is significantly lower than WT.Further demonstrate GmCRY1 can promote soybean blossoming, and GmCRY2 and GmCIB1 plays negative regulation effect to blooming.

GmCRY1 is overexpressed leaf presenility phenotypic results referring to Figure 12.

GmCRY2 overexpression blade evenings decline phenotypic results referring to Figure 13.

GmCRY2-RNAi is overexpressed leaf presenility phenotypic results referring to Figure 14.

GmCIB1 is overexpressed leaf presenility phenotypic results referring to Figure 15.

Leaf senile relevant parameter analysis result is referring to Figure 16.

GmCRYs and GmCIB1 is to the result of the regulation and control of soybean blossoming time referring to Figure 17.

GmFTs and GmTFLs in different genotype mRNA expressions referring to Figure 18.

The analysis of protein-protein interaction between 2.2 GmCRYs and GmCIB1

2.2.1 Yeast two hybrid assay analyzes protein-protein interaction between GmCRYs and GmCIB1

Research in arabidopsis shows that the CRYs and CIBs in the interaction that CRY2 and CIB1 has blue light to rely on, soybean is It is no also to have similar interactionWe are detected mutual between GmCRYs and GmCIBs by yeast two-hybrid assay first Effect.AtCRY2, GmCRY1 and GmCRY2 are building up to respectively on carrier pDEST32 (Bait), GmCIBs is building up to respectively On pDEST22 (Prey), difference cotransformation yeast cells, in (- His ,-Trp ,-Leu) deficiency culture medium, dark and blue light Screened under the conditions of (22 μm of ol m-2s-1).Find out that GmCRY2 and GmCIB1 have interaction under blue light from Figure 19 A-C, AtCRY2 and GmCIB1/4/8 has the interaction of blue light specifically.Betagalactosidase activity detection shows that (Figure 19 D) contains GmCRY2 and GmCIB1 yeast cells has very high betagalactosidase activity under blue light, further demonstrate that GmCRY2 with GmCIB1 has the interaction of blue light specifically.

The interaction of albumen is referring to Figure 19 between GmCRYs-GmCIBs.

The interaction of albumen is referring to Figure 20 between GmCRY2-GmCIB1.

Interaction of the domains (PHR domains and CCT domains) different GmCRY1＆2 between GmCIB1 is referring to figure 21 and 22.

Interaction between domains different GmCIB1 and GmCRY1＆2 is referring to Figure 23.

The protein-interacting between GmCRY1/2 and GmCIB1 is analyzed by yeast two-hybrid, BiFC, in vivo Experiment carries out step card and shows (Figure 24), and tobacco transient expression (Figure 20 F) display, GmCRY2 and GmCIB1 has in tobacco body The interaction that blue light is relied on.Using GmCIB1-OX be overexpressed plant carry out Pull down experiments, as a result show GmCRY1 with GmCRY2 can have the interaction of blue light specifically in soybean body with GmCIB1, and this interaction is with light application time Increase, action intensity constantly strengthens, and interaction strength is most strong during illumination two hours.GmCRY1 only has N-terminal in yeast cells PHR areas can be with GmCIB1 interactions, and GmCRY1 total lengths can be with GmCIB1 interactions under blue light in vivo, thus it is speculated that GmCRY1 exists Yeast and plant are likely to form different structures, therefore produce the different modes of action.

2.2.3 insect cell in vitro expression system Pull down analysis of experiments GmCRYs and GmCIB1 albumen phase interaction With

Using insect expression system, GmCRYs and GmCIB1-Flag albumen, external Pull down experiments are expressed respectively As a result (Figure 25) is shown, GmCRY1, GmCRY2 and GmCIB1 have the protein-interacting that blue light is relied on, and action intensity is with light Increase according to the time constantly strengthens.Compared with GmCRY2, action intensity is relatively low between GmCRY1 and GmCIB1.This experiment egg used It is in vain GmCRY1 full-length proteins, GmCRY1 total lengths can not be with GmCIB1 interactions in yeast body, and both can in soybean body Interaction, thus it is speculated that may be variant in different species GmCRY1 protein structure, so as to result in changing for the mode of working Become.

2.3 GmCIB1 protein DNA binding sites are detected

GmCIB1 albumen and DNA external interaction result show that GmCIB1 can be combined (Figure 26) with E-box.But CIB1 is combined with G-box (CACGTG) in vitro in arabidopsis, is combined to regulate and control the expression of downstream target gene in vivo with E-box.Push away Survey GmCIB1 in vivo may also by is combined with E-box regulation and control target gene expression, but GmCIB1 regulate and control target gene it is current It is not clear.

2.4 ChIP-qPCR detect that GmCIB1 and the interchromosomal of aging or regulation and control related gene of blooming interact

GmCIB1 is overexpressed the phenotype that genetically engineered soybean shows early ageing and evening flower, in order to find the downstream of GmCIB1 effects Target gene, tentatively to illustrate its mechanism of action.GmCIB1 and some candidate gene chromosomes are studied by ChIP-qPCR first The interaction in area.Candidate gene is selected in above-mentioned detected gene, some genes that mRNA expressions are changed greatly. GmFT3, GmFT4, GmTFL1, GmTFL3 in such as blooming, the related gene GmWRKY53 of aging.Experiment material is carried out The processing of dark and blue light, sees whether blue light can influence interaction therebetween.Experimental result is shown, no matter in blue light or dark Lower GmCIB1 albumen can be combined (Figure 27 a, 27b, 27e) with the E-box of GmFT3, GmFT4 and GmWRKY53 chromosomal region, be entered It is different that one step analysis finds that blue light and dark have identical also to have with reference to E-box down.As GmFT3 chromosomal regions are tied with GmCIB1 The E-box of conjunction is identical under blue light and dark, and a areas in GmFT4 are black dull lower specific bonds, and j areas are blue lights Lower specific bond.K areas containing two E-box in GmWRKY53 are significantly larger than dark with the binding abilities of GmCIB1 under blue light Under.And these regions that GmCIB1 is combined are not distributed only over gene promoter area, are also distributed, imply that down in genome The chromosome structure of trip target gene determines the binding site of transcription factor, while also speculating that regulation and control of the GmCIB1 to target gene are made With not being strict blue light dependence.The protein bound regions of GmCIB1 (figure is not detected in GmTFL1 and GmTFL3 27c, 27d), illustrate that GmCIB1 may not have regulating and controlling effect to the two.

3. discuss

The phenotypic analysis of 3.1 GmCRY1-OX, GmCRY2-OX, GmCRY2-RNAi and GmCIB1-OX transfer-gen plant

GmCRY1-OX, GmCRY2-OX, GmCRY2-RNAi and GmCIB1-OX transgenosis are obtained by Genetic Transformation of Soybean Soybean, is found during observing phenotype, under continuous illumination (LL), and aging correlation is had in different transfer-gen plants Phenotype.

The GmCRY1-OX under the conditions of LL, GmCRY2-OX and GmCIB1-OX transfer-gen plant show early ageing phenomenon, such as with Wild type (WT) turns yellow compared to cotyledon and single leaf and come off substantially in advance, the parametric measurement that leaf senile is characterized show WT also in When high light closes effect stage, these transgenic line Determination of Chlorophyll contents are reduced sharply, and photosynthetic rate is also greatly lowered, and declines simultaneously The mRNA level in-site of uneducated person correlation gene (GmWRYK53, GmSAGL12, GmAGL12, GmAPGL5) also substantially rises.And GmCRY2-OX But occurs opposite phenotype in transfer-gen plant, cotyledon and single leaf turn yellow and come off substantially to be postponed for WT, Ye Lv Cellulose content is not substantially reduced, and photosynthetic rate reduction speed is far below WT, and the expression of aging related genes is suppressed.Imply that GmCRY1 and GmCIB1 play a part of just regulating and controlling in leaf senile regulation process, and GmCRY2 may suppress declining for blade Always.The expression pattern of gene determines the function of gene to a certain extent.To GmCIB1 mRNA space-time table in embodiment 1 Expression patterns have carried out detailed analysis, as a result show that GmCIB1 is of a relatively high in the overall expression in single leaf period, are overexpressed In GmCIB1 Transgenic soybean plants, the phenotype shifted to an earlier date in single leaf period leaf senile has just shown, thus it is speculated that GmCIB1 is in list The concentration expression in leaf period promotes the performance of its function.And GmCIB1 photoperiod expression pattern analysis is shown, the gene MRNA level in-site is by the positive regulation and control of illumination, and as under the conditions of continuous illumination (Fig. 8 c a and c), GmCIB1 overall expression is in Existing growth trend, this expression pattern may be overexpressed plant with GmCIB1 and obvious senescence phenotype is shown under continuous illumination Correlation, the specific mechanism of correlation is not clear at present between gene expression pattern and its function, also needs to make further research.

Flowering phenotype analysis finds that GmCRY1-OX and GmCRY2-RNAi transfer-gen plants show early blossoming phenotype, and GmCRY2-OX and GmCIB1-OX genetically engineered soybeans show late bloom.FT and TFL are two work(in blooming in arabidopsis The opposite gene of energy, FT is that the integration factor in an approach of blooming integrates the signal from different flowering approach, Accelerate bloom. And TFL1 and FT function is completely on the contrary, be that one kind is bloomed inhibiting factor.TFL1 mainly passes through late blooming approach integrator gene LFY expression, and suppress floral meristem Gene A P1 and CAL expression, extend vegetative growth phase, suppress flowering transition, and Maintain the indeterminate growth state of inflorescence.FT 9 homologous gene GmFTs turn base in GmCRY1-OX and GmCRY2-RNAi in soybean Because the expression quantity of plant has a different degrees of raising, and expression quantity in GmCRY2-OX and GmCIB1-OX genetically engineered soybeans is all Maintain very low level.Arabidopsis bloom inhibiting factor TFL1 4 homologous gene GmFTs in GmCRY2-OX and GmCIB1-OX The expression quantity of transfer-gen plant has a different degrees of raising, and the table in GmCRY1-OX and GmCRY2-RNAi genetically engineered soybeans It is very low up to measuring.Result above shows that GmCRY2 and GmCIB1 may play identical regulating and controlling effect in regulation and control of blooming, and suppress out Flower.And GmCRY1 plays cone production effect.In embodiment 1 by by GmCIB1 gene overexpressions in arabidopsis to its work( Can be analyzed, the gene can promote AtFT mRNA expressions so as to Blooming in arabidopsis, its phenotype with The result that above-mentioned GmCIB1 genes suppress to bloom in soybean is opposite, thus it is speculated that be probably that result in gene due to the difference of species The difference of regulatory mechanism.Soybean is strict short-day plant, and arabidopsis is also drawn in long-day plant, and this research GmCIB1 expression light regulation and control, these factors, which can be, result in GmCIB1 genes and is shown not in arabidopsis and soybean Congenerous, also need to take off by the further investigation to GmCIB1 genes or even whole GmCIB gene families as specific mechanism Show.In summary, we can show that in leaf senile regulation and control GmCRY1 and GmCIB1 play facilitation, and GmCRY2 plays negative regulation.The GmCRY1 Accelerate blooms in regulation and control of blooming, and GmCRY2 and GmCIB1 suppresses to bloom.GmCRY1 and GmCRY2 plays antipodal effect in both regulation and control, and the two may play antagonism in every kind of regulation process.

The interaction of protein-protein between 3.2 GmCRYs and GmCIB1

In order to preferably annotate the function of GmCRYs and GmCIB1 in soybean, the interaction between them is carried out In-depth study.The interaction of GmCRY2 and GmCIB1 blue lights specifically is screened by yeast two-hybrid assay first, and made Increased with intensity with the enhancing of blue light light intensity and the increase of light application time, but GmCRY1 total lengths and GmCIB1 are no mutual Effect.There is the interaction of blue light specifically in GmCRY1/2 PHR areas and GmCIB1, imply that GmCRY PHR domains are albumen-eggs White interaction is required, and GmCRY1 CCT domains may influence its interaction between GmCIB1.To GmCIB1 Structural analysis show its N-terminal domain be also interactions between protein necessary to domain.

Pull-down experiments in vivo studies, tobacco transient expression and GmCIB1-OX transfer-gen plant bodies are further discussed The interaction of GmCRY2 and GmCIB1 blue lights dependence is demonstrate,proved.The GmCRY1 that is expressed respectively using insect protein expression system, GmCRY2 and GmCIB1 albumen and carry out Pull-down experiment also draw identical result.And between GmCRY1 and GmCIB1 Interaction is more interesting, although total length GmCRY1 in yeast and tobacco can not with GmCIB1 interactions between protein, but in soybean body There is the interaction that blue light is relied on both interior.The GmCRY1 and GmCIB1 albumen of insect expression also has weaker mutual under blue light Make.Speculate the interaction that GmCRY1 is likely to form in yeast, insect and plant between different structures, influence albumen.

3.3 GmCIB1 and the interchromosomal of aging or regulation and control related gene of blooming interact

Arabidopsis CIB1 is combined with the G-box (CACGTG) in E-box (CANNTG) in vitro, in vivo with FT promoter regions E-box combine to regulate and control to bloom.The in vitro test result that GmCIB1 and DNA interacts shows that GmCIB1 can be tied with E-box Close, ChIP experiments show that GmCIB1 and the E-box of arabidopsis FT homologous gene GmFT3 and GmFT4 chromosomal regions are combined, and also can Combined with the E-box in aging related genes WRYK53 homologous gene GmWRYK53 promoter region Matrix attachment regions, and it is this With reference to not strict blue light specificity.And suppressor TFL of blooming homologous gene GmTFL1 and GmTFL3 are not detected Calmodulin binding domain CaM.Speculate that GmCIB1 may be bloomed in soybean body by negative regulation GmFT expression inhibiting, promote senile correlation Because of the transcription such as GmWRYK53 and then promotion leaf senile.

4. brief summary

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Sequence table

<110>Institute of Crop Science, Chinese Academy of Agricultural Science

<120>Soybean GmCIB1 genes and GmCRY2 genes and its regulation and control bloom and aging effect

<160> 4

<210> 1

<211> 1263

<212> DNA

<213>Soybean

<400> 1

ATGTTGCATT GTCTCAACAC TTCGGGGAAT CTAGGAGCCA CCTGTTCGGA CATGACAGTG 60

TTGGAAAGAC AACGGGAAGC AACCATCAAG TGGCAGCAAC ACCTCCAGAA CAACCAACCA 120

CCCTACTTAA CGGACTTTAA CGTTAACGTT AACGCCGTCT TCTCTTCTTC TTCTTCTTCT 180

TCTCAGAGTC AGGGCCTGGT CATGAATATT TGCGCTGATT CCGCGCTCGG CGAGGTCCTA 240

ACTCACTCCG TCAAACCCGA CCCGGGTGTC TGGCCCGAAT TCGACGCCGG ATTCGGATCC 300

TCTTCCGGTT TTCTACCCAC CATTTCGCCA ACTTGTAGCA GAGGCGGAGA CTTGGTTTCA 360

CCCAAGGAAA ACATGGCCAG TGCGAAAGAA AACACTAAGA AGAGAAAACC TCAGAACTCC 420

AAGGTTGTTG CGGCGAGTGA TAATAAACAG GACAAGAGAG TCAAAGCAAG TGGTGAAGAA 480

GGAGAATCCA AAGTAACAGA GCAAACCAGC AACAAGAATG GCAAATCCAA CGCAAATAAA 540

AACAACAACA GAGAAACAAC CTCTGCGGAG ACTTCCAAGG ATAATTCGAA AGGCTCCGAG 600

GTTCAAAATC AGAAGCCAGA GTACATTCAT GTCCGAGCGC GTCGTGGACA AGCCACGGAT 660

AGTCATAGCT TAGCTGAAAG AGTTAGGAGG GAGAAGATTA GTGAGAGAAT GAAGTATTTG 720

CAAGATTTAG TACCGGGTTG CAACAAAGTT GCAGGGAAAG CTGGAATGCT TGATGAAATT 780

ATTAACTATG TTCAGTCTCT TCAACGCCAA GTTGAGTTCT TGTCAATGAA ATTAGCGGCT 840

GTAAACCCAA GGCTTGACTT CAACCTTGAC GAACTATTTA CCAAAGAGGT GTTTCCTTCT 900

TGTGCTCAAA GTTTTCCAAA CATTGGGATG CCATTAGATA TGAGTATGAG TAACAACCCT 960

TCGTATCTAC CATTTAATTC AGCGCAGCAA CTTGTATCGT GCTGTGGTGG ACTAATAAAC 1020

AACATGGGGA TAAGCCCTCC AAACATGGGA CTCCGAAGGA ACATTAGTAC TAGCCCTGTA 1080

CCTTTGCCCG AAACGTTTCT TGACTCGTCC TGTTTCACTC AAATTCTACC CTCCTCAAAT 1140

TGGGAAGGTG GTGATTTCCA AAGCCTTTAC AACGTTGCTT TTGATCAAGG GCGAACAGCA 1200

TCTTTTCCTT CTCAGCCATT TACAGGTCTA GTTGAAGCTA GCAATCTAAA GATGGAGATG 1260

TAA 1263

<210> 2

<211> 421

<212> PRN

<213>Soybean

<400> 2

MLHCLNTSGN LGATCSDMTV LERQREATIK WQQHLQNNQP PYLTDFNVNV NAVFSSSSSS 60

SQSQGLVMNI CADSALGEVL THSVKPDPGV WPEFDAGFGS SSGFLPTISP TCSRGGDLVS 120

PKENMASAKE NTKKRKPQNS KVVAASDNKQ DKRVKASGEE GESKVTEQTS NKNGKSNANK 180

NNNRETTSAE TSKDNSKGSE VQNQKPEYIH VRARRGQATD SHSLAERVRR EKISERMKYL 240

QDLVPGCNKV AGKAGMLDEI INYVQSLQRQ VEFLSMKLAA VNPRLDFNLD ELFTKEVFPS 300

CAQSFPNIGM PLDMSMSNNP SYLPFNSAQQ LVSCCGGLIN NMGISPPNMG LRRNISTSPV 360

PLPETFLDSS CFTQILPSSN WEGGDFQSLY NVAFDQGRTA SFPSQPFTGL VEASNLKMEM 420

*

<210> 3

<211> 1905

<212> DNA

<213>Soybean

<400> 3

ATGGGTAGCA ACAGGACTAT TGTTTGGTTT AGAAGGGACC TTAGAATTGA GGACAATCCT 60

GCATTAACTG CTGCTGCAAA GGAGGGTTCT GTACTCCCTG TGTATGTATG GTGCCCTAAA 120

GAGGAAGGAC AATTTTATCC TGGAAGAGTG TCAAGGTGGT GGCTGAAGCA GTCTCTTGCT 180

CACTTGGATC AATCACTGAA GTCTCTTGGA TCCAGACTTG TGCTCATCAA AACCCACAGT 240

ACTGCTGTGG CTCTCGTGGA ATGCGTAAAG GCCATTCAAG CAACCAAAGT AGTGTTTAAC 300

CATCTGTATG ATCCAGTTTC ACTTGTCCGT GATCACAACA TCAAAGAAAA GCTAGTGGAA 360

CAAGGCATAT CTGTTCAAAG CTACAATGGA GATCTGTTGT ATGAGCCATG GGAAGTAAAT 420

AGTGAGAGTG GACGTGCTTT TACTACCTTC AATGCTTTTT GGAAGAAATG CTTGCACATG 480

CAAATGGATA TTGTTTCAGT TGTTCCTCCA TGGCAATTAA TTCCAGCTGA AGGAAAAATT 540

GAGGAATGTT CATTGGAGGA ACTTGGTCTT GAAAATGAAT CAGAAAAACC AAGCAATGCA 600

TTGCTAGGAC GGGCATGGTC ACCGGGTTGG AGAAATGCTG ACAAGGCTTT AAGAGAATTT 660

GTGGAGCTTC ATCTACTTCA CTACTCCAAG AAAAGGCTGA AGGTTGGTGG AGAGTCCACC 720

TCACTCTTGT CCCCATATCT TCATTTTGGA GAATTAAGTG CAAGGAAAGT TTTTCAAGTG 780

ACTTGTATGA AGCAAATATT ATGGACAAAT GAAGGAAACA GTGCTGGTGA AGAGAGTGCA 840

AATCTTTTCC TCAGGGCCAT TGGACTTAGG GAGTACTCAC GCTATCTTTG TTTCAACTTT 900

CCCTTCACCC ATGAGAGAGC ACTTCTGGGG CACTTAAAGT TTTTTCCTTG GAATCCTGAT 960

CCTGATATCT TTAAGACTTG GAGACAAGGT AGGACTGGCT TTCCTTTGGT TGATGCAGGA 1020

ATGAGAGAAC TTTGGGCAAC AGGATGGATA CACAACAGAA TAAGAGTAAT AGTTTCCAGT 1080

TTTGCTGTGA AAATGTTGCT TCTACCCTGG AAATGGGGAA TGAAATATTT CTGGGACACA 1140

CTTCTGGATG CAGACCTTGA AAGTGATATC TTGGGTTGGC AATATATCTC AGGAGGCTTA 1200

CCAGATGGCC ATGAGCTTGA GCGCTTGGAC AATCCTGAGA TTCAAGGGGC TAAATTTGAT 1260

CCAGAAGGTG AATATGTGAG GCAATGGCTA CCTGAGTTGG CAAGAATGCC AACTGAGTGG 1320

ATCCATCATC CTTGGGATGC ACCACTTACT GTGCTTCGAG CAGCAGGAGT TGAGCTGGGC 1380

CAGAATTACC CAAAGCCAAT CATTGATATA GATTTGGCCA GAGAAAGACT CACTGAAGCT 1440

ATATTCAAGA TGTGGGAATC TGAAGCAGCA GCAAAAGCTG CTGGCTCTGA ACCAAGGGAT 1500

GAAGTTGTTG TAGACAACTC TCACACTGTT GAAAATTTGG ACACTCAAAA AGTTGTCGTC 1560

CTGGGAAAGG CTCCATGTGC TACTATTTCA GCTAATGATC AAAAGGTGCC TGCTCTTCAG 1620

GATTCCAAGA ATGAACCGCC TACGCGGAAG AGGCCAAAGC ACATGATAGA GGAGGGGCAG 1680

AACCAGGATC ACTCTCAAAA TCATAACAAA GATACTGGCT TGTCAAGCAT TGACCAAGAC 1740

ATTTGCTCCA CAGCTGATTC TTCATCATGT AAGAAGCAGT GTGCCAGTAC AAGCTCATAT 1800

TCATTTTCAG TTCCACAGCA GTGTTCCTCA TCTTCTAATC TGAAGTGGCC ATGGCAAGAG 1860

AAGATTGATA TGGAGCAGAG TTCAAGCAAA GATGGAGCTA TGTGA 1905

<210> 4

<211> 634

<212> PRN

<213>Soybean

<400> 4

MGSNRTIVWF RRDLRIEDNP ALTAAAKEGS VLPVYVWCPK EEGQFYPGRV SRWWLKQSLA 60

HLDQSLKSLG SRLVLIKTHS TAVALVECVK AIQATKVVFN HLYDPVSLVR DHNIKEKLVE 120

QGISVQSYNG DLLYEPWEVN SESGRAFTTF NAFWKKCLHM QMDIVSVVPP WQLIPAEGKI 180

EECSLEELGL ENESEKPSNA LLGRAWSPGW RNADKALREF VELHLLHYSK KRLKVGGEST 240

SLLSPYLHFG ELSARKVFQV TCMKQILWTN EGNSAGEESA NLFLRAIGLR EYSRYLCFNF 300

PFTHERALLG HLKFFPWNPD PDIFKTWRQG RTGFPLVDAG MRELWATGWI HNRIRVIVSS 360

FAVKMLLLPW KWGMKYFWDT LLDADLESDI LGWQYISGGL PDGHELERLD NPEIQGAKFD 420

PEGEYVRQWL PELARMPTEW IHHPWDAPLT VLRAAGVELG QNYPKPIIDI DLARERLTEA 480

IFKMWESEAA AKAAGSEPRD EVVVDNSHTV ENLDTQKVVV LGKAPCATIS ANDQKVPALQ 540

DSKNEPPTRK RPKHMIEEGQ NQDHSQNHNK DTGLSSIDQD ICSTADSSSC KKQCASTSSY 600

SFSVPQQCSS SSNLKWPWQE KIDMEQSSSK DGAM 634

Claims

1. purposes of a kind of gene of separation in regulating and controlling plant leaf blade aging and/or blooming, wherein the gene is with as follows Nucleotide sequence：

(1)SEQ ID NO：Sequence or its complementary series shown in 3；

(2) under stringent hybridization condition with SEQ ID NO：The sequence of 3 hybridization；

(3) with SEQ ID NO：3 have the sequence of at least 85%, 90%, 95% or 99% homogeneity, and it regulates and controls plant leaf blade and declined It is old and/or bloom；Or

(4) by SEQ ID NO：Sequence shown in 3 passes through the missing of wherein one or more nucleotides, displacement, insertion or addition And derivative resulting sequence.

2. the purposes described in claim 1, wherein the gene is contained in DNA molecular, the DNA molecular has GmCRY2 genes Biological function.

3. the purposes described in claim 1, wherein the gene is contained in recombinant vector.

4. the purposes described in claim 2, wherein the DNA molecular is contained in recombinant vector.

5. purposes of a kind of protein of separation in regulating and controlling plant leaf blade aging and/or blooming, wherein the protein has Following amino acid sequence：

(1)SEQ ID NO：Amino acid sequence shown in 4；

(2) as the sequence of the gene code described in claim 1；Or

(3) displacement comprising one or several amino acid residues and/or missing and/or the SEQ ID NO of addition：Ammonia shown in 4 Base acid sequence, the protein has regulation and control plant leaf blade aging and/or the function of blooming.

6. the purposes according to claim any one of 1-5, wherein the plant is monocotyledon or dicotyledon, it is excellent Elect arabidopsis or soybean as.

7. a kind of method for cultivating genetically modified plants, including a kind of gene of separation is introduced purpose plant to obtain transgenosis plant Thing, so as to regulate and control the leaf senile of the genetically modified plants and/or bloom, wherein the gene has following nucleotides sequence Row：

(1)SEQ ID NO：Sequence or its complementary series shown in 3；

(3) with SEQ ID NO：3 have the sequence of at least 85%, 90%, 95% or 99% homogeneity, and it regulates and controls leaf senile And/or bloom；Or

8. method as claimed in claim 7, further comprises the seed for obtaining the genetically modified plants.

9. method as claimed in claim 7, wherein the plant is monocotyledon or dicotyledon, preferably arabidopsis Or soybean.

10. it is a kind of regulate and control plant leaf blade aging method, including a kind of gene of separation is introduced into the plant so that its Expressed in plant, wherein the gene has following nucleotide sequence：

(1)SEQ ID NO：Sequence or its complementary series shown in 3；

(3) with SEQ ID NO：3 have the sequence of at least 85%, 90%, 95% or 99% homogeneity, and it regulates and controls leaf senile； Or

11. method as claimed in claim 10, wherein the gene is contained in recombinant vector.

12. method as claimed in claim 10, wherein the plant is monocotyledon or dicotyledon, preferably intends south Mustard or soybean.

13. a kind of method of regulation and control flowering of plant, including a kind of gene of separation is introduced into the plant so that it is in plant Middle expression, wherein the gene has following nucleotide sequence：

(1)SEQ ID NO：Sequence or its complementary series shown in 3；

(3) with SEQ ID NO：3 have the sequence of at least 85%, 90%, 95% or 99% homogeneity, and it regulates and controls flowering of plant； Or

14. method as claimed in claim 13, wherein the gene is contained in recombinant vector.

15. method as claimed in claim 13, wherein the plant is monocotyledon or dicotyledon, preferably intends south Mustard or soybean.