CN102234324A - Protein involving self-incompatibility and cross-compatibility control of phanerogam pollen, coding gene thereof and application - Google Patents

Abstract

The invention discloses protein involving self-incompatibility and cross-compatibility control of phanerogam such as Solanaceae plant pollen, coding gene thereof and an application of reducing the gene expression through transgene on aspects of preventing transgene flow and establishing a gene bank of a variety. The invention provides PhSSK1-RNAi carrier and a host cell containing the carrier. The gene of the present invention has an important theoretical significance in revealing molecule mechanism of self-incompatibility of the phanerogam such as Solanaceae plant. Through transgene RNAi, reducing the gene expression can cause pollen to loose compatibility in cross-pollination, having an important effect and meaning on preventing transgene flow of Solanaceae transgene plant and establishing a good trait control gene bank of Solanaceae economic crop such as potato, tomato and the like.

Description

Participate in albumen and the encoding gene and the application of the different friendship affinity control of polycarpeae pollen self-incompatibity

Invention field

The invention belongs to plant genetic engineering field.Specifically, the present invention relates to and polycarpeae, as the related protein and the encoding gene SSK1 thereof of the different friendship affinity control of Solanaceae class self incompatibility pollen, and the function of utilizing the different friendship affinity forfeiture of pollen that this expression of gene played in the transgenosis RNAi downward modulation pollen; Also relate to protein sequence comparative analysis to this coded by said gene etc. simultaneously.

Background technology

Transgenic technology is one of DNA recombinant technology, and transgenic plant are succeedd in existing so far 35 sections 120 various plants after nineteen eighty-three succeeds in tobacco first.Because transgenic technology can be by the importing of foreign gene, make crop have good quality and attribute that it does not have originally, therefore can improve crop yield, improve quality, reduce the usage quantity of weedicide and agrochemical agricultural chemicals, and then can save a large amount of labor forces, be important means that solve global food problem and agricultural sustainable development.Since the eighties in 20th century, along with biology gene engineering technology and development of technologies, genetically modified crops are large-area applying on producing." 2008 annual global biotechnology crop commercialization status report " statistical result showed according to year on the 23rd Agricultural biotechnologies International Service Organization February in 2009 (ISAAA) issue, the cultivated area of whole world genetically modified crops has 1.02 hundred million hm2, and plantation country has 22 ^[1]

But transgenic technology equally also is one " double-edged sword ".When the crop transgenic technology brings the huge scientific development of people, economic benefit and social benefit, the equally also potential danger that may cause to many-sides such as ecotope and human healths ^[2]These problems about the genetically modified organism security receive increasing concern.Think at present genetically modified crops the safety issue that may bring mainly show following several aspect.The first, genetically modified crops may be caused environment and healthy biological safety problem.Genetically modified crops have the resistance screening gene usually, and the someone thinks that these resistance markers might influence the result of treatment of human antibiotic medicine.Present in addition genetically modified crops have the target gene that changes over to and mostly are pest-resistant, disease-resistant genoid greatly, transgenic method also is generally pathogenic bacteria or virus-mediated, therefore has the pathogenic bacteria of these genes and the outflow of virus and might produce new pathogenic virus ^[3]The second, genetically modified crops may be caused the ecological security risk.Resistant gene with resistant transgenic crop of antiweed may flow by the gene between species and be transferred on weeds or the nearly edge wild-type kind, make these weeds and wild species have the resistance of antiweed, may become " superweed " after repeatedly shifting, and then destroy the ecosystem ^[4-6]In addition, genetically modified crops as adventive, usually have " selective advantage ", may influence the genetic construction in plant gene storehouse, cause losing of wild allelic and other genetic resources, and then influence the kind of species in the ecosystem and the quantity of population, make the forfeiture of species diversity ^[6]The 3rd, genetically modified crops might produce the problem of genetically modified food security.The prompting of existing research report, genetically modified crops can be caused potential harm when feeding mouse ^[7,8]Generally speaking, to point out us be necessary for the control of the effective security of transgenic experiments and genetically modified crops to the potential risks of this above-mentioned transgenic experiments and crop ^[9]

The chief reason that causes risk of transgenic experiments and genetically modified crops is that transgenosis flows and transfer with interracial between different plant species, and promptly gene drifts about.Gene drift (gene flow/dispersal) refers to the process that gene spreads between population by approach such as pollen pollination hybridization.The approach that the transgenic plant gene drifts about has three kinds.The first is by the seed of transgenic plant or the diffusion of tissue; It two is that pollen by transgenic plant is to of the same race or sibling species plant hybridization diffusion; Its three is by between non-biology of the same race, for example drift of the producers such as pathogenic bacteria of plant.In these three kinds of approach, it is fastest that the gene that is undertaken by pollen drifts about, and range of influence is also the widest.Therefore, be fertility and the affinity that changes its transgenosis pollen for the effective means of drift risk that reduces plant transgene.Thereby people such as Mariani utilize tapetum specific promoter TA29 promoters driven reorganization B-glycuronidase or ribonuclease gene (RNase T1 or barnase) in nineteen ninety express in the tapetum of transgene tobacco and rape and optionally destroy tapetum, to obtain male sterile plant ^[10]This technology has been applied to cash crop such as wheat, paddy rice and tomato ^[11-14]Subsequently, the research report that utilizes the RNAi technology to suppress the relevant gene of pollen development and then make up male sterile plant is also arranged ^[15]This shows, produce male sterile line by transgenic method and limit transgenic plant gene drift effective means.But, at present in having made up these strains systems of male sterile line, although the male sterile line that produces has limited genetically modified drift significantly, the transgenic line that also obtains the simultaneously selfing of having no idea, therefore also can't obtain corresponding homozygote, be unfavorable for breeding.Therefore, find and a kind ofly both can suppress genetically modified drift, but the method that can not influence selfing there is certain meaning for molecular breeding.

Self incompatibility is a reproduction sovereignty nuisance in the naturally occurring kind of occurring in nature, refers to the pollen that the gynoecium of polycarpeae oneself can specific identification oneself, finishes the process of pollinating and being fertilized thereby can suppress it ^[16]Discover that at present Solanaceae, Plantaginaceae, scrophulariaceae and rosaceous plant have self incompatibility mostly, the self incompatibility that they adopt is the gametophytic self-incompatibility of nuclease type.In these plants, the factor of the self incompatibility of control style and pollen is respectively S-nuclease (S-RNase) and SLF, what style factor S-nuclease played is Cytotoxic effect, thereby and the main effect of pollen factor S LF is to suppress the toxic action of dissident's S-nuclease to guarantee that pollen can not be suppressed and finishes the process of pollination fertilization when cross-pollination ^[16,17]The self incompatibility of plant just relates to the sprouting of pollen pollen in the pollination process and the growth of pollen tube, and do not influence other the character of the growth of pollen itself and plant, therefore the self incompatibility of utilizing transgenic method to change pollen on the one hand can produce the male sterile line of novel type, with the genetically modified drift of effective inhibition, reduce the risk that transgenic experiments and genetically modified crops may be caused; The self incompatibility that also can change style in addition on the one hand simultaneously makes the novel male sterile line of this class that produces can finish selfing and produces homozygote, therefore help setting up the gene pool of Solanaceae class cash crop one's best quality, proterties control, thereby be beneficial to the carrying out of breeding work by this method.

Summary of the invention

The present invention is from the research and the utilization of the gametophytic self-incompatibility of nuclease type, from the hybridization petunia (Petunia hybrida) of Solanaceae, discovered and cloned a factor that participates in control nuclease type gametophytic self-incompatibility, called after PhSSK1 (PhSLF-interacting-Skp1-Like1).Studies show that this gene is by acting on mutually with the pollen factor of self incompatibility, the assistance pollen factor is finished normally carrying out of the fertilization process of pollinating after cross-pollination, and this gene is the gene that pollen-specific is expressed, and does not have the genotype specificity.Significantly reduce this expression of gene in the pollen by RNAi and can effectively suppress to have the affinity of transgenic fragment pollen the time with hybridization petunia pollination with normal self incompatibility; Simultaneously, but do not suppress the affinity of the type pollen when the mutant petunia of pollination style self incompatibility afunction.Therefore this method is the new control techniques that can suppress the transgenosis drift of a class, and this method also is applicable to the technology of the male not affine system of novel structure of Solanaceae class simultaneously.

At foregoing, the purpose of this invention is to provide the gene of a new participation control Solanaceae class self incompatibility pollen-specific.

Gene provided by the present invention, name is called PhSSK1 (PhSLF-interacting SKP1-Like1), derives from hybridization petunia (Petunia hybrida), is one of following nucleotide sequences:

1) dna sequence dna;

2) polynucleotide of coded protein sequence;

3) nucleotide sequence that can under the rigorous condition of height, hybridize with the nucleotide sequence that limits;

4) have 75% above homology with the aminoacid sequence that limits, and the proteinic aminoacid sequence of coding identical function.

Another object of the present invention provides PhSSK1 coded by said gene protein, derives from the hybridization petunia, is one of following protein sequence:

1) by the protein sequence of PhSSK1 genes encoding;

2) with limit aminoacid sequence and have homology more than 75%, and have the proteinic aminoacid sequence of identical function.

3) have the protein that limits amino acid residue sequence, or will limit replacement, disappearance or the interpolation of one or several amino-acid residue of amino acid residue sequence process and have identical active by the protein that limits protein derived with the qualification amino acid residue sequence.。

The 3rd purpose of the present invention provides the transgene carrier of a kind of PhSSK1-RNAi, be used to make up a kind of transgenic line of male incompatibility of the affinity by suppressing pollen, this material can effectively avoid transgenosis to drift about, and is convenient to set up the germplasm gene pool of fine quality, property control.

Contain expression carrier of the present invention and clone or transgenic plant and all belong to protection scope of the present invention.Clone wherein can be eucaryon also can be protokaryon.

Specifically be expressed as follows:

1, a kind of related protein or its functional analogue that participates in the different friendship affinity control of polycarpeae pollen self-incompatibity, wherein said protein has the aminoacid sequence shown in the SEQ ID NO:2, described functional analogue is the aminoacid sequence shown in the SEQID NO:2 through replacing, lack or adding the homologous sequence of one or more amino acid or other species and have analogue with described protein identical functions, perhaps has the functional analogue of at least 75% homology with the aminoacid sequence shown in the SEQ ID NO:2.

2, according to above 1 described protein or its functional analogue, it has the aminoacid sequence shown in the SEQ ID NO:2.

3, a kind of gene of encode above 1 or 2 described protein or its functional analogue.

4, according to above 3 described genes, it has the nucleotide sequence shown in the SEQ ID NO:1.

5, a kind of gene, its be the nucleotide sequence of above 3 or 4 described genes through replacing, lack or add one or more Nucleotide and have mutant, allelotrope or the derivative of identical function with above 3 or 4 described genes.

6, a kind of above 3,4 or 5 described genes or its segmental carrier of containing.

7, above 6 described carriers, it is a plant expression vector.

8, above 7 described carriers, it is the PhSSK1-RNAi conversion carrier.

9, a kind of host cell, this cell contain above 3,4 or 5 described gene orders or its fragment.

10, a kind of host cell, this cell contain each described carrier among the above 6-8.

11, according to above 10 host cell, this cell is Bacillus coli cells, agrobatcerium cell or vegetable cell.

12, a kind of method that causes the forfeiture of the different friendship affinity of pollen in polycarpeae comprises with the carrier transformed plant cells described in above 8, and the plant transformed cell culture is become plant.

13, according to above 12 described methods, wherein said conversion comprises conversion RNAi interference method.

14, according to above 12 described methods, wherein said polycarpeae is a plant of Solanaceae, comprises the hybridization petunia.

Description of drawings

Below in conjunction with accompanying drawing the present invention is explained in further detail.

The dna sequence dna of Fig. 1 PhSSK1 gene.

The aminoacid sequence of Fig. 2 PhSSK1 genes encoding.

Fig. 3 PhSSK1 gene genotype is analyzed and expression pattern analysis.(A) the southern blotting technique hybridization analysis of PhSSK1 in the different genes hybridization petunia.DNA to be detected digests through EcoR V and EcoR I single endonuclease digestion respectively, and uses PhSSK1 full length gene and part fragment (1-789bp) to carry out hybridization analysis as probe respectively, and the numeral on right side is the molecular weight size of band, and unit is Kb.(B) RT-PCR that PhSSK1 expresses in each tissue in the hybridization petunia analyzes, and Tubulin is contrast.(C) the tissue specificity analysis that PhSSK1 expresses in each genotypic crossing petunia.Ordinate zou is the relative quantity of PhSSK1 mRNA.(D) the proteic immunoblotting of PhSSK1 detects in each genotypic crossing petunia different tissues.Antibody is the antibody of PhSSK1, and ponceau dyeing is last sample contrast.

The structure synoptic diagram of Fig. 4 PhSSK1-RNAi conversion carrier.Wherein the NOS transcription terminator is the transcription terminator on the original pBI101 carrier.The zone of white is two exons of PhSSK1 gene in the PhSSK1 genomic dna, gray regional intron.

Fig. 5 PhSSK1-RNAi transgenosis T ₀Analysis of molecules for plant.(A) T ₀Southern blotting technique hybridization analysis for plant.DNA to be detected uses the PhSSK1 full length gene to carry out hybridization analysis as probe through the digestion of EcoRV single endonuclease digestion, and the pointed band of black arrow is the endogenous PhSSK1 gene of acceptor plant, and the numeral on right side is the molecular weight size of band, and unit is Kb.(B) T ₀QRT-PCR for plant pollen PhSSK1 analyzes, and ordinate zou is the relative content (the mRNA amount of the PhSSK1 of wild-type receptor pollen is 1) of PhSSK1mRNA.PhSKP1-2 is that another one is cloned the SKP1 homologous gene that obtains from petunia pollen, at this experiment contrast as the PhSSK1 downward modulation.(C) left hand view is T ₀For the proteic immunoblotting assay of PhSSK1 in the plant pollen, Tubulin is last sample contrast.The histogram on right side is that the protein immunoblot result analyzes quantitative relative protein content through quantitation software Quantity One, and selecting Tubulin during calculating for use is with reference to albumen.

Fig. 6 PhSSK1-RNAi transgenosis T ₁Analysis of molecules for plant.(A) DNA that detects plant is that probe carries out the southern blotting technique hybridization analysis through the digestion of EcoRV single endonuclease digestion with the PhSSK1 full length gene.What black arrow was indicated is the endogenous PhSSK1 copy of plant, the size of the segmental molecular weight of numeral on each figure right side, and unit is Kb.(B) T ₁QRT-PCR for plant pollen PhSSK1 analyzes, and ordinate zou is the relative content of PhSSK1 mRNA.PhSKP1-2 is the contrast of experiment.

Fig. 7 PhSSK1-RNAi transfer-gen plant is male parent and S _3LS _3LThe gene type assay of filial generation.Include 4 little figure among the figure, every little figure is the analytical results of single transfer-gen plant.T-DNA is the PhSSK1-RNAi transgenic fragment, S _VAnd S _3LRepresent the different haplotype S-nucleases that detect respectively.Female parent that preceding two swimming lanes that detect are test cross and male parent contrast.。

Fig. 8 is affine kind of a petunia (S _OS _O) the functional analysis of style self incompatibility.(A) affine kind of petunia S _OS _ONot affine petunia S _IS _I, S _3LS _3LAnd S _VS _VThe nuclease glue of S-nuclease is analyzed in the style.The proteinic molecular weight size of numeral in left side, unit is KD, what black arrow was indicated is the band of active nuclease.(B) S _OS _OThe immunoblotting assay of middle S-nuclease.S _O-nuclease is intestinal bacteria (E.coli) expressing proteins, and all the other four roads are the detected result of the style total protein of a genotype plant.The proteinic molecular weight size of numeral in left side, unit is KD.What black box was indicated is detected S-nuclease (S-RNase), and shown in the asterisk is detected non-specific band in the style albumen.Last sample contrast is the albumin glue of coomassie brilliant blue staining.

Fig. 9 PhSSK1-RNAi transfer-gen plant is male parent and S _OS _OThe gene type assay of filial generation.Include 4 little figure among the figure, every little figure is the analytical results of single transfer-gen plant.T-DNA is the PhSSK1-RNAi transgenic fragment, S _O, S _VAnd S _3LRepresent the S-nuclease of the different haplotypes that detect respectively.Female parent that preceding two swimming lanes that detect are test cross and male parent contrast.。

The sequence explanation

SEQ ID NO:1 is the dna sequence dna of PhSSK1 gene; With

SEQ ID NO:2 is the aminoacid sequence of PhSSK1 genes encoding.

Embodiment

Following this paper will describe invention by specific embodiment, and still described embodiment illustrates for example, and is not limitation of the invention.

Embodiment 1

1, petunia material

The genotype of growing in the greenhouse is S _IS _I, S _3LS _3L, S ₃S ₃, S _VS _V, S _3LS _VAnd S _OS _OThe hybridization petunia, S representative here be the genotype in plant self incompatibility site (S site), subscript _I, _3L, ₃, _VWith _ORepresent different haplotypes respectively.S wherein _IS _I, S ₃S ₃, S _3LS _VAvailable from University of Illinois, U.S. north life science system and molecular biology of plants center, S _3LS _3LAnd S _VS _VFlower bud controlled pollination (promptly early stage at flower development, before the S-nuclease is expressed as yet, promptly be about about 3 centimetres in petunia bud length, its strip off is forced to receive powder) obtains genotype by the hybridization petunia is shelled for us, S _OS _OFor available from Beijing middle peasant Xintai City Agritech Laboratories Inc. of section.In above-mentioned petunia material, S _IS _I, S _3LS _3L, S ₃S ₃, S _VS _VAnd S _3LS _VFor having the hybridization petunia of normal self incompatibility, S _OS _OSelf-compatible hybridization petunia for style self incompatibility afunction.

2, the clone of PhSSK1 gene

Process is to international Solanaceae genome plan website (SGN, SOL Genomics Network, http://solgenomics.net/) issued the analysis of EST and the proteins encoded thereof of a petunia surplus in the of 10,000 on, find that one of them predicted single-gene encoded protein that derives from the stamen tissue is similar to a pollen factors A hSSK1 who identifies the self incompatibility that participates in the nuclease type that the clone obtains from the Plantaginaceae Common Snapdragon, contact is closely also arranged, with its called after PhSSK1 on phyletic evolution.Process PCR carries out the technology (RACE) of the terminal quick clone of cDNA, and we have obtained the complete encoding sequence of PhSSK1.Concrete steps are as follows, extract hybridization petunia S _3LS _3LTotal RNA of pollen, carry out reverse transcription and obtain cDNA, adopt then 5 ' special primer (primer sequence is: 5 '-ATAAAGCTTATGGCATCAGAAAAGAAAATG-3 ') and 3 ' CDSIII primer (primer sequence: 5 '-ATTCTAGAGGCCGAGGCGGCCGACATG-3 ') carry out pcr amplification, reaction conditions is 94 ℃ of 5min; 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 90s, 35 circulations; 72 ℃ of 10min cut glue recovery order-checking and have obtained the PhSSK1 full length gene.The PhSSK1 full length gene has 1135bp, and two exons and an intron are arranged, protein (Fig. 1 and Fig. 2 that 179 amino-acid residues of encoding are formed; SEQ ID NO:1 and 2).

3, the gene type assay of PhSSK1 and expression pattern analysis

Extracted S respectively _IS _I, S _3LS _3LAnd S ₃S ₃The genomic dna of genotype petunia, and then cut digestion with EcoRV and EcoRI enzyme, with PhSSK1 genome total length is that probe has carried out the southern blotting technique hybridization analysis, and detected result shows that the PhSSK1 gene does not have the haplotype specificity in each genotypic hybridization petunia.

Method by RT-PCR, qRT-PCR and protein immunoblotting hybridization detects the expression of PhSSK1 in each tissue of petunia.The primer of PhSSK1 RT-PCR is: forward primer 5 '-CAA TCG TAA GAC CATAGT AAA-3 ', reverse primer 5 '-GGA ATT ATT GGT CGA CCC TGT-3 ', reaction conditions is 94 ℃ of 5min; 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 90s, 30 circulations; 72 ℃ of 10min, electrophoresis detection.The primer of qRT-PCR is: forward primer 5 '-GAC ACT AAA ATC AAA CGA TGA CCA A-3 ', reverse primer is 5 '-TCA TGT TCT TGA GCA TTT CAG ATT G-3 ', reaction conditions is 95 ℃, 10min; 95 ℃, 15s, 60 ℃, 1min, 40 circulations.The antibody that protein immunoblotting hybridization is used is the antibody (available from Inst. of Genetics and Development Biology, CAS's animal center) of the PhSSK1 in rabbit source.The result of mRNA and protein detection shows that this gene is the gene (Fig. 3) that a pollen-specific is expressed.

Embodiment 2

1, the structure of PhSSK1-RNAi conversion carrier

In order to study the biological function of PhSSK1, we have made up and have been used for reducing the double source conversion carrier that petunia pollen PhSSK1 expresses by RNAi interferential method.The process that makes up is as follows: the initial carrier of employing is the pBI101 double source conversion carrier of Clontech company, and the size of this carrier is 12.2Kb; Be connected into the promotor of the expressing gene Lat52 of pollen-specific by HindIII and XbaI ^[18]Because PhSSK1 itself has the intron of a 500bp (265-765), therefore, when making up the RNAi carrier, keep PhSSK1 gene order 1-789, in ncbi database, carry out the BLAST comparison with its CDS sequence, choose PhSSK1 encoding sequence downstream from 1-289, the reverse complementary sequence of the special section of the long 289bp of being makes up the section that forms siRNA, and whole PhSSK-RNAi transcriptional domain inserts the pBI101 carrier by XbaI and ScaI double digestion; Transcription Termination uses the primary NOS transcription terminator (Fig. 4) on the carrier pBI101.The engineering strain that uses in the process of whole structure is E.coli DH5 α (available from Invitrogen biotech firm).

2, transgenosis T ₀Acquisition and evaluation for plant

The method that transforms by the leaf dish transforms the petunia with strict self incompatibility with the PhSSK1-RNAi carrier that builds, and the genotype of acceptor is S _3LS _3LConcrete step of converting is as follows: transform Agrobacterium with the PhSSK1-RNAi carrier, then so the hybridization petunia blade that is cut into small pieces after the Agrobacterium bacterium liquid that will have a transgene carrier and the sterilization cultivate altogether, and then induce, the screening callus is cultivated and is obtained transfer-gen plant.And then, extracted the genomic dna of wild-type receptor plant and transgenic line respectively, carry out completely enzyme with EcoRV and cut digestion, carry out hybridization analysis with the full length sequence of PhSSK1 gene as probe.Evaluation has obtained 7 independently transgenic lines, respectively called after B, E1, J2, K4, H6, J4 and M8.

And then we have detected the expression of PhSSK1 in the pollen of this 7 strain transgenic line.Show that by the qRT-PCR (required primer and reaction conditions are identical with qRT-PCR primer and reaction conditions among the embodiment 1) and the result of protein immunoblotting analysis (PhSSK1 antibody (available from Inst. of Genetics and Development Biology, CAS's animal center)) transgenosis PhSSK1-RNAi has significantly reduced T ₀Expression for PhSSK1 in the plant pollen.In addition, the gene of another one Skp1-like in the pollen, the expression of PhSKP1-2 have also been detected by qRT-PCR.The primer sequence that uses is: forward primer 5 ' TGG ATC TCA CAT GCC AGA CTG T 3 ', reverse primer 5 ' TTC TTAATG TTG AAC GTC TTC CTT ATC T 3 ', reaction conditions according to 96 ℃ 10 minutes; 96 ℃ 15 seconds, 60 ℃ 1 minute, 40 circulations are carried out.Detected result shows that this genetic expression is not subjected to genetically modified influence, shows that therefore transgenosis PhSSK1-RNAi carrier is the expression of specific downward modulation PhSSK1, does not have exist (Fig. 5) of " mistake is hit " phenomenon.

We are the transgenosis T to obtaining ₀Phenotype for plant is analyzed, and the result shows that the PhSSK1-RNAi transgenosis does not cause that the whole vine growth and development of transfer-gen plant takes place by significant the change, and style pollination behavior and solid situation are not affected yet.But, find in the detection for pollen pollination behavior, although pollen still shows as strict self incompatibility when pollination self, but under the situation of the affine pollination of normally different friendship, genetically modified pollen but shows the unusual of the remarkable pollination behavior that descends of not affine and solid situation, influenced the fertility or the self incompatibility of pollen after PhSSK1 expresses in the prompting transgenosis downward modulation pollen, perhaps the two has it concurrently.

3, transgenosis T ₁Acquisition and evaluation for plant

In order to detect after the PhSSK1-RNAi transgenosis for the influence of pollen affinity and fertility, we select transfer-gen plant J2 and K4 as research object.Selected genotype is S _VS _VThe not affine wild-type plant of selfing hybridization obtains T as male parent and above-mentioned two strain transfer-gen plants ₁For plant.Resulting T ₁Genotype for plant is S _3LS _VOur T subsequently to obtaining ₁The band plant has carried out the southern blotting technique hybridization analysis, has detected the offspring of 5 strain K4 and the offspring of 12 strain J2.Because these T ₁The male parent in generation is S _VS _V, so they distinguish called after VJ and VK.Be that probe detects with the PhSSK1 full length gene in the southern blotting technique hybridization analysis.Detected result shows has 9 strains (VJ2, VJ4, VJ5, VJ6, VJ7, VJ8, VJ10, VJ11 and VJ12) to have the transgenosis copy among the 12 strain VJ, 3 strains among the 5 strain VK (VK1, VK2 and VK3) have transgenic fragment, and these have the T of transgenosis copy ₁For plant with the banding pattern of transgenosis copy maternal consistent with it, the insertion that shows the transgenic fragment among K4 and the J2 is with a linkage group insertion of 4 transgenic fragments formation.

We are for the transgenosis T that obtains subsequently ₁Expression for PhSSK1 in the plant pollen is analyzed.Detected the mRNA content of PhSSK1 in these plant pollens with the method for qRT-PCR (method of detection and primer are identical with qRT-PCR among the embodiment 1).Results suggest, the transgenosis T that is detecting ₁This expression of gene has been subjected to significant downward modulation (Fig. 6) equally in the pollen in generation.

4, the expression that significantly reduces PhSSK1 in the pollen can cause the forfeiture of different friendship affinity of pollen or fertility

We are for the transgenosis T that obtains subsequently ₁Check and analysis have been carried out in pollination behavior for plant pollen.Transgenosis T in self-pollination and cross-pollination ₁The pollination behavior and the solid situation of the pollen of plant are not subjected to remarkable influence.But because the transgenosis T that we detect ₁Therefore for plant is heterozygote, and for the further pollination behavior of research transgenosis pollen, we are to being male parent and S with the transfer-gen plant _3LS _3LGene type assay has been carried out in filial generation after the affine pollination.Analytical procedure is that Auele Specific Primer carries out PCR, is divided into to have analysed 3 genes, and wherein T-DNA is the copy of transgenosis PhSSK1-RNAi, PhS _3L-RNase and PhS _V-RNase is respectively S _3LAnd S _VThe S-nuclease of haplotype is used for the S genotype identification of plant.Primer sequence is as follows: shown in:

T-DNA (PhSSK1-RNAi): forward primer 5 '-AGA CAC ACA CAA AGA GAA GGA G-3 ', reverse primer 5 '-CAA CCC AAC CCG TTC ATT TAC-3 '; PhS _3L-RNase (primer: forward primer 5 '-TGT GAC GAT CAC TGA AAT AAA T-3 ', reverse primer 5 ' CGA AGA AAG GAA TAT GTTCAT CC-3 '; PhS _V-RNase primer: forward primer 5 '-GAT TAC GGA CGA AGC TGA TTG-3 ', reverse primer 5 '-GAA ACT TAA TTA TGG GTC CAT AAT CC-3 '.

All PCR reaction conditionss are: 94 ℃ of 5min; 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 60s, 35 circulations; 72 ℃ of 10min.The PCR product detects through agarose gel electrophoresis then.

We have been total to check and analysis 4 transgenosis T ₁The offspring in generation, the number of individuals of each progeny population is 24 (satisfying statistical requirement), and then adds up for detected result.Analytical results shows, the individuality that has the transgenosis copy is seldom arranged in progeny population, therefore show that the pollen that has T-DNA in the transfer-gen plant can not normally finish the process of pollination fertilization when the affine pollination of different friendship, so T-DNA can not be delivered in the follow-on individuality.Can cause the decline of pollen affinity or fertility after PhSSK1 expresses and reduces in this results suggest self incompatibility petunia pollen; can show similar male sterile feature (Fig. 7 and table 1; only shown 20 strains in the 24 strain offspring individualities among Fig. 7, whole 24 strain offspring individualities carry out but the statistical analysis in the table 1 is based on).

Table 1 PhSSK1-RNAi transfer-gen plant T ₁On behalf of male parent and S _3LS _3LThe genotype of filial generation is separated statistics

A, the T of all detections ₂Female parent for colony is self-compatible S _3LS _3L, male parent is transgenosis T ₁For plant.Each number that detects colony is 24 strains.

B, T ₂If when meeting mendel's law for the separation of gene in the colony, the expection of transgenosis copy separates ratio.

C, T ₂Substantial sepn ratio for colony's transfer gene copy.

D, substantial sepn compares test of goodness of fit than separating with respect to expection.χ ² _0.01=6.635, χ ² _0.05=3.841, if calculated value is greater than χ ² _0.05, p＞0.05, then substantial sepn is separated more not remarkable than difference than with expection; If calculated value is less than χ ² _0.01, p＜0.01, it is remarkable that then substantial sepn is separated the ratio heteropole than with expection.

5, the detection of self-compatible hybridization petunia style self incompatibility

Usually the male sterile strain that uses is to be the fertility that has changed pollen, therefore also can't finish the process of normal self fertilizing when avoiding transgenosis to drift about, and therefore certain limitation is arranged on application in practice.Whether we also are similar situations by the similar male sterile line that PhSSK1-RNAi obtains so, be also the fertility that has influenced pollen after the PhSSK1 downward modulation whether promptly? in order to answer this problem, we must have the detection strain system of the mutant strain of strain style self incompatibility forfeiture as the test cross experiment.We identify from business-like hybridization petunia and obtain the self-compatible hybridization of strain petunia, prove that through gene type assay the genotype of this plant is S _OS _OWe have carried out detection of nuclease glue and protein immunoblotting detection to its style total protein subsequently.In nuclease glue detects, at first extract its style total protein, in the polyacrylamide gel that is added with yeast tRNA (available from Sigma-adrich biotech firm), carry out the SDS-PAGE electrophoresis then, after electrophoresis is finished with the Tris-HCl (pH 7.0) (available from Sigma-adrich biotech firm) of separation gel at 10mM, handle 2 times in 25% the Virahol (available from Sigma-adrich biotech firm), each 20 minutes, remove SDS, then at reaction solution (10mM Tris-HCl pH 7.4,100mMKCl, 40 μ M Zn (Ac) ₂, 1mM Cystine and 40 μ M Mg (Cl) ₂) in 37 ℃ the reaction 2 hours.After reaction was finished, albumin glue was through 0.1% toluidine blue (available from Sigma-adrich biotech firm) dyeing 5 minutes, photographic recording.Carried out the protein immunoblotting detection subsequently; extract each genotypic hybridization petunia style total protein and carry out the SDS-PAGE electrophoretic separation; then albumen is transferred on the pvdf membrane (available from Sigma-adrich biotech firm), detects with the antibody (available from Inst. of Genetics and Development Biology, CAS's animal center) of S-nuclease (S-RNase).More than two result of experiment all point out the style self incompatibility factor of determination S-nuclease of this strain system not express, therefore prove that this plant is the mutant of style self incompatibility afunction, be suitable for distinguishing detection has influenced pollen behind the PhSSK1 down-regulated expression in the pollen in the PhSSK1-RNAi transfer-gen plant fertility or self incompatibility (Fig. 8).

6, the expression that reduces PhSSK1 in the pollen has only influenced the different friendship affinity of pollen

The self incompatibility of transfer-gen plant pollen still is that fertility has been subjected to influence in order to detect actually, we with transgenosis T1 on behalf of male parent and S _OS _OCarried out the test cross experiment.The T that obtains behind the same test cross ₂Carry out gene type assay for colony, and the result is carried out statistical study.Analytic process is the same, also is to be transgenosis PhSSK1-RNAi copy, PhS by PCR reaction carrying out 4 gene: T-DNA of common detection _3L-RNase, PhS _V-RNase and PhS _O-RNase is respectively S _3L, S _VAnd S _OThe S-nuclease of haplotype is used for the S genotype identification of plant.For T-DNA, PhS _3L-RNase and PhS _VThe detection of-RNase is the same with primer.For PhS _OThe detection of-RNase is as follows with primer: forward primer 5 '-ATG TTT CAG TTT CAG CTC ACC TCA-3 ', reverse primer 5 '-TCA CTG TCG AAA CGTAAT ACC CG-3 '.The reaction conditions of PCR all with the term harmonization that detects above.The detected result of statistics shows that the pollen of transfer-gen plant is at pollination S _OS _OAfter, having the same process that can finish the pollination fertilization of the same and normal pollen of pollen of T-DNA, so show that the downward modulation of PhSSK1 does not influence the fertility of pollen, promptly genetically modified pollen itself still normally can be educated.And transgenosis has just influenced the self incompatibility of pollen, make that pollen is process (Fig. 9 and the table 2 that can not finish the pollination fertilization at the style with normal self incompatibility function of pollinating to, only shown 20 strains in the 24 strain offspring individualities among Fig. 9, whole 24 strain offspring individualities carry out but the statistical analysis in the table 2 is based on).

Table 2 PhSSK1-RNAi transfer-gen plant T ₁On behalf of male parent and S _OS _OThe genotype of filial generation is separated statistics

A, the T of all detections ₂Female parent for colony is self-compatible S _OS _O, male parent is transgenosis T ₁For plant.Each number that detects colony is 24 strains.

B, T ₂If when meeting mendel's law for the separation of gene in the colony, the genotypic segregation ratio of expection.N _T+For detecting the plant number that has the transgenosis copy in the colony; N _T-For detecting the plant number that does not have the transgenosis copy in the colony; N _S3LFor having the S site in the detection colony is S _3LThe plant number of haplotype; N _SVFor having the S site in the detection colony is S _VThe plant number of haplotype; N _T+S3LHave transgenosis copy and S in the colony simultaneously for detecting _3LThe plant number in haplotype S site; N _T-S3LDo not have the transgenosis copy in the colony but have S for detecting _3LThe plant number in haplotype S site; N _T+SVHave transgenosis copy and S in the colony simultaneously for detecting _VThe plant number in haplotype S site; N _T-SVDo not have the transgenosis copy in the colony but have S for detecting _VThe plant number in haplotype S site.

C, T ₂For colony's transgenosis copy and genotypic substantial sepn ratio.

D, substantial sepn compares test of goodness of fit than separating with respect to expection.χ ² _0.01=6.635, χ ² _0.05=3.841, if calculated value is greater than χ ² _0.05, p＞0.05, then substantial sepn is separated more not remarkable than difference than with expection; If calculated value is less than χ ² _0.01, p＜0.01, it is remarkable that then substantial sepn is separated the ratio heteropole than with expection.

Be defined as male not affine system accurately by our resulting male sterile line of PhSSK1-RNAi transgenic experiments.This male not affine system is different from traditional male sterile line, when we obtain the hybridization petunia of style self incompatibility afunction by transgenosis or other method for screening, this male not affine system can guarantee that on the one hand pollen can't unofficial biography and avoid genetically modified drift, finish selfing in the strain of the style self incompatibility afunction system more simultaneously, so that obtain homozygote, make up the gene pool of fine quality property control gene.All have self incompatibility in a lot of plants of Solanaceae, so this technology and experimental program can be promoted effectively in the breeding and transgenic experiments of Solanaceae cash crop.

Reference

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[2] Zhao Degang, Lv Litang, He Aigong, et al. " foreign gene removing " technology (Gene-Deletor) principle, characteristics and potential application foreground thereof [J]. Molecular Plant Breeding, 2008,6 (003): 413-8.

[3] money Haifeng county, Chen Zhehao, Fu Jie. the molecular strategy [J] that gene is escaped in the control transgenic plant. life science, 2004,16 (5): 288-91.

[4]Regal?P.Scientific?principles?for?ecologically?based?risk?assessment?of?transgenicorganisms[J].Molecular?Ecology?Notes，1994，3(1)：5-13.

[5]Raybould?A，Gray?A.Genetically?modified?crops?and?hybridization?with?wild?relatives：a?UK?perspective[J].Journal?of?Applied?Ecology，1993，30(2)：199-219.

[6]Warwick?S，Beckie?H，Small?E.Transgenic?crops：new?weed?problems?for?Canada？[J].PHYTOPROTECTION-QUEBEC-，1999，80(2)：71-84.

[7]Franck-Oberaspach?S，Keller?B.Consequences?of?classical?and?biotechnologicalresistance?breeding?for?food?toxicology?and?allergenicity?[J].Plant?Breeding，1997，116(1)：1-17.

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[9]Stewart?C，Richards?H，Halfhill?M.Transgenic?plants?and?biosafety：science，misconceptions?and?public?perceptions[J].Biotechniques，2000，29(4)：832-43.

[10]Mariani?C，Beuckeleer?M，Truettner?J，et?al.Induction?of?male?sterility?in?plants?by?achimaeric?ribonuclease?gene[J].Nature，1990，347(6295)：737-41.

[11] Zhou Xuerong, Fang Rongxiang. express the acquisition [J] of the male sterile rape of ribonuclease gene. Acta Genetica Sinica, 1997,24 (6): 531-6.

[12] Fu Rongzhao, Chen Zhankuan. artificial male sterility gene is imported the research preliminary study [J] of wheat with particle bombardment. Acta Genetica Sinica, 1997,24 (4): 358-61.

[13] Cao Guangcheng, Chen Zhankuan. artificial male sterility gene and the clone and the structure research [J] that recover gene. Scientia Agricultura Sinica, 1996,29 (3): 20-6.

[14] Li Shengguo, Liu Yule, Zhu Feng, the acquisition [J] of et al. genetically engineered male sterile tobacco. Botany Gazette, 1995,37 (8): 659-960.

[15]Napoli?C，Lemieux?C，Jorgensen?R.Introduction?of?a?chimeric?chalcone?synthase?geneinto?petunia?results?in?reversible?co-suppression?of?homologous?genes?in?trans[J].The?PlantCell?Online，1990，2(4)：279.

[16]Franklin-Tong?V?E.Self-Incompatibility?in?Flowering?Plants[M].Heidelberg：Springer，2008.

[17]Zhang?Y，Zhao?Z，Xue?Y.Roles?of?Proteolysis?in?Plant?Self-Incompatibility[J].Annual?review?of?plant?biology，2009，60：21-42.

[18] Xu Guandong, Luo Yuying. structure, regulation and control and the application thereof [J] of pollen specific gene Lat52. Capital Normal University's journal (natural science edition), 2000,21 (2): 47-51.

SEQUENCE?LISTING

＜110〉Inst. of Genetics and Development Biology, CAS

 

＜120〉participate in albumen and encoding gene and the application that the different friendship affinity of polycarpeae self incompatibility pollen is controlled

 

<130>IB100216

 

<160>2

 

<170>PatentIn?version?3.1

 

<210>1

<211>1135

<212>DNA

<213>Petunia?sp.

 

<400>1

atggcatcag?aaagaaaatg?gtgacactaa?aatcaaacga?tgaccaagag?tttcaagtgg 60

aggaagctgc?agttatccaa?tctgaaatgc?tcaagaacat?gattgaagat?gattgtgctt 120

ctagtgtcat?tccacttcct?aacatcgata?gcaaaacgtt?aagcaaagtc?atcgaatatc 180

tcaacaagca?cataacaaga?gacgaagatg?aggacgagga?acaggaggag?ggcaaggaca 240

aagggaagga?ggtggataca?ggtgcgtaca?agtagtaaat?gaacgggttg?ggttgaattt 300

tgatgggtaa?aattaattga?taagtcataa?ttcttttctt?ttctcgtcga?cacataactt 360

aactcgtcca?aattttggtt?ggttctgtat?atgtcaatta?accccttata?gcctaacctg 420

acaatccaat?ttatctcact?ttttctgttt?gacttaaatt?cccttgttct?tccaagtaga 480

tgtaattttt?aatatcaaaa?ttttgatttt?gtatgcttgg?gtaattgggt?ttgtattgga 540

ttgtggccaa?ttagattagg?ctgttgtgac?tcacttattc?caatattttg?gttggtcact 600

ttggttaaac?tcattgagtt?aagtcaataa?acgagtcata?actaaacata?tttatgctta 660

ggcgagttaa?acccttttgt?catgttgtaa?acatatcatg?atccaaacca?tttcatgtta 720

gacgaattat?aaatgattga?gttgattttt?cctcgtgtgc?aggtgaagaa?gatgatctca 780

aggaattcga?cgaacaattc?gtgaacgtag?gatttgaaga?gctctttgac?ataataatgg 840

cagcaaatta?tttgaacatt?cacgagttga?tggaattatg?ttgccaatct?gcagcagata 900

gattgaagaa?caaaagtgtt?agagctgttc?gtgaaatgtt?aaaaattact?aatgatctta 960

ctgaagaaga?agagcaagag?attattaatg?atgctccatg?ggcatttgaa?ggtcctgaaa 1020

ttgatgatac?tgtcaattag?gttaataaca?ataatatatc?tttcattttt?tttcggtaga 1080

aaaattatct?agtatctaat?ttacatagct?atatacaggg?tcgaccaata?attcc 1135

 

<210>2

<211>179

<212>PRT

<213>Petunia?sp.

 

<400>2

Met?Ala?Ser?Glu?Lys?Lys?Met?Val?Thr?Leu?Lys?Ser?Asn?Asp?Asp?Gln

1 5 10 15

Glu?Phe?Gln?Val?Glu?Glu?Ala?Ala?Val?Ile?Gln?Ser?Glu?Met?Leu?Lys

20 25 30

Asn?Met?Ile?Glu?Asp?Asp?Cys?Ala?Ser?Ser?Val?Ile?Pro?Leu?Pro?Asn

35 40 45

Ile?Asp?Ser?Lys?Thr?Leu?Ser?Lys?Val?Ile?Glu?Tyr?Leu?Asn?Lys?His

50 55 60

Ile?Thr?Arg?Asp?Glu?Asp?Glu?Asp?Glu?Glu?Gln?Glu?Glu?Gly?Lys?Asp

65 70 75 80

Lys?Gly?Lys?Glu?Val?Asp?Thr?Gly?Glu?Glu?Asp?Asp?Leu?Lys?Glu?Phe

85 90 95

Asp?Glu?Gln?Phe?Val?Asn?Val?Gly?Phe?Glu?Glu?Leu?Phe?Asp?Ile?Ile

100 105 110

Met?Ala?Ala?Asn?Tyr?Leu?Asn?Ile?His?Glu?Leu?Met?Glu?Leu?Cys?Cys

115 120 125

Gln?Ser?Ala?Ala?Asp?Arg?Leu?Lys?Asn?Lys?Ser?Val?Arg?Ala?Val?Arg

130 135 140

Glu?Met?Leu?Lys?Ile?Thr?Asn?Asp?Leu?Thr?Glu?Glu?Glu?Glu?Gln?Glu

145 150 155 160

Ile?Ile?Asn?Asp?Ala?Pro?Trp?Ala?Phe?Glu?Gly?Pro?Glu?Ile?Asp?Asp

165 170 175

Thr?Val?Asn

Claims (14)

1. one kind participates in related protein or its functional analogue that the different friendship affinity of polycarpeae pollen self-incompatibity is controlled, wherein said protein has the aminoacid sequence shown in the SEQ ID NO:2, described functional analogue is the aminoacid sequence shown in the SEQID NO:2 through replacing, lack or adding the homologous sequence of one or more amino acid or other species and have analogue with described protein identical functions, perhaps has the functional analogue of at least 75% homology with the aminoacid sequence shown in the SEQ ID NO:2.

2. protein according to claim 1 or its functional analogue, it has the aminoacid sequence shown in the SEQ ID NO:2.

3. gene of claim 1 or 2 described protein or its functional analogue of encoding.

4. gene according to claim 3, it has the nucleotide sequence shown in the SEQ ID NO:1.

5. gene, its be the nucleotide sequence of claim 3 or 4 described genes through replacing, lack or add one or more Nucleotide and have mutant, allelotrope or the derivative of identical function with claim 3 or 4 described genes.

6. one kind contains claim 3,4 or 5 described genes or its segmental carrier.

7. the described carrier of claim 6, it is a plant expression vector.

8. the described carrier of claim 7, it is the PhSSK1-RNAi conversion carrier.

9. host cell, this cell contains claim 3,4 or 5 described gene orders or its fragment.

10. host cell, this cell contains each described carrier among the claim 6-8.

11. according to the host cell of claim 10, this cell is Bacillus coli cells, agrobatcerium cell or vegetable cell.

12. a method that causes the forfeiture of the different friendship affinity of pollen in polycarpeae comprises with the carrier transformed plant cells described in the claim 8, and the plant transformed cell culture is become plant.

13. in accordance with the method for claim 12, wherein said conversion comprises conversion RNAi interference method.

14. in accordance with the method for claim 12, wherein said polycarpeae is a plant of Solanaceae, comprises the hybridization petunia.

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