CN101792770B - Photoperiodic tolerant mutant hd1-3 gene for regulating and controlling flowering time of rice and application thereof - Google Patents

Abstract

The invention relates to a photoperiodic tolerant mutant hd1-3 gene for regulating and controlling the flowering time of rice. The nucleotide sequence of the gene is SEQ ID No.1, and the corresponding hd1-3mRNA sequence of gene is SEQ ID No.2. The invention discovers a photoperiodic tolerant mutant in a constructed rice insertion mutant pool. The mutant provides an extremely good base material for researching the flowering mechanisms of short day plants under the photoperiodic conditions; by the research of the invention, an important functional gene which has specific functions and is used for regulating and controlling the flowering time of the rice is expected to be obtained, which not only provides evidences and the base material for proving the flowering mechanisms of the short day plants under the photoperiodic conditions but also has important practical significance and economic benefit for guiding the breeding of the rice (particularly the breeding of two-series hybrid rice), regulating the growth period of the rice, further improving the yield of the rice and solving the grain problems of the world population. The relevant research provides the theoretical basis for the molecular design of the breeding of the rice and provides references for the production development and the theoretical research of other crops.

Description

Photoperiod insensitiveness mutant hd1-3 gene and application thereof that adjusting and controlling rice is bloomed

This research obtains project of national nature science fund project (30871328), Tianjin Normal University's academic innovation of young and middle-aged teacher advances plan (52X09039, subsidy 5RL082).

Technical field

The invention belongs to the paddy rice integral body improved technical field of situation of growing, relate to paddy rice photoperiod insensitiveness mutant hd1-3 gene and application thereof, this gene is bloomed most important for the heading of adjusting and controlling rice.

Background technology

" bread is the staff of life ", grain have critical role in national economy.Paddy rice is one of most important food crop in the world, and it is main food with paddy rice that the whole world has 2/3rds population approximately.Its cultivated area accounts for China's grain area 27%, and output but accounts for 39% of total grain output.The accurate figure bit sequence of paddy rice was finished comprehensively and was delivered in 2005, and this clone for the paddy rice functional gene provides advantageous conditions.To experience two periods of vegetative growth phase and reproductive stage all one's life of paddy rice, most important to blossoming and bearing fruit of paddy rice from nourishing and growing to the transformation of reproductive growth, this transition process is determined by the paddy rice internal gene, also be subjected to the influence of external environment factor, determine that one of most important environmental factors of this transition process is exactly the photoperiod.Paddy rice is a short day plant, can realize under the condition on short from nourishing and growing to the transformation of reproductive growth, thereby blossom and bear fruit, and finishes the whole life history of paddy rice, understands fully that therefore the mechanism of blooming of paddy rice under the photoperiod condition has important meaning for the breeding of paddy rice.Take a broad view of rice genetic breeding development course, people forget the huge grace that twice Green Revolution brought for the people of the world never, and from the discovery that is found to the Yebai cytoplasm male sterility line in the sharp short source of short pin crow, rice yield increases substantially.Breeding of hybridized rice has been realized three series mating, is that parent's " bilinear method " simplified the cross-breeding program with photoperiod-temperature sensitive male sterility, and the quality of hybrid rice is also had clear improvement.Studies show that the photoperiod not only plays a decisive role to the basic vegetative period of paddy rice, and most important to the conversion of photoperiod-temperature sensitive male sterility line fertility.

Vernalization is most important for winterness crop, and for example winter wheat must just can blossom and bear fruit through vernalization stage, finished its life history, but more even more important than vernalization to crop (for example paddy rice) accumulated temperature of southern spring and summer sowing.Illumination condition is very extensive to the long-living development impact of plant, the winterness crop that has passed through vernalization stage also must just can blossom and bear fruit by the illumination condition that is fit to, and some plants that not influenced by vernalization also must ability normal development under suitable illumination condition.The approach that illumination mainly relies on by the photoperiod influences blooming of plant, photoperiodism is to the adaptation of changes of seasons and observed the earliest from plant, plant can be divided into three types according to photoperiodic reaction type: long day plant, short day plant and day-neutral plant (Garner and Allard, 1920,1923; Vergara et al.1985).Long day plant is looser to long reaction of day, and the long day is handled the energy early flowering, but also can bloom in certain short day condition, only postpones flowering period.And short day plant is tighter relatively to the light application time requirement, and a short day condition can promote it to bloom, and a long day condition then can be postponed it and be bloomed.But the short day plant kind that some photosensitivity are stronger can not be blossomed and beared fruit under the long day condition, and its growth only rests on vegetative growth phase.The investigator had confirmed by disconnected reaction during dark that short day plant mainly made a response to the length of dark phase afterwards, rather than was decided by illumination length (Hamner andBonner, 1938 of every day; Thomas and Vince-Prue, 1997).Short day plant can discern certain dark phase length, and when the dark phase reached certain threshold value, no matter day how much long, it can both be converted to generative growth phase from vegetative growth phase, thereby blossoms and bears fruit.Day-neutral plant gegenschein length is reaction not, can both normally blossom and bear fruit under long day condition and short day condition.

The research of the molecule mechanism of flowering of plant has in recent years had considerable progress, comparatively deep in the research of model plant Arabidopis thaliana especially, Arabidopis thaliana is a long day plant, and related idiotype network mainly comprises optical signal genes involved (Light signal related genes), flowering time gene (Flowering-time genes) and floral meristem characteristic gene (Floral meristem identity genes) three classes under the known photoperiod condition.The optical signal genes involved can perceived light stimulation, ambient light stimulated changes the signal that vegetable cell can discern into and inwardly transmit, thereby make plant that a series of biochemical reactions take place.The optical signal genes involved mainly comprises Photoreceptors gene and clock gene.Known Photoreceptors gene mainly contains phytochrome gene (PHYA～PHYE) and cryptochrome gene (CRY1 and CRY2).Plant can be by the light of this two classes Photoreceptors gene perception different zones, by bloom (the Lin et al.2000a) of 3 Photoreceptors approach (ruddiness approach, blue light approach and far-red light approach) regulation and control plant.Known clock gene comprises ELF3, ZTL, CCA1, LHY, TOC1, ELF4, GI (Hicks et al 1996; Schaffer et al.1998; Wanget al.1998; Fowler et al.1999; Somers et al.2000; Strayer et al.2000; Alabadiet al.2001; Kikis et al.2005), the daily rhythmicity that these genes all have on the expression level changes, and is bringing into play important effect for the physiological clock circulation that plant keeps correct.

Arabidopis thaliana is bloomed in the regulatory gene network, and known flowering time gene mainly contains CO, FT, SOC1 and FD.The CO zinc finger protein of encoding is mainly expressed in leaf, can experience a day long signal, promotes (the Putterill et al.1995 of blooming under the long day condition; Samach et al.2000).CO crosses to express and makes the plant early flowering, knocks out the delay of can not blooming or bloom of this gene plant.A similar phosphotidylethanolabinding binding protein of FT genes encoding (PEBP) also can promote flowering of plant (Kardailsky et al.1999) under the long day condition.FT mainly expresses at the leaf phloem, and it directly is subjected to the regulation and control of CO gene.Studies show that recently, the activity of FT gene is subjected to the regulation and control of FD gene, the transcription factor of a bZIP type of FD coding, the yeast two-hybrid test shows that FD and FT interact, further experiment shows, it is essential that FD regulates for the activity of FT, and same FT also is essential (Abe et al.2005 for the activation of FD; Wigge et al.2005).Though the CO gene can induce FT to express at the leaf phloem, but induce into colored initial position in the stem apical meristem, and studies show that the promotor that does not have specifically expressing in the stem apical meristem, how do CO and FT regulate flowering time so? studies show that recently, the mRNA of FT gene can be by being delivered in the stem apical meristem in leaf texture's phloem sieve aperture, thereby regulate the expression of its downstream floral meristem characteristic gene, cause flowering of plant, infer that the mRNA of FT gene may be " flowering hormone " (Florigen) main moiety of signal (Huang et al.2005).The SOC1 MADS-box transcription factor (Lee et al.2000) of encoding, it can regulate the flowering of plant time under the regulation and control of FT gene, the activity (for example it can regulate the LFY activity) that also can regulate and control the floral meristem characteristic gene makes plant change to reproductive growth from nourishing and growing.

The floral meristem characteristic gene becomes to spend most important for plant, the defective of these genes will make plant can not form normal flower, show the phenotype of imperfect floral organ.Known floral meristem characteristic gene has LFY, AP1/CAL, FUL and TFL1.Former three is mainly expressed in floral meristem, promotes the merismatic formation of plant flowers, promotes to bloom.TFL1 is the gene of the floral meristem characteristic of another type, opposite with the several genes in front, tF11 mutant inflorescence meristem is replaced by floral meristem, plant performance early flowering, therefore TFL1 has check effect by the stem apical meristem to the floral meristem transformation to plant, suppresses to bloom (Gustafson-Brown et al.1994; Ohshima et al.1997; Liljegren et al.1999; Ferrandiz et al.2000; Pelaz et al.2000; Blazquez et al.2006).Can interact between the above-mentioned floral meristem characteristic gene, mutually regulation and control, at first, the expression that AP1/CAL can forward regulates LFY makes plant form new floral meristem.Secondly, LFY and AP1/CAL can suppress the expression of TFL1, and LFY can also raise AP1 and express the expression that further suppresses TFL1, thereby plant is normally bloomed.In addition, TFL1 also can play the negative regulation effect to LFY, AP1 and CAL gene.Mutual regulation and control between these floral meristem characteristic genes have guaranteed plant from nourishing and growing to the stability of this transformation of reproductive growth, if there are not the mutual regulation and control between these genes, this transition process might be replied.By the mutual regulation and control of above-mentioned flowering time gene and floral meristem characteristic gene, thereby plant is finished by the stable transformation of nourishing and growing to reproductive growth.

People just know that blade is the photoperiodic initial organ of plant perception before 50 years, and may there be a kind of " flowering hormone " that impels flowering of plant in deduction, but just open the mysterious veil of flowering of plant process gradually up to people in recent years, under extraneous environmental factors and plant hormone effect, said gene network three genoids interact, regulation and control mutually, optical signal is delivered to the stem apical meristem from plant leaf through stem, just finally causes flowering of plant.But up to the present, the idiotype network that relates to this process is imperfection still, still has the gene of many branch roads indeterminate, needs further to replenish perfect.

Though known floral genes has certain conservative property in the Arabidopis thaliana in paddy rice, but paddy rice is a short day plant, just in time opposite with Arabidopis thaliana in photoperiodic reaction, compare with long day plant, paddy rice belongs to the plant at long night, be that can it bloom and be decided by relatively dark phase length, rather than be decided by light application time length.For short day plant, the photoperiod mechanism of blooming of mediation is also very indeterminate at present, related genes involved great majority or the unknown, it is still unclear how short day plant discerns dark phase length.

Aspect paddy rice optical signal genes involved (Photoreceptors and clock gene) research, Izawa etc. (Izawa et al.2000) utilize the se5 mutant clone to go out to relate to phytochrome chromophoric group synthetic heme oxidase gene SE5, the se5 mutant be the photoperiod insensitiveness but show tangible early blossoming effect, on function, the synthetic defective of se5 mutant performance phytochrome, therefore the SE5 gene plays an important role in the blooming of the paddy rice of phytochrome mediation, and the restraining effect of under long day condition paddy rice being bloomed is particularly remarkable especially.3 kinds of phytochrome genes are arranged in the paddy rice, be respectively PHYA, PHYB and PHYC.(Takano et al.2005) such as Japan scholar Takano formulates out different double-mutant (phyAphyB, phyBphyC and phyAphyC double-mutant) with different combinations the function of 3 kinds of phytochromes studied, the result shows that phyA and phyC can mediate the far-red light approach and go yellow, on the time that the adjusting paddy rice blooms, single phyA mutant is to the almost not influence of paddy rice flowering time, and phyB and phyC mutant plant early flowering, phyAphyB and phyAphyC double-mutant plant then show intensive early blossoming effect.In blue light receptor research, the Shanghai full study group of Yang Hong of plant physiology ecological Studies institute cloned rice Os CRY1 gene and analyzed its function (Zhang et al.2006), disclosed this gene suppresses rice seedling coleoptile and blade under the blue light mediation elongation.Paddy rice cryptochrome gene OsCRY1 and OsCRY2 (Hirose et al.2006) have also cloned in Japanese Takano of while study group, find that OsCRY1 mainly expresses in the green plant tissue, under the blue light mediation, work in the elongation of inhibition rice seedling coleoptile; And OsCRY2 mainly expresses in plant embryos bud scale, flower and callus, can promote that paddy rice blooms under long day and short day condition.In addition, the OsGI relevant with physiological clock also cloned, the CI gene of the phraseology of its rhythmicity and Arabidopis thaliana is closely similar, but its function is opposite with the CI of Arabidopis thaliana, it is inhibited to blooming of paddy rice, can be used as a repressor (Hayama et al.2003) of regulating the paddy rice flowering time.

Paddy rice is under secular cultivation condition, some mutational varieties that adapt to local photoperiod condition have obtained preservation by natural selection, in addition, some photoperiod covariation materials in the breeding process of paddy rice, can have been produced, this hereditary network research of blooming material that provides the foundation for paddy rice under the photoperiod condition.The study group of Japan scholar Yano identifies the QTL site that 14 rice ear sprouting periods are correlated with (Hd1～Hd14) (Lin et al.1998,2002,2003 with the different groups of indica-japonica hybrid; Yamamoto et al.1998,2000; Yano 1997,2001), the discovery in these QTL sites and analyze not only provides the foundation to the bloom research of mechanism of paddy rice, and natural variation, domestication and the breeding research for paddy rice simultaneously provides foundation.At present existing several QTL site has been cloned and has been disclosed its critical function in paddy rice blooms, for example Hd1, Hd3, Hd6 and Edh1.Hd1 is the main effect site of QTL of blooming, and is the homologous gene of Arabidopis thaliana CO gene, and the zinc finger protein of encoding is mainly expressed by day, and night expression amount very low, show the expression pattern on daytime.Hd1 has dual-use function in the regulation and control of blooming, suppress blooming of paddy rice under long day condition, but promotes paddy rice bloom (Yano et al.2000) under short day condition.These are different with the CO gene, do not find that so far CO is inhibited to Arabidopis thaliana blooming under the photoperiod condition.Izawa etc. (Izawa et al.2002) have analyzed SE5 and the Hd1 interaction relationship shows, the sudden change of SE5 does not influence the expression pattern of Hd1, the more approaching phenotype with the se5 single mutant of se1se5 double-mutant.Ehd1 be with figure clone from the japonica rice variety platform 65 and the hybridization segregating population of African cultivated rice IRGC104038 (Oryzaglaberrima Steud.) in isolated, the allelotrope that in T65, contains hd1 and two afunction of ehd1, the phenotype of having showed late heading.Contain the Ehd1 that function is arranged and show the phenotype of early earing and O.glaberrima IRGC104038 and Japan are fine.The Ehd1 B-type effect regulon of encoding can not rely on Hd1 and independently exercises its function, induces FT-like genetic expression and promotes blooming of paddy rice in a short day condition, and do not express (Doi et al.2004) under long day condition.Ehd1 is a distinctive gene in the paddy rice, in Arabidopis thaliana, there is not homologous gene, different with Arabidopis thaliana, Hd1 of paddy rice and Ehd1 can promote blooming of paddy rice in regulation and control FT-like genetic expression under short day condition, and Arabidopis thaliana only promotes to bloom by CO gene regulating FT gene under long day condition.In addition, Ehd1 does not have activity for where growing a day condition, and is not clear at present, and supposition may be the approach that is different from the regulation and control flowering of plant of another photoperiod mediation of Arabidopis thaliana in the paddy rice.In addition, studies show that recently the Ghd7 gene suppresses the expression of Ehd1 under long day condition, the paddy rice heading that has also participated in the photoperiod regulation and control is bloomed.The Ghd7 protein that contains the CCT structural domain of encoding is postponed heading stage of paddy rice under the long day condition, increased the size of plant height and fringe, has important effect (Xue et al.2008) for the output that increases paddy rice.

Hd3a is a QTL in paddy rice the 6th dyeing, the similar phosphotidylethanolabinding binding protein of encoding has higher homology with FT, and it is activation that paddy rice blooms, it has higher expression under the short day condition, but expression amount is very low under the long day condition.Under the regulation and control of Hd1, Hd3a promotes bloom (the Kojima etal.2002) of paddy rice under short day condition.In Arabidopis thaliana, CI is activation of CO, the activity that CO can positive regulation FT.But Hayama etc. (Hayama et al.2003) find the mRNA level of Hdd1 and the mRNA level performance positive correlation of OsGI when analyzing the mRNA level of Hd1 in the OsGI transfer-gen plant and Hd3a under long day and short day condition, and the mRNA level of the mRNA level of Hd3a and OsGI shows negative correlation.This has hinted that the expression of Hd1 may suppress the expression of Hd3a, the expression that Hd1 can negative regulation Hd3a.Simultaneously show that also OsGI is positioned the upstream of Hd1 and Hd3a, can regulate and control the latter's expression.In addition, studies show that OsMADS51 has also participated in should the photoperiod regulatory pathway, and itself and OsGI can regulate and control Hd1 and Hd3a expresses, thus the heading stage of adjusting and controlling rice (Kim et al.2007).In fact, paddy rice be one long night plant, utilize dark during disconnected reaction experiment can to prove that paddy rice can discern night long.The molecule mechanism of disconnected reaction was because Hd3a expression when disconnected during dark is subjected to the result (Ishikawa et al.2005) that intensive suppresses during nearest research was verified dark.In addition, the expression to Hd3a under the long day condition of Ghd7 gene has very strong restraining effect, but does not influence the expression (Xue et al.2008) of Hd3a under the short day condition.

Hd6 is another QTL that relates to the photoperiod approach, the α subunit (CK2 α) (Takahashi etal.2001) of a protein kinase C K2 of coding, and it postpones paddy rice under long day condition blooms, and its definite regulatory pathway is not clear.Zhang Qifa academician study group of nearest Hua Zhong Agriculture University has cloned an induced flowering and has regulated and control the transcription factor RID1 of the transformation of flower, it inserts mutant rid1 clone from paddy rice T-DNA, mutant rid1 is the mutant that can not ear and bloom of an evaluation, and it is grown and only rests on vegetative growth phase.The zinc finger protein of a Cys-2/His-2 type of RID1 coding, it is not having corresponding homologous gene at Arabidopis thaliana, has important regulation (Wuet al.2008) for the flowering time of paddy rice.

In the double-linear hybrid rice breeding, the conversion of photoperiod for fertility plays crucial effects.Shi Mingsong has found light sensitive nuclear sterility paddy rice (stone bright loose 1985) in the seventies in last century, performance was male when the young fringe of this paddy rice was grown under short day condition normally can educate, and performance male sterile (Lu Xing osmanthus 2003) when its young fringe is grown under long day condition.In the fertility conversion for land-reclaimable 58S, Ye Jianrongs etc. (2004) find that paddy rice lower molecular weight gtp binding protein gene OsRACD has participated in the fertility conversion of light sensitive nuclear sterility paddy rice under the photoperiod condition, the OsRACD transgenosis 58S plant fertility of justice has recovery to a certain degree, and the OsRACD gene of antisense has reduced the fertility of 58N.Up to the present, the photoperiod is still unclear to the mechanism of action of paddy rice light sensitive nuclear sterility, therefore understands fully that as early as possible the idiotype network that adjusting and controlling rice is bloomed under the photoperiod condition has important directive significance for rice breeding.

Summary of the invention

The photoperiod insensitiveness mutant hd1-3 gene and the application thereof that the object of the present invention is to provide adjusting and controlling rice to bloom.Above-mentioned purpose of the present invention is to be achieved by following method:

The nucleotides sequence of paddy rice photoperiod insensitiveness hd1-3 gene is classified SEQ ID No.1 as, and its pairing hd1-3mRNA sequence is SEQ ID No.2.

The aminoacid sequence of the photoperiod insensitiveness mutant hd1-3 gene that adjusting and controlling rice is bloomed is SEQ ID No.3.

The present invention further discloses paddy rice photoperiod insensitiveness hd1-3 gene in the adjusting and controlling rice heading application aspect blooming.The present invention inserts in the mutant library in the paddy rice that makes up and has found a photoperiod insensitiveness mutant, this mutant was at long day condition (14.5h light, 9.5h dark) and short day condition (11.5h light, 12.5h dark) performance heading simultaneously, and wild-type (in spend 11) was at short day condition (11.5h light, 12.5h dark) ear simultaneously with mutant down, under long day condition (14.5h light, 9.5h is dark), postpone and earing than mutant.This mutant provides fabulous base mateiral for the short day plant mechanism of blooming under the research photoperiod condition, and we have isolated goal gene from mutant, and this gene is the critical function gene that an adjusting and controlling rice is bloomed.This gene not only produce evidence and base mateiral in order to verify that short day plant is bloomed under the photoperiod condition by mechanism, and for the breeding of instructing paddy rice (particularly double-linear hybrid rice breeding), regulate rice growing season, further improve the output of paddy rice, the food problem that solves world population has important practical significance and economic benefit, relevant research will provide theoretical foundation for the molecular designing of rice breeding, and offer reference for the production development of other crop and theoretical investigation.

Description of drawings:

Fig. 1 is that the phenotype of mutant is described.Annotate: A: mutant and wild-type phenotype, M is mutant 1f1132, WT spends 11 in the wild-type; B: the spike length of mutant and wild-type and each panel length, investigated 15 strains altogether, 5 repetitions, each repeats 3 individual plants; C: mutant and the heading stage of wild-type at different seedtimes; D: the photoperiodic variation tendency during the different seedtimes; E: the variation tendency of the medial temperature during the different seedtimes, red block are illustrated in this section period because the low temperature due to the continuous rainy weather.

Fig. 2 is for heading stage of mutant under different photoperiods and the temperature condition and wild-type and go out number of sheets speed.Annotate: A: the heading stage of mutant and wild-type under different photoperiods and the temperature condition; B: mutant and wild-type goes out number of sheets speed under different photoperiods and the temperature condition.1f1132 is a mutant, and WT spends 11 in being.Each handles investigation 10 strains.

Fig. 3 represents the sequential analysis in mutant and wild-type Hd1-3 site.Annotate: A: mutant 1f1132 mutational site is analyzed.Fragment is inserted in the representative of black triangle; On behalf of base, vertical fine rule replace; Blue vertical fine rule and numeral have been indicated the relative position in hd1-3; The position of SEF shown in the arrow and SER primer, the insertion fragment of the 315bp that can in mutant, increase.B: the segmental PCR of the insertion of 315bp detects among the mutant 1f1132.The expression of C:Hdd1-3 in mutant and wild-type.D: the proteinic aminoacid sequence of the Hd1 of deduction and 1f1132.The line of black is represented Zinc finger domain, and the proteinic amino acid replacement of the 1f1132 of Japanese fine Hd1 albumen and deduction represented in asterisk.E: the linkage analysis in mutant and hd1-3 site.P ₁For in spend 11; P ₂Be 1f1132.

Fig. 4 represents the expression pattern of Hd1-3 under different photoperiods and the condition of different temperatures.

Fig. 5 represents the expression pattern of Hd3a under different photoperiods and the condition of different temperatures.Annotate: M is 1f1132; WT spends 11 in being.A, B, C, D represent Hd3a under 27 ℃ of conditions of high temperature and 23 ℃ of conditions of low temperature under express spectra, the expression pattern of wild-type under the A:LD condition; The expression pattern of wild-type under the B:SD condition; The expression pattern of mutant under the C:LD condition; The expression pattern of mutant under the D:SD condition.E: be the express spectra of wild-type under 27 ℃ of conditions of high temperature under the different photoperiods; F: be the express spectra of mutant under 27 ℃ of conditions of high temperature under the different photoperiods; G: wild-type and mutant Hd3a expression pattern under 27 ℃ of conditions of short day high temperature; H: wild-type and mutant Hd3a expression pattern under 27 ℃ of conditions of long day high temperature.

Embodiment

For above and other objects of the present invention, feature and advantage can be become apparent, preferred embodiment cited below particularly, and conjunction with figs. elaborate.Following specific embodiment is only used for explanation rather than restriction the present invention.

Embodiment 1

1 material

1.1 vegetable material

The material of participating in the experiment is to spend 11 (Oryza sativa L.ssp.Japonica) and mutant 1f1132 thereof in the japonica rice variety, and the paddy rice T-DNA that 1f1132 comes from structure inserts mutant library, and genetic background is spent 11 backgrounds in being.Spend in about 500 strains 11 and 500 strain 1f1132 plant in the China Paddy Rice Inst experimental plot summer in 2007 by stages (N30 ° 05 ', E119 ° 05 '), the sowing time is respectively May 15, May 28, June 12, June 23, July 10 and July 21.Transplanting seedlings during 20 days length of time rice seedlings grow, reach 50% o'clock record by colony's stem heading rate heading stage.In spend 11 and F1 generation of 1f1132 hybridization be planted in Sanya, Hainan (N18 ° 15 ', E109 ° 43 '), the F2 segregating population of hybridization is planted in Beijing (39 ° 48 of N ', E116 ° 28 ').Above material all keeps the field Routine Management.

1.2 plantation of artificial climate box material and growth conditions

Evaluation under the control condition is carried out in daylight type growth cabinet (KOITOTRON S-153W-Special, Japan is little to be that company makes), and shared 4 growth cabinets supply the processing of examination material, comprise that two temperature settings and two photoperiods are provided with.Temperature is set to 23 ℃ of day weighted mean temperatures (low temperature) and 27 ℃ (high temperature), and light application time is set to short day condition (SD): 11.5h light, and 12.5h is dark; Long day condition (LD) 14.5h light, 9.5h is dark.4 growth cabinet growth conditionss are set to LD, 27 ℃; LD, 23 ℃; SD, 27 ℃; SD, 23 ℃.Spend 11 to be seeded in the rice seedling bed in 1f1132 and the contrast, it is in the nutrition pot of 12cm to diameter that the shoot transplanting equipment of uniformity is selected in cartonning the last week respectively, every basin 2 strains, mark stem leaf age, the rice seedlings of two week sizes respectively select 20 basins to be transferred to the growth cabinet of setting, every case 5 basins.The stem leaf age was added up in per 4 days in the back, put down in writing the heading time and the ripening stage of every strain.Short day promotion rate and high temperature promotion rate formula to heading stage are calculated as follows:

Short day promotion rate (%)=(heading fate-weak point of long day day heading fate)/long day heading fate * 100%

High temperature promotion rate (%)=(cryogenic heading fate-high temperature heading fate)/low temperature heading fate * 100%

2 methods

2.1 the trace of oryza sativa genomic dna extracts

Adopt simple and easy CTAB method to extract.Concrete steps are as follows:

1) get the fresh paddy rice tender leaf of about 300mg with liquid nitrogen flash freezer after grind into powder, be transferred to rapidly in the 1.5mL Enpdoff pipe;

2) add 650 μ L through the CTAB of 65 ℃ of preheatings extraction buffer (100mM Tris-HCl pH 8.0,20mM EDTA, 1.4MNaCl, 2.0%CTAB, 1%PVP), 65 ℃ of water-bath 40min shake mixing every 10min;

3) add isopyknic chloroform isoamyl alcohol mixed solution and (contain 76: 4: 20 chloroform: primary isoamyl alcohol: ethanol (V/V)), shake mixing 3min, leave standstill 3min;

4) the centrifugal 8min of 10000rpm carefully shifts supernatant in another clean 1.5mL centrifuge tube;

5) add 0.8 times of volume Virahol, put upside down mixing gently, room temperature leaves standstill and a moment occurs white flocks;

6) the centrifugal 5min of 10000rpm abandons the supernatant collecting precipitation;

7) the washing with alcohol precipitation of adding 800 μ L75%;

8) abandon supernatant behind the centrifugal 3min of 12000rpm;

9) drying up post precipitation on the super clean bench, to add 200 μ L TE (pH8.0) dissolving standby.

2.2 the extraction of the total RNA of rice plant

The total RNA of plant adopts TRIzol (Invitrogen) reagent to extract, and concrete steps are as follows:

1) after about 1g plant leaf grinds even oar in liquid nitrogen, shifts the 1.5mL centrifuge tube with DEPC handled of 100mg rapidly in prior precooling;

2) add 1mLTRIzol extracting solution vibration mixing;

3) after room temperature leaves standstill 5 minutes, add the chloroform of 0.2mL, shake mixing after 3 minutes, room temperature is placed 15min;

4) under 12,000 * g condition, 4 ℃ of centrifugal 15min shift supernatant to the special-purpose 1.5mL centrifuge tube of RNA;

5) add the 0.5mL Virahol, leave standstill 10min in room temperature behind the mixing gently;

6) 12,000 * g, 4 ℃ of centrifugal 10min precipitated rnas;

7) abandon supernatant, add twice of 1mL 75% alcohol washing precipitation;

8) it is standby that the ddH2O dissolving that adding 20 μ L DPEC handle after the seasoning in the air is stored in-70 ℃ of refrigerators.

2.3 the mutational site is detected

Fine from Japan, spend 11 and the blade of mutant LF1132 in isolation of genomic DNA owing in mutant, inserted the fragment of 279bp, therefore can detect mutational site in the mutant with PCR.It is SEF:5 '-AGAGGAACAGGAGAAGACGC-3 ' and SER:5 '-ACCACTATGCTGCTGCTCAC-3 ' that PCR detects the primer.Amplification condition is: 1min/95 ℃; 30cycles (30sec/94 ℃, 30sec/58 ℃, 1min/72 ℃); 5min/72 ℃.Reaction system following (cumulative volume 20 μ L):

DNA 10-30ng

10 * Buffer (contains 20mM Mg ²⁺) 2 μ L

dNTP(2.5mM) 0.2mM

SEF 0.2μM

SER 0.2μM

ExTaq enzyme (TaKaRa) 1U

DdH ₂O mends to 20 μ L

2.4 the expression analysis of Hd1-3 in mutant and wild-type

In order to analyze the expression of Hd1-3 in mutant and wild-type, natural condition sampling in following 24 hours in field since sampling in 9: 30 morning, every sampling in 3 hours once, is got sample 9 times altogether.Total RNA separates the back from the plant leaf of 30 days sizes (Invitrogen USA) handles digested genomic dna with DNaseI.(China) cDNA is synthesized in reverse transcription to total RNA of 1 μ g for TaKaRa, Dal ian with the M-MLV ThermoScript II.Reverse transcription program following (cumulative volume 20 μ L):

Total RNA 1 μ g

10μM?Ol?igo(dT) 5μL

70 ℃ of sex change 10min

dNTP(10mM) 1μL

RNA enzyme inhibitors (TaKaRa) 1U

M-MLV ThermoScript II (200U/ μ L) 0.6 μ L

DdH ₂O mends to 20 μ L

1h/42℃；15min/70℃。After be stored in-20 ℃ of refrigerators standby.

Synthetic cDNA carries out PCR reaction amplification with the special primer of Hd1-3 gene, does confidential reference items with rice Os Actinl and analyzes the Hd1-3 expression of gene relatively.

Genetic expression special primer sequence is: HD1F:5 '-GGTTATGGAGTTGTGGGAGCAGAC-3 ' and HD1R:5 '-AGTGAAGGGACATCTGAAGCGAGG-3 '.OsActin1 confidential reference items primer sequence is: ActinF:5 '-GACTCTGGTGATGGTGTCAGC-3 ' and ActinR:5 '-GGCTGGAAGAGGACCTCAGG-3 '.The pcr amplification condition is as follows:

For Hd1-3 gene: 1min/95 ℃; 35cycles (30sec/94 ℃, 30sec/60 ℃, 30sec/72 ℃); 5min/72 ℃.

For OsActinl:1min/95 ℃; 26cycles (30sec/94 ℃, 30sec/60 ℃, 30sec/72 ℃); 5min/72 ℃.

Reaction system following (cumulative volume 20L):

cDNA 1μL

10 * PCR Buffer (contains 20mM Mg ²⁺) 2 μ L

dNTP(2.5mM) 0.2mM

Hd1F/ActinF 0.2μM

Hd1R/ActinR 0.2μM

ExTaq enzyme (TaKaRa) 1U

DdH ₂O mends to 20 μ L

2.5Hd1-3 and Hd3a real-time quantitative PCR (Real-time PCR) is analyzed

For analyze under controlled conditions Hd1-3 and the accurate expression of Hd3a, the blade that moves into the material in 4 about two weeks of growth cabinet is collected, 24h takes a sample round the clock, gets sample one time every 4h, and extracts total RNA.With DNase I (Invitrogen, USA) digested genomic dna.(China) cDNA is synthesized in reverse transcription to total RNA of 1 μ g for TaKaRa, Dalian with the M-MLV ThermoScript II.The reverse transcription program is described with 2.2.3.The cDNA that counter-rotating records is used for the Real-timePCR template.(China) test kit description operation is at Stratagene Mx3000P for Tiangen, Beijing according to SYBR Green PCR master mix for Real-time PCR ^TM(STRATAGENE USA) carries out amplified reaction on the PCR instrument to Thermal System.Hd1-3 gene the primer sequence is described with 2.2.3.Hd3a gene primer sequence is: HD3aF:5 '-TTGGTAGGGTTGTGGGTGATGTGC-3 ' and HD3aR:5 '-AGGTTAGGGTCACTTGGGCTTGGT-3 '.Each reaction is carried out 3 times and is repeated, and with OsActin1 as the confidential reference items relative quantitative assay, 2 of Livak description is adopted in data analysis ^{-Δ Δ Ct}Method is carried out (Livak et al.2001).Real-time pcr amplification condition is: 2min/95 ℃; 40cycles (20sec/95 ℃, 30sec/60 ℃, 30sec/68 ℃).Reaction system following (cumulative volume 20 μ L):

cDNA 1μL

2.5×PCR?master?mix 9μL

Primer?F 0.2μM

Primer?R 0.2μM

DdH ₂O mends to 20 μ L

3 interpretations of result

3.1 the phenotype of mutant is described and genetic analysis

The paddy rice T-DNA that the 1f1132 mutant derives from structure inserts mutant library, under the experiment Tian Zhengji of China Paddy Rice Inst plantation (photoperiod is approximately: 14h light, 10h is dark) condition, and the performance phenotype (Figure 1A) of significantly early earing.Mutant 1f1132 is approximately than spending 11 early to ear 18 days in the wild-type.In addition, compare with wild-type, the increment of mutant also descends to some extent, and for example spike length shortens, plant height and tiller number also descend (Figure 1B).In order to study the genetic development of mutant, we with 1f1132 and in spend 11 hybridization, the F2 segregating population that is obtained is planted in Beijing (long day condition), at 1020 F2 individual plants of plantation, the 253 strains plant that early ears is arranged, heading in after planting 57 days; 254 strains heading in evening plant, heading in after planting 76 days; 513 strain intermediate phenotypes, but more approach late heading phenotype plant its heading stage, heading in after planting 70 days.Show that the phenotype that mutant is early eared controlled by single-gene.

Analyze the heading stage of mutant for a nearlyer step, exploration causes the possible environmental factors of heading early, we adopt the interval sowing method with mutant and in spend 11 kinds to plant in China Paddy Rice Inst experiment field, be divided into 6 sowings in period, be respectively May 15, May 28, June 12, June 23, July 10 and July 21.Record is seeded into the weather condition between heading stage simultaneously.The result shows that mutant is respectively 61d, 50d, 49d, 43d, 41d and 47d at the heading fate (fate from sowing to heading) of 6 dates of seeding, respectively than spending 11 to do sth. in advance 14d, 18d, 17d, 16d, 18d and 11d (Fig. 1 C) in the wild-type.Substantially, the heading fate of mutant and wild-type is along with the postponement in sowing time is all shortened, but sowed sowing on relative July 10 on July 21,1f1132 prolongs breeding time to some extent, this may be relevant with low temperature between heading stage (medial temperature is lower than 24 ℃, shown in the red frame of Fig. 1 E) according to the photoperiod variation tendency, begin to last September 17 heading stage from first May 15 sowing time, the duration of day approximately is increased to maximum value 14h (June 23) from 13.3h, drops to 12.1h (September 20) (Fig. 1 D) afterwards gradually.Yet temperature raises along with the postponement in sowing time basically, except July 21 after planting, (medial temperature is lower than 24 ℃, and minimum is 19 ℃ owing to the continuous rainy weather in Hangzhou descends temperature in for some time that is about to ear; Fig. 1 E).From top data, we infer that tentatively the phenotype of mutant may be closely related with photoperiod or temperature.

3.2 mutant and wild-type are analyzed heading stage under the growth cabinet control condition

In order accurately to study photoperiod and temperature effect to mutant and wild-type, we plant two materials in the growth cabinet of 4 different photoperiods and temperature setting, 4 growth cabinets by 2 photoperiods handle (long day condition LD:14.5h light, 9.5h is dark; Short day condition SD:11.5h light, 12.5h is dark) combine with 2 Temperature Treatment (23 ℃ of 27 ℃ of high temperature and low temperature).Under 27 ℃ of conditions of high temperature, the heading fate of mutant 1f1132 under SD and LD condition is (from the time that sowing is extremely eared and experienced, be respectively 45 days and 45.8 days down together), and in spend 11 heading fates under SD and LD condition to be respectively 48 days and 69 days (Fig. 2 A, table 1), show that mutant shows insensitiveness to the photoperiod.The Zao 3 days heading stage of mutant under the SD condition than wild-type, but under the LD condition than wild-type heading in 14 days ahead of time.Short day promotion rate of wild-type and mutant is respectively 30.4% and 1.7%, shows that the short day condition has tangible accelerating effect to wild-type, but mutant is not had tangible accelerating effect (table 1).

Under 23 ℃ of conditions of low temperature, the heading fate of mutant 1f1132 under SD and LD condition was respectively 65.2 days and 54.6 days, and in spend 11 heading fates under SD and LD condition to be respectively 67.6 days and 81 days (Fig. 2 A, table 1), the Zao 2.4 days heading stage of mutant under the SD condition than wild-type, but under the LD condition than wild-type heading in 27 days ahead of time.Paddy rice is a short day plant, and the SD condition can promote the heading of paddy rice, and the LD condition suppresses its heading.Yet in this research, the SD condition can not promote the heading of mutant, otherwise the LD condition has promoted the heading of mutant, and short day promotion rate of mutant shows as negative (19.4%, table 1).But wild-type is acted normally, and the promotion rate was 16.5% in short day, shows that the short day condition has tangible accelerating effect to wild-type, but mutant is not had accelerating effect (table 1).Hinted that the photoperiod has the regulating effect of negative sense, meeting at heading stage the quilt shortening rather than the common prolongation in long day of mutant to the heading of mutant under 23 ℃ of conditions of low temperature.In addition, with compare under 27 ℃ of conditions of high temperature, all postponed the heading stage of mutant and wild-type under cold condition, wild-type and the mutant high temperature promotion rate under the SD condition is respectively 29% and 30.7%, high temperature promotion rate under the LD condition is respectively 14.8% and 16.1% (table 1), under the different photoperiods, high temperature all has the effect that promotes heading, and promoter action is more obvious under short day condition.Show the heading stage that temperature can the positive regulation paddy rice, cold condition is inhibited to the heading of paddy rice.

3.3 under differing temps and the illumination condition to the influence of the stem number of blade

Usually, the plant stem goes out the number of sheets can react the time difference that plant heading is bloomed, so the stem that we have analyzed mutant and wild-type goes out the number of sheets and leafing speed.Go out the number of sheets (table 2) as can be seen under growth cabinet differing temps and the photoperiod, under 27 ℃ of conditions of high temperature, the stem number of blade of mutant 1f1132 under SD and LD condition is 9, and spend 11 under same condition, to be respectively 8 and 11 in the wild-type, photoperiod does not influence the stem number of blade of mutant, but the wild-type stem number of blade increases under the LD condition to some extent, and indirection table is understood sudden change to the photoperiod insensitiveness, and wild-type is to photoperiod-sensitive.No matter under the SD condition still under the LD condition, the leafing speed of mutant is higher than wild-type leafing speed (Fig. 2 B), the heading stage that shows mutant ahead of time, this with above-mentioned different photoperiod conditions under heading stage of mutant consistent heading stage early than wild wild-type.

Under 23 ℃ of low temperature, the stem number of blade of mutant 1f1132 under SD and LD condition is 10 and 9, and spend 11 under same condition, to be respectively 9 and 11 (table 2) in the wild-type, mutant lacks 1 in the stem number of blade under the LD condition under than the SD condition, leafing speed under SD and the LD condition is respectively 0.20/day and 0.25/day, and the leafing speed under the LD condition is faster than leafing speed under the SD condition (Fig. 2 B).This show mutant LD condition following heading stage ahead of time, to do sth. in advance about 11 days result following heading stage than under the SD condition in the LD condition consistent with the said mutation body.Lacked than long day in short day and spend 11 the stem number of blade to show in the wild-type, and leaf emergence rate basically identical every day under SD and LD condition is 0.17/day (Fig. 2 B).But no matter under the SD condition still under the LD condition, mutant and the wild-type leafing under 23 ℃ of low temperature also rate has all been slowed down, and shows that low temperature has suppressed growing of paddy rice, has postponed the heading stage of paddy rice.

Different photoperiods of table 1 and temperature are to the effect at mutant and wild-type heading stage

Different photoperiods of table 2 and temperature go out the number of sheets to mutant and wild-type influence

3.4 1f11132 mutational site sequential analysis

In the research, se1 mutant HS66 and HS110 showed the phenotype of early earing in the past, and performance photoperiod insensitiveness.Proved afterwards that Se1 and Hd1 were homotopic (Yano et al.2000).Sequential analysis finds that HS66 and HS110 contain a 43bp disappearance respectively in the Hd1 site and a 433bp inserts.Compare with Japan is fine in addition, HS66, HS110 and their wild-type Ginbouzu contain a 36bp insertion in the Hd1 site and a single base is replaced (Yano et al.2000).In this research, 1f1132 mutation type surface and se1 mutant are closely similar, thus we to the 1f1132 mutant and in spend 11 Hd1 site to carry out sequencing analysis, see that whether containing sudden change in the Hd1 site of mutant takes place.Sequencing result shows, compare with Japanese fine sequence, in spend 11 Hd1 site to contain that a 36bp inserts and single base replacement (being same as the mutational site of Ginbouzu) (Fig. 3 A).But in the 1f1132 mutant, except with in spend 11 same position contain that 36bp inserts and single base replacement, also containing 6 new single bases replaces and two new insertion fragments, the insertion fragment of a 129bp and a 150bp insert fragment, these two are inserted first exon that fragment is arranged in the Hd1 site, repeat to form (Fig. 3 A) by the genome sequence in this site own.We infer that the phenotype of earing the morning of mutant may be to be replaced and two new insertion fragments cause by 6 new single bases because in spend 11 with the Ginbouzu of forefathers' research in contain all that identical 36bp inserts and a single base replacement.PCR detects proof, can successfully amplify two new sites of inserting in mutant 1f1132, but in spend 11 and the Japan insertion fragment and the 150bp that can not amplify 129bp in fine insert fragment (Fig. 3 B).Above result shows that this gene is a new allelotrope, shines the heading that can promote paddy rice under the condition in length, names to be hd1-3.

Whether caused the variation of this gene on transcriptional level in order to detect the hd1-3 sudden change, we took a sample in 24 hour photoperiod, got once every 3 hours, from the rice plant blade of 30 days sizes, extract total RNA, analyze the expression level of this gene with RT-PCR after the reverse transcription at mutant and wild-type sample, the result shows that in the mutant plant hd1-3 expression level all sharply descends (Fig. 3 C) on each time point, express but still can detect hd1-3 at some time points.Although contain 2 new insertion fragments and 6 new single bases replacements in the 1f1132 mutant, these sudden changes do not cause terminator codon, so hd1-3 still can translate into a protein (Fig. 3 D).In addition, these sudden changes do not occur in the Zinc finger domain.These sudden changes may only influence the proteic activity of Hd1-3, so at the 1f1132 mutant, hd1-3 still has low-level expression.

In order to prove that the 1f1132 mutant phenotype is caused by the hd1-3 site mutation, 1f1132 and in spend 11 to backcross and produce the F2 segregating population, and according to judging phenotype heading stage phase.1020 F2 individual plants have the 253 strains plant (mutation type surface) that early ears, 254 strains heading in evening plant, 513 strain intermediate phenotype plant.We extract genomic dna from the blades of plant, 32 strains heading plant in evening and 64 strain osculant plant is early eared in 253 strains, carry out genetic linkage analysis with molecule marker, PCR detects and to show that the 253 strains plant that early ears has all amplified the banding pattern of 1f1132 mutant, 31 strains heading in evening plant is spent 11 banding patterns in having showed, heterozygosis banding pattern (Fig. 3 E) has been showed in 65 strains.This result has hinted that the 1f1132 mutant phenotype is caused by the hd1-3 site mutation.

3.5Hd1-3 with the expression analysis of Hd3a under different photoperiods and condition of different temperatures

Above analytical proof morning of mutant 1f1132 ear mutation type surface owing to the Hd1-3 site mutation causes, in order to disclose the mechanism of rice ear sprouting period from molecular level, we have analyzed the expression rule of wild-type Hd1-3 under different photoperiods and condition of different temperatures with real time quantitative PCR method.The result shows, Hd1-3mRNA has showed similar expression pattern under different photoperiods and condition of different temperatures, higher expression level was arranged at night, and expression amount descends (Fig. 4) by day, shows the Hd1-3 expression pattern at night.Generally speaking, photoperiod and temperature are less to the Hd1-3mRNA level affects, but the Hd1-3mRNA level is slightly high under the LD condition, and especially the expression level under low temperature long day condition improves (Fig. 4).CO expression of gene pattern similarity (Putterill et al.1995 in this expression level of Hd1-3 and the Arabidopis thaliana; Bl á zquez et al.2003).

Research has in the past shown that Hd3a is activation that the paddy rice heading is bloomed, and is subjected to the regulation and control of Hd1.High-caliber Hd3a expression amount can promote the heading of paddy rice under the short day condition, and low-level Hd3a expression amount suppresses heading (the Izawa et al.2002 of paddy rice under the long day condition; Kojima et al.2002).So we have analyzed under different photoperiods and condition of different temperatures the expression level of Hd3a in the mutant and wild-type.The result shows, no matter at SD still under the LD condition, subzero treatment all greatly reduce mutant and wild-type Hd3a mRNA level (Fig. 5 A, B, C, D).It is consistent that the result of study at top heading stage shows that cold condition has suppressed the heading stage (table 1) and the Hd3a expression of results under the cold condition of mutant and wild-type.So it is because due to low-level Hd3a expresses that this result shows the heading in evening of mutant and wild-type under the cold condition.

Hd3a has higher expression level by day, and night, expression level reduced, and had shown the expression pattern on daytime.Under the SD condition, Hd3a has higher expression level, but under the LD condition, the expression of Hd3a is suppressed, and consistent with former research results (Kojima et al.2002) (Fig. 5 E, F).In addition, under the SD condition, Hd3a is different with expression peak value in the wild-type at mutant, wild-type begins to reach peak expression in the photophase, but mutant begins to reach peak value (Fig. 5 E in the dark phase, F), but be higher than mutant at photophase later stage Hd3a expression level in mutant apparently higher than wild-type in the expression of Hd3a in photophase early stage in wild-type.Under the LD condition, the expression of Hd3a in wild-type be difficult to detect and to obtain, and expresses but may obviously detect Hd3a in mutant, hinted Hd1-3 under the LD condition to the negative regulation effect of Hd3a (Fig. 5 G, H).

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Sequence table

＜110〉Tianjin Normal University

＜120〉the adjusting and controlling rice photoperiod insensitiveness mutant hdl-3 gene and the application thereof of blooming

<160>2

<210>1

<211>5129bp

<212>DNA

＜213〉gene order

<220>

<221>gene

<222>(1)..(5129)

<400>1

aaagcaaaga?tgaacagagg?tggactgttc?tccttacaaa?tttattgcaa?aaaaaaaagt 60

agattctcta?tggagaggca?tatttaacgt?tatagcaggg?ctataataag?aaacagatgg 120

ctcaaagaaa?atgataagca?acaaataacc?cataaaggac?ccatgtcata?tatagatagg 180

ccttgcaaac?aaacaaaaaa?aaggtatgca?aggaaataat?agttccttca?gcagttgaga 240

aatcatataa?cagaagaaaa?aaaacacatt?ggtaataatt?tgacttctcc?actagaatag 300

atatttttgg?atgagggagg?ccggcaaaga?aactatttag?tgacatggca?ggccgctttg 360

gaacttttct?catgtatctg?cactggctac?ctcacaccgg?aggttcgtaa?aggcggaacg 420

gctaaggggg?agtgccaatg?actggcacaa?aaccgcatgc?tttggaatcc?cagagaaccc 480

aaccctccct?ttctcttttg?caaaagaatg?tgcataaaga?gagagaggca?gcatctttcc 540

tctgctcttt?tttgtttttc?cctttatttg?tacatcatca?cagtggcttc?caattctccc 600

cctttgggct?tagttccatc?taagagatgt?ccattgaatt?gtttagggac?aatcctagga 660

atgcatatgg?gaggattcta?ttgatccctg?taaaatctct?ttagggacag?tcctaagtta 720

aaatggaaca?tgattacgac?aaaagagaat?cagtgtactc?atcactaaaa?tgcaaagctt 780

ttttgcgcaa?aagcgcgatt?gctttggctt?aaaaggtatt?gacatcagtt?caggttagat 840

acttagattt?agctcaaggg?cgccaattct?ccataggacc?cgccaaagtg?cacaaaatct 900

ttatctgaaa?acgacccctc?cattgaggat?tggcatcagg?gggtgagaag?agaaacgctg 960

aagcagcaaa?catccaaagg?caaccacaag?acaggcattg?cagccgccgc?tcgccccccc 1020

cccccccccc?cccggggatc?gcgccaagtg?tcaatcgctg?gattcgactt?gacaccccct 1080

tactattagt?atactctaca?ctcaaactcc?ccaggacaaa?aacaccgtga?ctttcccctc 1140

cctagctcct?tccaaaaaac?actcacaaaa?ttccacaaga?gccatgcgag?gtagaggaac 1200

aggagaagac?gcatacacac?acgacacata?gagagagagg?acaaacacaa?tagcttggat 1260

cgatagactt?gtccatgtgg?tgcaagctaa?agctactact?accacaagca?aggctacttc 1320

gttcatgaat?tataattttg?gtggcaacgt?gttcgaccag?gaggttggag?ttggaggcga 1380

aggaggagga?ggaggagagg?ggagcggctg?cccatgggcg?cggccgtgcg?acgggtgccg 1440

cgcggcgccg?agcgtggtgt?actgccgcgc?ggacgcggcg?tacctgtgcg?cgtcgtgcga 1500

cgcgcgggtg?cacgcggcca?accgcgtggc?gtcccgccac?gagcgcgtgc?gggtgtgcga 1560

ggcctgcgag?cgcgccccgg?ccgcgctcgc?gtgccgcgcc?gacgccgccg?cgctgtgcgt 1620

ggcgtgcgac?gtgcaggtgt?actccgcgaa?cccgctcgcc?aggcgccacc?agcgcgtccc 1680

cgtcgcgccg?ctcccggcca?tcaccatccc?ggccacctcc?gtcctcgctg?aggcggtggt 1740

ggccaccgcc?accgtcctcg?gcggcaagga?cgaggaggtg?gactcttgga?ttatcctctc 1800

caaagattcc?aacaacaaca?acaacaataa?caacagcaac?agcagcaaca?acggcatgta 1860

ttttggtgaa?gtcgatgagt?actttgatct?tgtcaggtac?aattcgtact?acgacaacaa 1920

caataacgac?aacagcaaca?gcaacagcag?caacaacgac?aacgacaacg?acaacgacaa 1980

taacaacaac?agcaacagca?gcaacaacgg?catgtatttt?ggtgaagtcg?atgagtactt 2040

tgatcttgtc?aggtacaatt?cgtactacga?caacaacaat?aacgacaaca?gcaacagcaa 2100

cagcagcaac?aacgacaacg?acaacgacaa?cgacaataac?aacaacagca?acagcagcaa 2160

caacggcatg?tattttggtg?aagtcgatga?gtactttgat?cttgtcgggt?acaattcgta 2220

ctacgacaac?cgcatcgaaa?acaaccaaga?tcagcagtat?gggatgcatg?aacagcaaga 2280

gcagcagcag?cagcagcagg?agatgcaaaa?ggagtttgca?gagaaggaag?ggagcgagtg 2340

tgtggtacct?tcacagatca?caatgctgag?tgagcagcag?catagtggtt?atggagttgt 2400

gggagcagac?caggccgcct?ccatgaccgc?cggcgtcagt?gcttacacag?attccatcag 2460

caacagcgtg?agttcatcta?ttactagctg?caactatttt?tttttcagag?aatgaacatc 2520

tattactgtt?gttagttagt?tgttactaca?tgccacgttg?tcaatgtttt?agagttcata 2580

ctagtacttt?tgagtggaaa?aacattctcc?aaacaaaagc?tactgtctaa?caaaatgaag 2640

ggataaataa?acagatctca?acaagaaaac?aaagatactt?ttctacttcc?aagctgcgat 2700

ctttaggctg?attaaatgga?accgataaaa?aaaatacttt?aaagaaaagt?acacaattga 2760

tctttaggca?gaccagttga?ctacttcctg?tatttctaag?catatacgat?ccatgctaac 2820

tcactaattg?aaaagaagtg?agtttgttaa?ccttttatgt?acacagcaat?caccacacga 2880

aagacctcat?gaaaagtagg?ataagtgtaa?gtgtaattca?tattttatcc?cagtgcataa 2940

atttaaaata?tcttactttt?gcgacagtaa?aaaagatatt?ggaagttttt?cttatgtatg 3000

taaaattaaa?ttaagcccat?ctatatatca?ttgcagggtc?tctgacacct?gcaatctcct 3060

tatgattcgc?atatttcagt?gaccatttgc?cgattccatc?tcagatatct?ttctcatcaa 3120

tggaggcggg?tatagtacca?gacagcacgg?tgatagatat?gccaaattcc?agaatcctga 3180

cacctgctgg?agcaatcaat?ctcttctcag?gtccctcgct?tcagatgtcc?cttcacttca 3240

gctccatgga?cagggaggcc?agggtgctca?ggtacaggga?gaagaagaag?gccaggaagt 3300

ttgagaagac?aatacgttat?gaaacaagaa?aggcgtatgc?agaggcacga?ccccggatca 3360

agggccgttt?cgccaagaga?tcagatgtgc?agatcgaagt?ggaccagatg?ttctccactg 3420

cagctctatc?tgacggtagc?tatggtactg?ttccatggtt?ctgatgggac?tcatgagacg 3480

ctatcttata?ggcatatata?tggggactta?ctgagtagca?ataacatcga?tccagtggga 3540

gtagttctag?acaatctgtg?ttatgaataa?tagtgtgttg?tttgcgactt?aaaattgatc 3600

aagtacctta?gctttttaaa?gttttgcttt?gtaatttccg?gatagcagat?atatattgtt 3660

ggtacttgct?cagtagcttt?aagtttttga?agtaagcaaa?gagcagtgat?gagatgaaat 3720

gagtatgtgt?ataactgtat?atagataatt?ctagggtacc?ttggccaaca?atcacagtag 3780

caacaatgct?ttaggggttt?aggtgacgaa?ttgggggttt?agttgtttac?tatgaagtag 3840

caccaaaatg?gtggaacata?tatattccta?ttttcgttcc?atcatgacat?ataactgctg 3900

tctaaccagc?ctatgttgac?tgaaaacaaa?gctcgtttca?ttacaaaata?aaagatggaa 3960

ccctgattaa?gtgtgtccac?tggccagata?gtatcatagt?agtataataa?tgaactggag 4020

ttcaagtctt?ttatattcca?cttggatctc?ggtgccattt?tcttaatgat?gtatcttagt 4080

gatgggctat?cattgtatcc?ggttggaatt?tcagcagagg?tgaaggtgat?ggtgttcact 4140

cagcttttaa?atatccatta?ttcttacagc?cctccagatc?attctggatg?aaaagaaaag 4200

cagtgacaaa?gaatttactg?ttgctttaac?catgagaatc?atatctttcc?agcacggcct 4260

atcttctcac?taaaccatga?gctatcggtg?atcagtaact?cgaatgatag?ttgtagggaa 4320

tataggaaca?ttgcctgata?ctgataggta?caccattgtg?gttgggtgat?ataagtacaa 4380

caaactcaca?gaaaagtttc?cctattattt?tgtgaatcaa?gagagcataa?ataaggaagc 4440

tctttctcat?tctcagtgca?ttcctcttcc?ctttcactag?caactagtgg?catggatctt 4500

attgcctttt?gttttgagat?ggagcattca?ccaaatatct?aaggcatctc?agggcaccaa 4560

ccgcatatac?aggaatacag?gatacatcta?atcgtttttg?gctaagcttg?actgtatcgg 4620

ttagatattg?cacaaaaaac?agagttagga?attaagccct?aagagatggt?aattggaaac 4680

tggaaagtga?acttttcatt?tcaaatatca?acaaagagag?gtcaaaaaag?taaagtgaaa 4740

taaagcacac?gggagataca?gatccatatt?ttgaccgaaa?ctgacgacat?ataccactct 4800

agtatggata?gagagaacaa?atcaaagttc?tgcagaagat?aaaactagac?atagttgact 4860

agtaacagaa?gagattcctg?aactttctca?ctgaaactat?caagcaaata?gataaaactc 4920

gtggtgatat?ttcatccaca?tcagcactga?gaacagaaca?gcaagcaagc?agtttgattg 4980

tgatggaggg?agctcctagc?acatcatcat?attatgaagt?aatatttaat?aatcatggaa 5040

gtatgatgaa?ggtatttttc?tggcaacagt?tcttgtttga?tgcatcagaa?tacctgatta 5100

acgtggaatt?aatcaagcat?cagaatcca 5129

<210>2

<211>1503bp

<212>mRNA

＜213〉artificial sequence

<220>

＜221〉mRNA sequence

<222>(1)..(1503)

<400>2

atgaattata?attttggtgg?caacgtgttc?gaccaggagg?ttggagttgg?aggcgaagga 60

ggaggaggag?gagaggggag?cggctgccca?tgggcgcggc?cgtgcgacgg?gtgccgcgcg 120

gcgccgagcg?tggtgtactg?ccgcgcggac?gcggcgtacc?tgtgcgcgtc?gtgcgacgcg 180

cgggtgcacg?cggccaaccg?cgtggcgtcc?cgccacgagc?gcgtgcgggt?gtgcgaggcc 240

tgcgagcgcg?ccccggccgc?gctcgcgtgc?cgcgccgacg?ccgccgcgct?gtgcgtggcg 300

tgcgacgtgc?aggtgtactc?cgcgaacccg?ctcgccaggc?gccaccagcg?cgtccccgtc 360

gcgccgctcc?cggccatcac?catcccggcc?acctccgtcc?tcgctgaggc?ggtggtggcc 420

accgccaccg?tcctcggcgg?caaggacgag?gaggtggact?cttggattat?cctctccaaa 480

gattccaaca?acaacaacaa?caataacaac?agcaacagca?gcaacaacgg?catgtatttt 540

ggtgaagtcg?atgagtactt?tgatcttgtc?aggtacaatt?cgtactacga?caacaacaat 600

aacgacaaca?gcaacagcaa?cagcagcaac?aacgacaacg?acaacgacaa?cgacaataac 660

aacaacagca?acagcagcaa?caacggcatg?tattttggtg?aagtcgatga?gtactttgat 720

cttgtcaggt?acaattcgta?ctacgacaac?aacaataacg?acaacagcaa?cagcaacagc 780

agcaacaacg?acaacgacaa?cgacaacgac?aataacaaca?acagcaacag?cagcaacaac 840

ggcatgtatt?ttggtgaagt?cgatgagtac?tttgatcttg?tcgggtacaa?ttcgtactac 900

gacaaccgca?tcgaaaacaa?ccaagatcag?cagtatggga?tgcatgaaca?gcaagagcag 960

cagcagcagc?agcaggagat?gcaaaaggag?tttgcagaga?aggaagggag?cgagtgtgtg 1020

gtaccttcac?agatcacaat?gctgagtgag?cagcagcata?gtggttatgg?agttgtggga 1080

gcagaccagg?ccgcctccat?gaccgccggc?gtcagtgctt?acacagattc?catcagcaac 1140

agcatatctt?tctcatcaat?ggaggcgggt?atagtaccag?acagcacggt?gatagatatg 1200

ccaaattcca?gaatcctgac?acctgctgga?gcaatcaatc?tcttctcagg?tccctcgctt 1260

cagatgtccc?ttcacttcag?ctccatggac?agggaggcca?gggtgctcag?gtacagggag 1320

aagaagaagg?ccaggaagtt?tgagaagaca?atacgttatg?aaacaagaaa?ggcgtatgca 1380

gaggcacgac?cccggatcaa?gggccgtttc?gccaagagat?cagatgtgca?gatcgaagtg 1440

gaccagatgt?tctccactgc?agctctatct?gacggtagct?atggtactgt?tccatggttc 1500

tga 1503

<210>3

<211>500AA

<212>PRT

＜213〉artificial sequence

<220>

<221>CHAIN

<222>(1)..(500)

<400>3

Met?Asn?Tyr?Asn?Phe?Gly?Gly?Asn?Val?Phe?Asp?Gln?Glu?Val?Gly?Val

1 5 10 15

Gly?Gly?Glu?Gly?Gly?Gly?Gly?Gly?Glu?Gly?Ser?Gly?Cys?Pro?Trp?Ala

20 25 30

Arg?Pro?Cys?Asp?Gly?Cys?Arg?Ala?Ala?Pro?Ser?Val?Val?Tyr?Cys?Arg

35 40 45

Ala?Asp?Ala?Ala?Tyr?Leu?Cys?Ala?Ser?Cys?Asp?Ala?Arg?Val?His?Ala

50 55 60

Ala?Asn?Arg?Val?Ala?Ser?Arg?His?Glu?Arg?Val?Arg?Val?Cys?Glu?Ala

65 70 75 80

Cys?Glu?Arg?Ala?Pro?Ala?Ala?Leu?Ala?Cys?Arg?Ala?Asp?Ala?Ala?Ala

85 90 95

Leu?Cys?Val?Ala?Cys?Asp?Val?Gln?Val?Tyr?Ser?Ala?Asn?Pro?Leu?Ala

100 105 110

Arg?Arg?His?Gln?Arg?Val?Pro?Val?Ala?Pro?Leu?Pro?Ala?Ile?Thr?Ile

115 120 125

Pro?Ala?Thr?Ser?Val?Leu?Ala?Glu?Ala?Val?Val?Ala?Thr?Ala?Thr?Val

130 135 140

Leu?Gly?Gly?Lys?Asp?Glu?Glu?Val?Asp?Ser?Trp?Ile?Ile?Leu?Ser?Lys

145 150 155 160

Asp?Ser?Asn?Asn?Asn?Asn?Asn?Asn?Asn?Asn?Ser?Asn?Ser?Ser?Asn?Asn

165 170 175

Gly?Met?Tyr?Phe?Gly?Glu?Val?Asp?Glu?Tyr?Phe?Asp?Leu?Val?Arg?Tyr

180 185 190

Asn?Ser?Tyr?Tyr?Asp?Asn?Asn?Asn?Asn?Asp?Asn?Ser?Asn?Ser?Asn?Ser

195 200 205

Ser?Asn?Asn?Asp?Asn?Asp?Asn?Asp?Asn?Asp?Asn?Asn?Asn?Asn?Ser?Asn

210 215 220

Ser?Ser?Asn?Asn?Gly?Met?Tyr?Phe?Gly?Glu?Val?Asp?Glu?Tyr?Phe?Asp

225 230 235 240

Leu?Val?Arg?Tyr?Asn?Ser?Tyr?Tyr?Asp?Asn?Asn?Asn?Asn?Asp?Asn?Ser

245 250 255

Asn?Ser?Asn?Ser?Ser?Asn?Asn?Asp?Asn?Asp?Asn?Asp?Asn?Asp?Asn?Asn

260 265 270

Asn?Asn?Ser?Asn?Ser?Ser?Asn?Asn?Gly?Met?Tyr?Phe?Gly?Glu?Val?Asp

275 280 285

Glu?Tyr?Phe?Asp?Leu?Val?Gly?Tyr?Asn?Ser?Tyr?Tyr?Asp?Asn?Arg?Ile

290 295 300

Glu?Asn?Asn?Gln?Asp?Gln?Gln?Tyr?Gly?Met?His?Glu?Gln?Gln?Glu?Gln

305 310 315 320

Gln?Gln?Gln?Gln?Gln?Glu?Met?Gln?Lys?Glu?Phe?Ala?Glu?Lys?Glu?Gly

325 330 335

Ser?Glu?Cys?Val?Val?Pro?Ser?Gln?Ile?Thr?Met?Leu?Ser?Glu?Gln?Gln

340 345 350

His?Ser?Gly?Tyr?Gly?Val?Val?Gly?Ala?Asp?Gln?Ala?Ala?Ser?Met?Thr

355 360 365

Ala?Gly?Val?Ser?Ala?Tyr?Thr?Asp?Ser?Ile?Ser?Asn?Ser?Ile?Ser?Phe

370 375 380

Ser?Ser?Met?Glu?Ala?Gly?Ile?Val?Pro?Asp?Ser?Thr?Val?Ile?Asp?Met

385 390 395 400

Pro?Asn?Ser?Arg?Ile?Leu?Thr?Pro?Ala?Gly?Ala?Ile?Asn?Leu?Phe?Ser

405 410 415

Gly?Pro?Ser?Leu?Gln?Met?Ser?Leu?His?Phe?Ser?Ser?Met?Asp?Arg?Glu

420 425 430

Ala?Arg?Val?Leu?Arg?Tyr?Arg?Glu?Lys?Lys?Lys?Ala?Arg?Lys?Phe?Glu

435 440 445

Lys?Thr?Ile?Arg?Tyr?Glu?Thr?Arg?Lys?Ala?Tyr?Ala?Glu?Ala?Arg?Pro

450 455 460

Arg?Ile?Lys?Gly?Arg?Phe?Ala?Lys?Arg?Ser?Asp?Val?Gln?Ile?Glu?Val

465 470 475 480

Asp?Gln?Met?Phe?Ser?Thr?Ala?Ala?Leu?Ser?Asp?Gly?Ser?Tyr?Gly?Thr

485 490 495

Val?Pro?Trp?Phe

500

Claims (3)

1. the adjusting and controlling rice photoperiod insensitiveness mutant hd1-3 gene of blooming, its nucleotide sequence is shown in SEQ ID No.1.

2. the albumen of the adjusting and controlling rice photoperiod insensitiveness mutant hd1-3 genes encoding of blooming, its aminoacid sequence is shown in SEQ ID No.3.

3. the described adjusting and controlling rice of the claim 1 photoperiod insensitiveness mutant hd1-3 gene of blooming is in the application of adjusting and controlling rice aspect blooming.

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