CN104292319B - Application of OsGSL5 protein in controlling plant fertility - Google Patents

Abstract

The invention discloses an application of an OsGSL5 protein in controlling plant fertility. The invention provides the application of a substance for inhibiting the expression of an OsGSL5 encoding gene in regulating and controlling the plant fertility. The amino acid sequence of the OsGSL5 protein is the sequence 2 in the sequence table. Experiments prove that sterile rice can be obtained by silencing the OsGSL5 gene expression in the rice by use of an antisense nucleic acid, and thus prove that the OsGSL5 gene is capable of regulating and controlling the fertility of the rice; the role that the OsGSL5 gene plays in the pollen development and formation process has an important application prospect especially in creating manually controllable male sterility, creating hybrid rice and creating an acceptor material of genetically modified organism biosafety.

Description

Application of the OsGSL5 albumen in control plant fertility

Technical field

The present invention relates to biological technical field, more particularly to application of the OsGSL5 albumen in control plant fertility.

Background technology

Oryza sativa L. is one of most important cereal crops in the world, and it supports the population of global half, how to improve Oryza sativa L. Yield be the major issue for being related to national economy.At present, the rice of China's overwhelming majority is produced by hybrid rice, by control Pollen fertility processed cultivates the key that sterile line is hybrid rice production.Some karyogenes of the production and application of hybrid rice or cell The sterile line of matter male, in paddy rice cross breeding breeding and volume increase important function has been played.

Pollen development is a very complicated cellular processes, relate to all links of pollen development, such as subtrahend point Split, mitosiss, the development of tapetal cell, the formation of anther wall etc. (Liu et al.2005).Any one process or ring Saving the abnormal plant fertility that is all likely to result in of appearance reduces, possibly even completely sterile when serious.China male sterility research and Using maintaining the leading position in the world.Modern cell biology technology, hereditism and molecular biology method are applied in recent years In-depth study has been done to the male sterility process of model plant Oryza sativa L. and arabidopsiss, some new results of study has been achieved, such as： EMS1 (Zhao et al.2002), CER1 (Aarts et al.1995) in arabidopsiss, AtGSL2 (Dong et Al.2005) gene and Oryza sativa L. UDT1 (Jung et al.2005), MSP1 (Onomura et al.2003), GAMYB (Kaneko Et al.2004), UDPG (Chen et al.2007), WDA1 (Jung et al.2006) gene etc., the mutation of said gene Directly results in the fertility of plant reduces, the complete infertility having.Ems1 encodes a receptor kinase gene, and the gene is primarily involved in The development of pollen mother cell, tapetum.UDT1 genes are the key genes of microspore development, and the gene mainly acts on subtrahend point Period is split, it works for the development of tapetal cell, the meiosis of microsporocyte and the degraded in middle level. GAMYB is required for microsporocyte be close to tapetal cell to absorb nutrition and then carry out normal meiosis. WDA1 is required for paddy pollen wall wax coat is formed.The reason to plants male sterility mechanism is deepened to their research Solution.

During angiosperm anther development, callose (callose) is many sugars for being widely present in plant cell Son, constitutes the important component of plant cell wall.Callose layer is mainly between cell wall and plasma membrane to the report of callose Callose participates in dynamic change (the Waterkeyn et al.1962 during pollen development；Steiglitz.1977).Little spore The early stage of primary and secondary reductional cell division starts to synthesize callose, and the callose of synthesis surrounds microsporocyte.Due to calluss Matter can reduce the permeability of cell wall, so as to create a kind of relatively independent environment separated with surrounding tissue.Sporidiole Blast cell meiosis first metaphase, the callose wall of sporidiole starts to disappear, and to increase cell permeability, enters field headquarters Conveying is supported, tetrad initial stage callose wall reaches most thick.Postmeiotic is completed in microsporocyte, tapetum secretes callosity in good time Zhi matter enzyme discharges sporidiole to decompose the quadrantal callose of parcel.Callose has mainly played barrier or molecule mistake Filter is acted on.Corpus callosum enzymatic synthesiss and secretion time have decisive action (Stanley and for the normal development of pollen Linskens.1974；Waterkeyn and Beinfait.1970；Zhang et al.2002).Fei etc. (1999) is to intending south Mustard malesterile mutants ms32 Ultrastructural observation shows that tapetal cell has been prematurely formed synthesis and has secreted the thick of calluss enzyme Rough endoplasmic reticulum, result in parcel pollen mother cell and quadrantal calluss wall is degraded in advance, cause pollen abortion.Worrall etc. Etc. (1992) will proceed in Nicotiana tabacum L. through the special promoter fusion of the corpus callosum gene of modification and arabidopsiss Meiosis. Result of study shows, the transfer-gen plant of corpus callosum enzyme is expressed in reduction division phase, occurs in that part or completely infertility Phenotype.Seem normal on the meiosis surface of the pollen mother cell for lacking calluss body wall, but the formation of pollen wall is not high The orderly deposition of degree, sporopollenin is in the random distribution of pollen wall surface.It is possible thereby to infer, calluss body wall in advance or prolong Degraded late will all make the growth course of sporidiole be interfered, and cause the exception that outer wall is deposited to cause plant infertility.But It is little with regard to the molecular regulation mechanism report of callose degraded during pollen development.Additionally, the normal synthesis and degraded of calluss With plant disease-resistant, degeneration-resistant, g and D, auxin signal transduction and fertility it is closely related.

The content of the invention

It is an object of the present invention to provide suppressing the purposes of the material of OsGSL5 encoding genes expression.

Application of the purposes of the material of the suppression OsGSL5 encoding genes expression that the present invention is provided in regulation and control plant fertility；

The aminoacid sequence of the OsGSL5 albumen is sequence 2 in sequence table.

In above-mentioned application, the regulation and control plant fertility is reduction plant fertility.

It is an object of the present invention to provide suppressing the other new application of the material of OsGSL5 encoding genes expression.

Application of the material of the suppression OsGSL5 encoding genes expression that the present invention is provided in culture male sterile rice.

The aminoacid sequence of the OsGSL5 albumen is sequence 2 in sequence table.

In above-mentioned application, the material of the suppression OsGSL5 encoding genes expression is DNA molecular A, the DNA molecular A's Sequence is sequence 1 in sequence table from the reverse complementary sequence of 5 ' end 5171-5713 positions nucleotide or containing the DNA molecular The recombinant vector of A.

In above-mentioned application, the recombinant vector is the DNA molecular A to be inserted in expression vector pCambia1390-Ubi The recombinant vector for obtaining, in an embodiment of the present invention, recombinant vector OsGSL5i-pCambia1390-UBI is by DNA molecular A The recombinant vector obtained between SpeI the and BamHI sites in insertion expression vector pCambia1390-Ubi, wherein DNA molecular A's Sequence is reverse complementary sequence of the sequence 1 from 5 ' end 5171-5713 positions nucleotide in sequence table.

Expression vector pCambia1390-Ubi is by the Ubiquitin promoteres insertion plant shown in sequence in sequence table 4 Between HindIII the and PstI sites of binary expression vector pCambia1390, intermediate carrier pCambia1390-Ubi is constituted.

In above-mentioned application, the plant is monocotyledon or dicotyledon, and the monocotyledon is specially Oryza sativa L..

The 3rd purpose of the present invention is to provide a kind of method that cultivation fertility is reduced or cultivates sterile plants.

The method that the present invention is provided, comprises the steps：The material for suppressing the expression of OsGSL5 encoding genes is imported into purpose In plant, transgenic plant is obtained, the fertility of the transgenic plant is less than the purpose plant；

The material of the suppression OsGSL5 encoding genes expression is DNA molecular A, and the sequence of the DNA molecular A is sequence table Reverse complementary sequence of the middle sequence 1 from 5 ' end 5171-5713 positions nucleotide.

In said method, the DNA molecular A in above-mentioned recombinant vector by importing in purpose plant.

In said method, the plant is monocotyledon or dicotyledon, and the monocotyledon is specially Oryza sativa L..

4th purpose of the invention is to provide a kind of DNA molecular.

The DNA molecular that the present invention is provided, be it is following 1) or 2)：

1) sequence is that sequence 1 is divided from the DNA of the reverse complementary sequence of 5 ' end 5171-5713 positions nucleotide in sequence table Son；

2) under strict conditions with 1) hybridize and encode same protein DNA molecular.

Above-mentioned stringent condition can be that with 0.1 × SSPE (or 0.1 × SSC), the solution of 0.1%SDS is miscellaneous in DNA or RNA Hybridize and wash film at handing over 65 DEG C in experiment.

The experiment proves that, the present invention is obtained not using OsGSL5 gene expressions in antisensenucleic acidses silence Oryza sativa L. body Oryza sativa L. is educated, so as to prove that OsGSL5 genes can be with adjusting and controlling rice fertility, work of the gene played in pollen development forming process With particularly with the receptor for creating human controllable's male sterility, initiative hybrid rice and creation GMO bio-safety There is important application prospect on material.

Description of the drawings

Fig. 1 is the mutant phenotype of Oryza sativa L. sterile mutant gsl5-1

Figure 1A：Period of maturation Oryza sativa L. WT lines compare with sterile plant form：A left side is wild type, and the right side is infertility mutation Body；Figure 1B：Spike of rice after plant maturation a, left side is wild type, and the right side is sterile line；Fig. 1 C：Wild type flower pesticide and mutant during heading Flower pesticide form compares, and the left side is wild type flower pesticide, and the right is mutant flower pesticide.Fig. 1 D：The pollen of WT lines (WT) is in Jing Observe after starch-kalium iodide dyeing；Fig. 1 E：Mutant pollen is being observed Jing after starch-potassium iodide dyeing；

Fig. 2 is OsGSL5 gene analysiss in mutant gsl5-1

A is analyzed for the structure and Tos17 insertion points of OsGSL5 genes, and initiation codon (ATG) and termination codon (TGA) are Jing is marked, single exon of box indicating OsGSL5 genes, the intron of gene on horizontal line table；Two represent respectively to triangle The position of Tos17 insertions in gsl5-1.P1, P2, represent two Genome Primers across T-DNA, and P3 represents prominent positioned at gsl5-1 Border primer in variant on T-DNA；

B is the genotype detection that T-DNA inserts heterozygous plant offspring.Plant genotype when really settled P1 and P3 are matched Target product can be amplified, because T-DNA Insert Fragments are too big, the product of P1 and P2 pairing amplifications cannot be obtained；Wild type Plant is due to the insertion without T-DNA, so P1 and P3 pairing amplifications are without product, but can be expanded using primer P1 and P2 pairings Increasing obtains target product；And OsGSL5 heterozygosis T-DNA insertion transfer-gen plant when then P1 and P2 pairings and P1 and P3 are matched all Can expand and obtain product.Plant phenotype：20 T1 for the 2nd, 5,9,13 plants in individual plant be mutant phenotype, the gene of mutant plant Type is the insertion of OsGSL5 homozygosis T-DNA, and the genotype of normal plant is wild (being represented with W) or heterozygous (with He tables Show), this result shows that mutant phenotype T-DNA insertions intragenic with OsGSL5 are isolated；

As a result C shows to be expression of the RT-PCR analysis OsGSL5 in wild type (WT) and mutant gsl5-1 Expression of the OsGSL5 in mutant is knocked out completely.With Actin as internal standard.

Fig. 3 is the comparison of wild type and mutant flower pesticide paraffin section.

Fig. 3 a-h are wild type flower pesticide in microspore stage, vacuolization pollen period, mitosiss period and mature pollen The flower pesticide transverse section of phase.Fig. 3 i-p are expression mutant flower pesticide in microspore stage, vacuolization pollen period, mitosiss period With the flower pesticide transverse section of mature pollen phase.Bars=25 μm.

Fig. 4 is to suppress the expression of the endogenous OsGSL5 of wild rice to reduce rice fertility using antisensenucleic acidses

Fig. 5 is the gene expression analysis figure of 3 plants of RNA interference systems

Fig. 6 is OsGSL5i-pCambia1390-UBI carrier structure figures

Specific embodiment

Experimental technique used in following embodiments if no special instructions, is conventional method.

Material used, reagent etc. in following embodiments, if no special instructions, commercially obtain.

Embodiment 1, OsGSL5 gene mutation bodies gsl5-1 are obtained and phenotype

First, the acquisition of gsl5-1 mutants

1st, the acquisition of gsl5-1 mutants

T-DNA-inserted Mutant Pool in Rice used utilizes carrier by Scientia Agricultura Sinica ground force biotechnology research PFX-E24.2-15R it is built-up (creation method of mutant library has published paper, sees etc. and to see Wan etc., Activation tagging,an efficient tool for functional analysis of the rice genome.Plant Mol Biol.2009Jan；69(1-2):69-80).

Pick out 10000 parts of Transgenic Rice kinds " fine (Jing kept by Chinese Academy of Agricultural Sciences's biotechnology of Japan Rice is sequenced kind) " seed, be sowed at rice seedling bed Jing seed soaking, after accelerating germination, transplant to land for growing field crops after 35 days.Every part of row of material kind two, often goes 10 plants, be a family, and it is the experimental plot in the village the Chinese Academy of Agricultural Sciences high-tech industrial park of Langfang in Hebei Province ten thousand to plant place, by normal The paddy rice planting method of rule carries out field management.The screening through land for growing field crops receives altogether the low mutation family of 300 infertility or fertility.Its In, an original number is fertility extremely low (such as Figure 1A-E) for the mutation type surface of S78, and mutant is named as into gsl5-1.

2nd, the flanking sequence of gsl5-1 mutant T-DNA insertion points is separated

Using PCR Walking methods (Siebert etc., An improved PCR method for walking in uncloned genomic DNA.Nucleic Acids Res,1995,23:1087-1088), this mutant has been separated Flanking sequence.Insertion point is determined according to the matched position of flanking sequence and rice genome, the flanking sequence position is as a result shown On the 6th chromosome of Oryza sativa L., insertion point annotates website (http corresponding to rice genome:// Rice.plantbiology.msu.edu/ a gene (accession number on)：LOC_Os06g08380), OsGSL5 is named as, Its nucleotides sequence is classified as sequence 1 in sequence table, and the albumen of coding is named as OsGSL5, and the aminoacid sequence of the albumen is sequence 2. Tos17 is inserted in the 6th exon of the gene (see Fig. 2A).

The flanking sequence of the isolated gsl5-1 mutant T-DNA insertion points of PCR Walking methods is sequence 3.

3rd, Tos17 insertion flanking sequences and mutant phenotype isolate detection

In order to the mutant phenotype for confirming pollen abortion is that the present invention is right because the intragenic Tos17 insertions of OsGSL5 cause 19 T₁For transfer-gen plant (wherein, the 2nd, 5,9,13 plants for mutation individual plant) carried out Tos17 insertion points flanking sequence with Mutant phenotype isolates detection.Isolate concretely comprising the following steps for detection：(1) in OsGSL5 gene Tos17 insertion points both sides Design one pair of genes group primer P1 (5 ' ATT TGG GAC CTC AGC GAA GC 3 ') and P2 (5 ' TGA CTC AAG GGC TCT GTT GC 3 '), a vector primer LB1 (P3 is designed on Tos17:5’-CGG TTA CAT CTT CTC AAA CTC AAT GTG G-3 '), such as Fig. 2 B.In T1 in plant, OsGSL5 homozygosis T-DNA inserts plant ability only when P1 and P3 is matched Target product can be amplified；And because Tos17 insertions make the product that P1 and P2 matches amplification reach 17kb, in the PCR of setting Response procedures cannot obtain corresponding PCR primer (method of so identification plant genotype is the universal method in field).It is wild Type plant so P1 and P3 pairing amplifications are without product, but can be matched due to the insertion without T-DNA using primer P1 and P2 Amplification obtains target product.And OsGSL5 heterozygosis T-DNA insertion transfer-gen plant is when then P1 and P2 pairings and P1 and P3 are matched Can expand and obtain product.

Using CTAB methods from 19 plants of T1 for STb gene is extracted in individual plant respectively as template, with primer combination P1+P3 in (1) Enter performing PCR amplification with P1+P2.Reaction system is 20 μ L, is specifically included：The μ L of DNA profiling 1；The Taq enzyme reaction buffering of 10 times of volumes The μ L of liquid 2；2mM dNTP1.5μL；The μ L of 25mM magnesium ions 1.2；10 μM of μ L of primer 0.2；0.3 unit Taq enzyme, plus distilled water is to 20 μ L.Response parameter arranges as follows：94℃5min；94 DEG C of 45sec, 55 DEG C of 45sec, 72 DEG C of 1.5min, 28cycles；72 DEG C of 5min, 25℃1min.(3) by product Jing agarose gel electrophoresiies, the genotype of each individual plant is judged according to the banding pattern on gel.

Experimental result shows, during 19 plants of T1 are for individual plant, the genotype of 4 plant mutant body surface type plant is OsGSL5 homozygosis T- DNA is inserted, and other normal plant genotype of 15 plants of phenotypes are wild or heterozygous (such as Fig. 2 B).This shows that gsl5-1 is mutated The mutant phenotype of body is caused because the intragenic Tos17 of OsGSL5 are inserted, and the mutant shows as recessive Mutants homozygous.

2nd, the paraffin section observation of wild type and mutant different times flower pesticide

RT-PCR analyzes expression of the OsGSL5 in wild type (WT) and mutant gsl5-1, as a result shows OsGSL5 prominent Expression in variant is knocked out (Fig. 2 C) completely.With Actin as internal standard.

The flower pesticide from wild type and mutant of different development stage is taken in Carnoy fixative (dehydrated alcohol:Ice Acetic acid:Chloroform=6:1:3) (anther development is divided into 8 periods, and details refer to Feng, wait .2001.Pollen to fix 24 hours development and its stages in rice(Oryza sativa L.).Chinese J.Rice Sci 15(1)： 21-28.).Material after fixation is dehydrated that (70% ethanol, 90% ethanol, 95% ethanol, anhydrous alcohol are always each in serial ethanol Order is placed 30 minutes).Material waxdip embedding after dehydration, section and opens up piece, the rearmounted dimethylbenzene of 37 DEG C of oven for drying (4-7d) In molten wax, dyeing, neutral gum mounting, do after microscopy, micro- sem observation is placed in after natural air drying, take pictures.

As a result as Fig. 3 shows, front 4 periods of mutant and wild type flower pesticide do not have notable difference (note：Front 4 periods No difference), from the 5th period of anther development, the anther tapetum of wild type starts degraded, to finally disappearing completely Lose, produce normal pollen.And the anther tapetum of mutant does not have normal cleavage when should degrading, until development Final period, ultimately results in the abortion of pollen.

Embodiment 2, suppress OsGSL5 expression material cultivate Oryza sativa L. sterile material in application

1st, the preparation of DNA molecular

Primer is designed according to the CDS sequences (sequence 1) of OsGSL5 genes as follows：5’-GCT TAT CTG AAA TCA TCT TGT CTC TC-3 ' and 5 '-CCA AGA TGC GGG AGA TCT G-3 '.

The fine seedling total serum IgE of Oryza sativa L. Japan is extracted, RNA is synthesized into cDNA with reverse transcriptase.It is with the cDNA that reverse transcription is obtained Template, with above-mentioned primer performing PCR amplification is entered, and 0.8% agarose gel electrophoresiies detection is carried out to PCR primer, obtains molecular weight about For the band of 540bp.The fragment is reclaimed with agarose gel QIAquick Gel Extraction Kit (TIANGEN).By the recovery fragment and pMD19- TSimple (Takara) connects, and connection product is converted into bacillus coli DH 5 alpha competent cell, is carried according to pMD19-T Simple Carboxylic Bian penicillin resistance label screening positive colony on body, obtains containing the recombiant plasmid for reclaiming fragment, with the recombiant plasmid Universal primer M13 on carrier carries out nucleotide sequencing to it.

Through sequencing, the PCR primer on the recombiant plasmid is sequence 1 in sequence table from 5 ＇ ends 5171-5713 positions cores Thuja acid.

2nd, the acquisition of Ubiquitin promoteres

With corn gene group DNA as template, P1 and P2 is primer, enters performing PCR amplification, and the PCR primer for obtaining 2Kbp is Ubiquitin promoteres, nucleotides sequence is classified as sequence 4 in sequence table.

Primer used by amplification ubiquitin promoteres：P1:5’-GCCCAAGCTTCTAGTGCAGTGCAGCGTGAC-3’ And P2:5’-GCAACTGCAGGTACCTAGTGCAGAAGTAACACCA-3 ',

3rd, the acquisition of the recombinant vector of the expression of OsGSL5 is suppressed

Ubiquitin promoteres insertion plant binary expression vector in the sequence table that above-mentioned 2 are obtained shown in sequence 4 Between HindIII the and PstI sites of pCambia1390 (carrier is purchased from Cambia, Queensland.Australia), structure Into intermediate carrier pCambia1390-Ubi；

Reverse complemental fragment of the sequence 1 from 5 ＇ ends 5171-5713 positions nucleotide in above-mentioned 1 is obtained sequence table again (OsGSL5i) fragment between SpeI the and BamHI sites of intermediate carrier pCambia1390-Ubi is replaced, recombinant vector is obtained, is ordered Entitled OsGSL5i-pCambia1390-UBI (Fig. 6, wherein, HPT represents hygromycin selectable marker.35S represents Caulimovirus Promoter.UBI represents corn promoter UBI.HindIII, PstI, SpeI, BamHI represent each restriction enzyme site).

OsGSL5i-pCambia1390-UBI be suppress OsGSL5 expression recombinant vector, sequence 1 wherein in sequence table Reverse complemental fragment from 5 ＇ ends 5171-5713 positions nucleotide is the DNA molecular A of the expression for suppressing OsGSL5.

Through sequencing, recombinant vector OsGSL5i-pCambia1390-UBI is that DNA molecular A is inserted into expression vector The recombinant vector obtained between SpeI the and BamHI sites in pCambia1390-Ubi, the wherein sequence of DNA molecular A are sequence table Reverse complementary sequence of the middle sequence 1 from 5 ' end 5171-5713 positions nucleotide.4th, the transgenic paddy rice of OsGSL5 expression is disturbed Acquisition

The recombinant vector electricity for building proceeds to that (electric conversion instrument is eppendorf Products, and applied voltage of the present invention is 1800V, operation instructions of the concrete operations with reference to the instrument) Agrobacterium (Agrobacterium tumefaciens) bacterial strain EHA105, the Strain Designation after conversion is OsGSL5i-pCambia1390-UBI.

OsGSL5i-pCambia1390-UBI is proceeded to into fine (the A Draft Sequence of of Oryza sativa L. Jing rice varieties Japan the Rice Genome(Oryza sativa L.ssp.japonica)Stephen A.Goff,et al.Science 296, 92 (2002), hereinafter also referred to wild rice) Seed inducement calluss in, obtain regenerating strain.It is specific as follows：

1) seed callus induction is as follows：Choose it is full, without going mouldy ripe rice paddy seed, rice husk (note is sloughed by hand Meaning keep embryo it is complete), by the shelling shell grain of rice go to the aseptic triangular flasks of 50mL → plus appropriate amounts of sterilized water wash once → abandon water, pour into 75% ethanol is appropriate, shake stirring, places 30 seconds (during appropriate jog is dynamic mix) → abandon ethanol → plus appropriate amounts of sterilized water is washed Once → and water → pour appropriate 2%NaCLO into is abandoned, shake stirring frequently, coprocessing 30 minutes → abandon NaCLO → wash 3 with aseptic It is secondary, stop every time 1 minute → remove sterilized water, seed is gone in the sterile petri dish with aseptic filter paper from triangular flask and is blotted. Inducing culture：MS+2,4-D 2mg/L+ sucrose 30g/L+3.6g/L plant gels (pH5.8).

2) Agrobacterium-medialed transformation

(1) the subculture embryo callus of about 7 days are selected as transformation receptor material.

(2) above-mentioned Agrobacterium restructuring thalline OsGSL5i-pCambia1390-UBI is collected, liquid is resuspended in and is co-cultured culture Base (adds final concentration of 100mg/L AS (acetosyringone), the liquid for adjusting PH5.2 to obtain on the basis of YEP fluid mediums Body culture medium) in, 28 DEG C, 200rpm concussion and cultivate 1-2 hours；OD values are adjusted to 0.2.

(3) by step (2) obtain embryo callus soaking step 2) obtain Agrobacterium bacterium solution, then carry out bacterium solution Immersion, processes 30min.

(4) calluss are blotted on aseptic filter paper, is transferred to the solid co-cultivation culture for being covered with one layer of sterilizing filter paper Base (adds 5mg/L 2,4-D, 0.4g/L CH (hydrolysis acid anhydride casein), 0.1mg/L 6- benzyl ammonia on the basis of MS minimal mediums Base adenine (6-BA), 10g/L glucoses, 20mg/L As (acetosyringone) and 3.6g/L plant gels (phytagel), adjust The solid mediums that obtain of sterilizing of PH to 5.2) on, 19 DEG C of light cultures 2-3 days.

(5) step (4) calluss that obtain of culture are produced, rinsed with sterile water 3-4 time, then with Cef containing 50mg/L The rinsed with sterile water of (head born of the same parents' thiophene mycin), is then put into calluss on the filter paper of sterilizing.

(6) calluss blotted with aseptic filter paper that step (5) is obtained are transferred to into Selective agar medium (to train substantially in MS Add on the basis of foster base 5mg/L 2,4-D, 0.4g/L CH (hydrolysis acid anhydride casein), 250mg/L Cef (head born of the same parents' thiophene mycin), 50mg/L HPT and 3.6g/L plant gels (phytagel), the solid medium for adjusting the sterilizings of PH to 5.8 to obtain) on, 25 DEG C are dark Culture, goes to every two weeks on subculture medium subculture once.

(7) Jing after step (6) 3 successive transfer culture, eugonic resistant calli is transferred to into differentiation culture Base (on the basis of MS minimal mediums, addition 1mg/L 6- benzyl aminoadenines (6-BA), 0.1g/L CH (hydrolysis acid anhydride cheese Element), 250mg/L Cef (head born of the same parents' thiophene mycin), 50mg/L HPT and 3.6g/L plant gels (phytagel), adjust PH to 5.8 The solid medium that sterilizing is obtained) on, 25 DEG C, 16h, 2000Lx illumination cultivation start on two weeks or so fresh calluss There is green bud point to occur, twice after subculture, grow seedling per 15 days subcultures once, be transferred to root media and (train substantially in MS Add 3g/L plant gels (phytagel) on the basis of foster base, the solid medium for adjusting the sterilizings of PH to 5.8 to obtain) carry out training of taking root Support.

(8) under growth root culture grows complete seedling, and seedling is transferred to into strong seedling culture base, and (1/2MS minimal mediums add Plus 3g/L plant gels (phytagel), adjust the solid mediums that obtain of sterilizing of PH to 5.8) on culture acquire seedling.

(9) by the seedling on step (8) strong seedling culture base, seedling exercising one week, by culture medium rinsed clean in clear water, is first moved Plant to greenhouse, then be transplanted to big Tanaka and obtain turning OsGSL5i-pCambia1390-UBI T0 for plant.60 plants of independences are obtained Transformation seedlings.

It is that MS minimal mediums add heterogeneity that table 1 is used medium, specific as follows：

3) RNA interference system detection

Reverse transcription is carried out to regeneration strain blade and obtains cDNA as template, with pGSL5f (5 '-CTC TCC GGT TCT TCA TCT TCC AA-3 '), pGSL5r primers (5 '-ATT ACC AGC CAC GAC AGA CCA TA-3 ') carry out QRT- PCR is expanded, and with Actin as internal reference, internal control primer is 5 '-TGC TAT GTA CGT CGC CAT CCA G-3 ' and 5 '-AAT GAG TAA CCA CGC TCC GTC A-3’.Using Oryza sativa L., Japan is fine as wild type control (WT).

As a result Fig. 5 is seen, compared with Oryza sativa L. Japan is fine, OsGSL5 genes in 44 plants of independent transformation Seedlings in 60 plants of independent transformation Seedlings Expression is decreased obviously, and selecting 3 expressions to decline higher regeneration strain Ri-1, Ri-2, Ri-3 carries out following being to test, It is named as and turns OsGSL5RNAi Oryza sativa L. Ri-1, Ri-2, Ri-3.

Using same method by empty carrier pCambia1390-UBI Introduced into Rice Jing rice varieties Japan it is fine in, turned Empty carrier Oryza sativa L..

5th, the fertility detection of the transgenic paddy rice of OsGSL5 expression is disturbed

Turn OsGSL5RNAi Oryza sativa L. Ri-1, Ri-2, Ri-3, Japanese fine (WT) and turn empty carrier Oryza sativa L. (UBI-1390) training Support, gather Oryza sativa L. mature anther, take a flower pesticide on microscope slide, plus one and drip distilled water, smashed to pieces in flower pesticide with tweezers, make pollen grain Release, then Jia 1~2 drop I2-KI solution, covered is examined under a microscope.

I2-KI solution：Take 2g KI to be dissolved in 5~10ml distilled water, add 1g I2, until completely dissolved, then add distillation Water 300ml.Store in standby in brown bottle.It is every be dyed to blueness be the stronger pollen grain of the vigor containing starch, in yellowish-brown Color for hypogenetic pollen grain.2~3 slice, thin pieces of observation, 5 visuals field are taken per piece, the rate of dyeing of pollen are counted, with rate of dyeing Represent the Pollen Activity of pollen.

Turn OsGSL5RNAi Oryza sativa L. Ri-1, Ri-2, Ri-3, Japanese fine (WT) and turn empty carrier Oryza sativa L. (UBI-1390) maturation The setting percentage of each material tassels of Shi Tongji.10 plants of each strain, experiment is repeated 3 times, results averaged.

As a result as shown in table 2 and Fig. 4,

Table 2 is fertility testing result

	Setting percentage	Pollen Activity
			WT	98%	95%
gsl5-1	1%	1%
			Ri-1	0	5%
Ri-2	0	3.5%
			Ri-3	0	2%
UBI-1390	97%	94%

Altogether in 44 plants of the transgenic paddy rice of detection interference OsGSL5 expression, fertility less than 30% have 30 plants it is (wherein complete Complete sterility has 10 plants), account for the 33.3% of sum.

As can be seen here, suppress to be caused after the expression of endogenous OsGSL5 genes in wild rice by antisense technology The obvious reduction of the fertility of rice plant, it is the gene for controlling rice fertility to further demonstrate OsGSL5 genes, and thus side Method can regulate and control the fertility of plant, for actual production.

Claims (3)

1. it is a kind of to cultivate the method that fertility is reduced or cultivates sterile plants, comprise the steps：OsGSL5 coding bases will be suppressed Because the material expressed is imported in purpose plant, transgenic plant is obtained, the fertility of the transgenic plant is planted less than the purpose Thing；

The aminoacid sequence of the albumen of the OsGSL5 encoding genes coding is sequence 2 in sequence table；

The material of the suppression OsGSL5 encoding genes expression is DNA molecular A, and the sequence of the DNA molecular A is sequence in sequence table Reverse complementary sequence of the row 1 from 5 ' end 5171-5713 positions nucleotide；

The plant is Oryza sativa L..

2. method according to claim 1, it is characterised in that：

The DNA molecular A in recombinant vector by importing in purpose plant；

The recombinant vector is that the DNA molecular A is inserted the recombinant vector obtained in expression vector.

3. a kind of DNA molecular A, is reverse complementary sequence of the sequence 1 from 5 ' end 5171-5713 positions nucleotide in sequence table DNA molecular.