CN104805100B - Paddy gene OsS μ 2 applications in plant leaf blade aging is delayed of BP - Google Patents

Abstract

The invention discloses paddy gene OsS μ 2 applications in plant leaf blade aging is delayed of BP, the nucleotide sequence of the paddy gene OsS μ BP 2 is as shown in SEQ ID NO.1.Described application, including：The paddy gene OsS μ BP 2 are connected into expression vector, structure obtains recombinant expression carrier；By the recombinant expression carrier transformation receptor plant.The present invention is cloned by Tula clone technology from rice premature mutant oss μ bp 2 obtains gene OsS μ BP 2；And prove that gene OsS μ BP 2 can delay plant leaf blade aging by function complementation experiment；Gene OsS μ BP 2 can be applied to field of plant breeding, and the screening and seed selection to resistance to aging plant variety are significant.

Description

Applications of the paddy gene OsS μ BP-2 in plant leaf blade aging is delayed

Technical field

The present invention relates to plant genetic engineering and Rice molecular breeding technical field, more particularly to a kind of paddy gene OsS μ Applications of the BP-2 in plant leaf blade aging is delayed.

Background technology

Leaf senile is a kind of performance that plant adapts to environment, is the only stage which must be passed by grown, in aging course generally With the restructuring of carbohydrate and to tender leaf or the transhipment related substances of seed, significant (the Nam H of procreation to offspring G.The molecular genetic analysis of leaf senescence.Curr Opin Biotechnol, 1997,8：200-207).However, the heading stage functional leaf that is especially in the milk in rice growth process crosses presenility, will lead Cause that setting percentage is low, sterile grain rate is higher and the consequence such as quality variation.Research shows that rice milking stage heading stage functional leaf is often postponed 1 day and declined Always, 2% or so can be increased production in theory, actually increasing production 1% or so, (the aging plant physiology of the grand plant leaf blades in Liu road leads to News, 1993,2：14-19；Ma Yuefang and Lu Dingzhi irrigation methods are to hybrid rice aging and late growth stage some physiologically actives Influence, rice in China science, 1990,2：56-6), and rice quality (Thomas H and Smart can also be improved CM.Crops that stay green.Ann Appl Biol, 1993,123：193-219).Hybrid rice has root system hair Reach, tillering ability is strong, leaf area is big, big panicle many grains per panicle and the features such as high yield and high quality, used in China's large area, to ensure China's grain Food safety has made significant contribution, but the later stage of partial hybridization rice especially indica Hybrid Rice (such as two line system) is easier to early ageing It is a kind of common phenomenon, this will cause its later stage Dry-matter production rale to decline rapidly, and then influence kernel grouting process, finally Yield potentiality is influenceed to play.In recent years, although scientist is made great efforts to solve this problem and taken by tradition and modern breeding means Important achievement is obtained, but the leaf presenility problem of indica Hybrid Rice remains important the asking of Genetic and breeding in rice worker's urgent need to resolve Topic.

On the origin cause of formation of aging, angularly carried out from the morphosis and molecular physiology characteristic of blade both at home and abroad in decades Numerous studies, it is proposed that radical damage is said, gene regulation is said, Photo -carbon imbalance is said, Nutrient Stress is said and hormonal balance is said Hypothesis in theory (Wei Daozhi, with new guest, know several hypothesis GUIHAIAs of bright plant leaf blades Aging Mechanism perhaps, 1998,18：89- 96；Brutovska E, Samelova A, Dusicka J and Micieta K.Ageing of trees：Application Ofgeneral ageing theories.Ageing Research Reviews, 2013,12：855-866).Although these are false Say that all the aging startup to blade, senescence process have done relatively more rational explanation, but declined apart from real solution to a certain extent Old mechanism problem also has certain gap, it is also necessary to carries out more thoroughgoing and painstaking researchs.To find out its cause, it is because each is false Say and all put undue emphasis on some factors for causing leaf senile, wherein gene regulation say order of the exaggeration gene in growing, Stage expression it is important；Photo -carbon imbalance, which is said, to be focused on the transmission of luminous energy, is injured using with active oxygen；Nutrient Stress, which is said, focuses on Yuan Ku Between the equilibrium of supply and demand；Hormonal balance say focus on be underground ground correlation and hormon between balance.But leaf Piece aging can be determined by simple factor, in senescence process senile cell in structure, on physiological acoustic signals with And all there occurs significant changes on molecular level, be one be related to that many factors such as ion, hormone and gene regulate and control jointly answer Miscellaneous systematic procedure.

Substantially clearly, abscisic acid, salicylic acid and jasmonic etc. are the important hormones for promoting plant leaf blade aging；And Ca²⁺、K⁺It is considered as then with N and the closely bound up mineral nutrient element of crops leaf senile such as rice and cotton；Liu Deng (Liu L, Zhou Y, Zhou G, Ye R, Zhao L, Li X, Lin Y.Identification of early Senescence-associated genes in rice flag leaves.Plant Mol Biol, 2008,67：37-55) Sword-like leave using two line system is material, and the related differential expression base of 533 agings is obtained by the identification of Subtractive hybridization method Cause, its function are related to macromolecular substances metabolism, regulation protein synthesis, energetic supersession, regulatory gene, removing toxic substances, pathogenicity and inverse Border, cytoskeleton composition and flower development etc..(Wu X Y, Kuai B K, Jia J Z, the Jing H C.Regulation of such as Wu Leaf senescence and crop genetic improvement.J Integr Plant Biol, 2012,54：936- 952) leaf senile correlation gene is divided into by 7 classes, including transcription factor, protease or kinase gene, participation according to gene function Gene, substance transportation related gene, the gene for participating in catalytic action, binding-protein gene and the biology for being metabolized processing degradation close Into related gene.

S μ BP-2 at first in human genome clone (Mizuta TR, Fukita Y, Miyoshi T.Shimizu A, Honjo T.Isolation of cDNA encoding a binding protein specific to 5’- phosphorylated single-stranded DNA with G-rich sequences.Necleic Acids Res.1993,21：1761-1766), research is thought, it belongs to Upf1-like unwindases (Jankowsky E.RNA helicases at work：Binding and rearranging.Trends Biochem Sci.2011,36：19-29), The mutation of the gene will cause people's I types distal end spinal muscular atrophy disease (Lim SC, Bowler MW, Lai TF, Song H.The Ighmbp2helicase structure reveals the molecular basis for diseace-causing Mutations in DSMAl.Nucleic Acids Res.2012,40：11009-92) so far the genoid in rice Function is not yet related to.

Rice is one of most important cereal crops, in the world more than half population using rice as staple food.In China, with The reduction being continuously increased with land area of population, requires more and more urgent for rice yield is increased, national food peace Full strategy is also just particularly important, therefore it is significant to cultivate the rice of resistance to aging.It is but also not resistance in rice at present The transgenic paddy rice of non-irrigated and resistance to aging, thus the gene for cloning resistance to aging is found, the kind of resistance to aging is cultivated to improving rice production Amount, improvement rice quality are significant.

The content of the invention

The invention provides a kind of applications of paddy gene OsS μ BP-2 in plant leaf blade aging is delayed, the gene can Delay plant leaf blade aging.

Applications of the paddy gene OsS μ BP-2 in plant leaf blade aging is delayed, the nucleosides of the paddy gene OsS μ BP-2 Acid sequence is as shown in SEQ IDNO.1.

Paddy gene OsS μ BP-2 belong to Upf1-like unwindase genes, therefore are named as OsS μ BP-2.In rice leaf In aging course, OsS μ BP-2, can be with Delaying Leaf-Senescence as negative regulatory factor.Utilize leaf senile specific expression gene Promoter starts the expression of OsS μ BP-2 genes, conversion plant, can be in the hair of plant reproductive Later growth Delaying Leaf-Senescence It is raw, improve the photosynthesis of plant.The amino acid sequence such as SEQ ID of the protein of the paddy gene OsS μ BP-2 codings Shown in NO.2.

Specifically, described application, including：

(1) the paddy gene OsS μ BP-2 are connected into expression vector, structure obtains recombinant expression carrier；

(2) by the recombinant expression carrier transformation receptor plant.

Wherein, the recipient plant can be rice or arabidopsis, preferably, the recipient plant is rice.

Present invention also offers a kind of recombinant expression carrier, including initial carrier and the purpose base for inserting the initial carrier Cause, the base sequence of the target gene is as shown in SEQ ID NO.1.

The construction method of recombinant expression carrier is conventional method.Preferably, the original described in the recombinant expression carrier Beginning carrier is pCAMBIA1300 or pSB326-Actin-NOS.Wherein, pSB326-Actin-NOS is overexpression vector, can be shown Write the expression quantity for improving target gene.

Present invention also offers a kind of transformant for including the recombinant expression carrier.During transformation receptor plant, it can use The method of agrobacterium mediation converted, specifically, described Agrobacterium are Agrobacterium EHA105.

Compared with prior art, the invention has the advantages that：

The present invention is cloned by Tula clone technology from rice premature mutant oss μ bp-2 obtains gene OsS μ BP-2； And prove that gene OsS μ BP-2 can delay plant leaf blade aging by function complementation experiment；Gene OsS μ BP-2 can be applied to Field of plant breeding, the screening and seed selection to resistance to aging plant variety are significant.

Brief description of the drawings

Fig. 1 is the phenotype of mutant oss μ bp-2 different growings of the present invention；

(A) tillering stage；(B) heading stage；

Fig. 2 is the OsS μ BP-2 assignments of genes gene mapping of the present invention and sequencing analysis result；

(A) gene just positions；(B) gene finely positioning；(C) BAC in section is positioned；(D) ORF in section is positioned；(E) dash forward Become structure and the mutational site of gene；(F) Sequencing chromatogram of wild type site；(G) Sequencing chromatogram in mutant mutational site；It is prominent Become site as shown in arrow；

Fig. 3 is mutant oss μ bp-2 of the present invention genetic complement the result.

Embodiment

Molecular biology used in following examples and biochemical method are known technology, are compiled in Ausubel Write by John Wiley and Sons companies publish Current Protocols in Molecular Biology and The Molecular published by Cold Spring Harbor Laboratory Press (2001) that J.Sambrook etc. writes Cloning：A Laboratory Mannual, 3^rdED. document is waited to be discussed in detail.Experiment used in example is applied below Material is commercially available products unless otherwise specified.

The separation and genetic analysis of the mutant of embodiment 1

By EMS mutagenesis rice varieties Zhejiang extensive 7954, leaf presenility mutant oss μ bp-2 are filtered out from offspring, through more Generation backcrossing and selfing obtain the stable strain of mutant character.Early ageing character starts from the tillering stage (figure of oss μ bp-2 mutant plants When 1A), to full heading time, there is different degrees of early ageing symptom (Figure 1B) in all blades.Female parent is made with oss μ bp-2, point Inextensive with Zhejiang 7954 and japonica rice material 02428 carry out respectively hybridization obtain F₁, F₁Plant is acted normally；F₁It is selfed the F obtained₂Afterwards , then there is wild type and mutation type surface in generation, its segregation ratio wild type：Mutant=3: 1, so as to illustrate Senescence by list Recessive gene controls.

The aging gene OsS μ BP-2 of embodiment 2 finely positioning

Utilize oss μ bp-2/02428 F₂For colony as target group, 215 plants of early ageing individual plants are obtained.Choose uniform The 500 pairs of SSR primers and InDel being distributed on 12 chromosomes of rice are marked one by one to japonica rice material 02428 and mutant oss μ bp-2 carry out polymorphism analysis, and normal gene pond and mutator pond are analyzed using the polymorphism primer screened.As a result find SSR marker R3M23 and RM168 on No. 3 chromosome of rice exists bright between mutant gene pond and normal gene pond Aobvious inclined separation, using more than 215 early ageing individual plant checkings marking, tentatively OsS μ BP-2 are positioned between RM168 and R3M23 (Fig. 2A).

20 pairs of primers are searched for and devise again near R3M23 and RM168, wherein 6 show well parent Polymorphism, respectively SSR marker RM5488, RM3646, RM15361, RM6266, RM3513, RM15363 and InDel mark R3M30, marked using this 7 Dui, most at last OsS μ BP-2 are positioned between RM15363 and RM15361 (Fig. 2 B), and physical distance is 121kb, region include two BAC, share 14 annotation genes (table 1, Fig. 2 C and 2D).

The molecular labeling of the OsS μ BP-2 gene finely positionings of table 1

The aging mutator of embodiment 3 is predicted and sequencing compares analysis

It is special using 12 pairs after carrying out functional analysis to 12, section annotation gene according to the positioning result of embodiment 2 Property primer pair wherein 3 candidate genes be sequenced, as a result find candidate gene LOC_Os03g38990 ORF regions hair A base (G) (Fig. 2 F) is given birth to and has been mutated into A (Fig. 2 G), the shearing in turn resulting in gene maturation mRNA changes, amino Frameshift mutation occurs for acid encoding area.In order to further verify that the mutational site whether there is, we are in target group F2 generations Normal phenotype and senescence phenotype individual plant are sequenced respectively, as a result find that senescence phenotype individual plant all has the mutational site, and just The mutational site is then not present in normal phenotype individual plant.

The OsS μ BP-2 of embodiment 4 genetic complement checking

The structure of gene complementation carrier：Full-length cDNA (the GenBank of OsS μ BP-2 genes in GenBank accession number：AK099721 sequences Design pair of primers)：

Sense primer：OsSμBP-2F：5’-ATGGCGGGGCGGAGTGGC-3’；

Anti-sense primer：OsSμBP-2R：5’-CAGTGGTACCThe TCAGCTCTGGTATTCTGAT-3 ' (digestions containing KpnI Site)；

The cDNA of OsS μ BP-2 genes is obtained from Nipponbare amplification using RT-PCR technology, and Purified in electrophoresis obtains OsS μ BP-2-cDNA fragments；

Simultaneously by high-fidelity enzyme PrimerSTAR with following primer：

OsSμBP-2PF：5’-CAGTTCTAGATAGCCACAGAACCGTTAGTC-3 ' (restriction enzyme site containing XbaI)；

OsSμBP-2PR：5’-GCCACTCCGCCCCGCCAT-3’；

PCR reaction systems：

Response parameter：98 DEG C are denatured 10 seconds, and 55 DEG C are annealed 15 seconds, and 72 DEG C extend 2 points, totally 35 circulations.

72 DEG C extend 5 minutes.

The promoter that Nipponbare genome obtains OsS μ BP-2 genes is expanded, and Purified in electrophoresis obtains to obtain OsS μ BP-2- Promoter fragments；Fusion DNA vaccine technology is recycled, by OsS μ BP-2-cDNA fragments and OsS μ BP-2-Promoter segment compositions And be extended using primer OsS μ BP-2PF and OsS μ BP-2R, finally obtain the OsS μ BP-2 pieces containing promoter and cDNA Section, carries out digestion to PCR primer after purification with KpnI and XbaI, is connected into Agrobacterium plasmid pCAMBIA1300-Nos and is formed PC1300-OsS μ BP-2, selection are inserted correct plasmid and are sequenced to determine to be properly inserted fragment.Utilize Agrobacterium simultaneously Mediated method converts Agrobacterium EHA105 and is used for untransformed mutants oss μ bp-2.

Mutant oss μ will be directed respectively into using Agrobacterium tumefaciens-mediated Transformation with complementing vector pC1300-OsS μ BP-2 Bp-2 obtains transfer-gen plant and is planted in field.As a result show, the transgenic progeny for turning pC1300-OsS μ BP-2 is recovered just Often, do not occur early ageing phenomenon (Fig. 3), so as to confirm that candidate gene is exactly target gene.

The transgenic paddy rice of resistance to aging of the marker-free of embodiment 5 is cultivated

Gene overexpression vector is built：Full-length cDNA (the GenBank of OsS μ BP-2 genes in GenBank accession number：AK099721 sequences Design pair of primers)：

Sense primer OsSuBP-2-1F：5′-CAGTCTAGACCATGGCGGGGCGGAGTGGC-3 ' (the enzymes containing XbaI Enzyme site)；

Anti-sense primer OsS μ BP-2-1R：5′-CAGTGGTACCTCAGCTCTGGTATTCTGAT-3 ' (the enzymes containing KpnI Enzyme site)；

Using round pcr OsS μ BP-2 genes, and electricity are obtained from the plasmid pC1300-OsS μ BP-2 amplifications in embodiment 4 Swimming purifying obtains OsS μ BP-2 fragments, with XbaI and KpnI double digestion OsS μ BP-2 fragments, and is inserted into through XbaI and KpnI enzymes Exceeded reach cut obtains pSB326-Actin-OsS μ BP-2-NOS in carrier pSB326-Actin-NOS, selection insertion is correct Plasmid is sequenced to determine to be properly inserted fragment.

PCR reaction systems：

Response parameter：98 DEG C are denatured 10 seconds, and 55 DEG C are annealed 15 seconds, and 72 DEG C extend 2 points, totally 35 circulations.

72 DEG C extend 5 minutes.

The preparation method of transgenic paddy rice is to use existing technology (PanG., et al., Map-based cloning of a novel rice cytochrome P450genes Cyp81A6that confers resistance to two Different classes ofherbicides.Plant Molecular Biology, 2006,61：933-943) chooses Full rice varieties Nipponbare seed, shells, and induction produces callus as converting material.By electrization by embodiment The T-DNA carrier pSB326-Actin-OsS μ BP-2-NOS obtained in 3 import Agrobacterium EHA105.Take carrier containing T-DNA PSB326-Actin-OsS μ BP-2-NOS Agrobacterium draws plate, chooses single bacterium colony and is cultivated in LB culture mediums, prepares for rice conversion Agrobacterium.

Rice Callus to be transformed is somewhat blotted on aseptic filter paper, callus is put into OD₆₀₀For 0.5 (contain acetosyringone, 200 μm of ol/L) in Agrobacterium bacterium solution, bacterium solution is abandoned after placing 40 minutes at room temperature, then by rice callus group Knit to be placed on aseptic filter paper and suck unnecessary bacterium solution, callus is transferred to co-culture 50-55 hours are cultivated on base, surface is not had The callus for having many Agrobacteriums is transferred on micro-organisms base and cultivated 5-7 days, and surface then is not had into the callus of Agrobacterium again Tissue, which is transferred on screening and culturing medium, to be cultivated 6 weeks (every two weeks subculture once).The kanamycin-resistant callus tissue obtained after screening is transferred to pre- point Change on culture medium (first light culture 5-7 days, the differentiation of illumination in then 16 hours germination) 4-6 weeks, be transferred to after resistance seedling grows up to Taken root on root media, regeneration plant finally is washed away into culture is cultivated based in greenhouse or field, until harvest seed.With The primer PCR of Nos terminators and hygromycin phosphotransferase gene (hereinafter referred to as HPH genes) identification T0 contains for plant, harvest There is the T0 of OsS μ BP-2 genes and HPH genes for the seed on plant.T0 is planted into T1 for transgenosis for the seed on plant Seedling, Nos terminators and HPH gene primers identification transgenic seedling, selection harvest only have the kind of the transgenic seedling of OsS μ BP-2 genes Son.

By above-mentioned transgenic paddy rice seed and after not being transferred to rice paddy seed culturing and transplanting seedlings to the field of OsS μ BP-2 genes, see Its tillering stage, the growing state of full heading time blade are examined and detect, the rice for finding to be overexpressed OsS μ BP-2 genes is being eared The chlorophyll total amount of sword-like leave is significantly higher than control after 28 days, and up to 14.13% (table 2), this indicates that the gene has and delays blade to decline Old function.

Sword-like leave chlorophyll content after table 2 is overexpressed transfer-gen plant and the control heading same day and eared 28 days

Claims (2)

1. paddy geneOsSμBP-2Application in Senescence of Rice is delayed, it is characterised in that the paddy geneOsSμ BP-2Nucleotide sequence as shown in SEQ ID NO.1.

2. application as claimed in claim 1, it is characterised in that including：

（1）By the paddy geneOsSμBP-2It is connected into expression vector, structure obtains recombinant expression carrier；

（2）By the recombinant expression carrier rice transformation.