CN105039398A - Method for preparing male sterile peppers - Google Patents

Abstract

The invention belongs to the technical field of pepper breeding, and discloses a method for preparing male sterile peppers. The method includes the following steps that S1, a reconstructed RNAi interference vector containing the CaMS1 gene shown as SEQ ID NO:1 and SEQ ID NO:2 is constructed; S2, the reconstructed RNAi interference vector obtained in S1 is used for converting agrobacteria to obtain reconstructed agrobacterium; S3, the reconstructed agrobacterium is used for transfection of a pepper explant, a transformation plant is obtained through cultivation, and the male sterile peppers are obtained through screening. The reconstructed vector containing the anther-specific gene is built, so that a material capable of reducing activity of pollen is prepared; by improving and optimizing the materials, a pepper male sterile material is further prepared, and the method has important value in selection of new varieties of the peppers.

Description

A kind of method preparing male sterile pepper

Technical field

The invention belongs to pepper breeding technical field, particularly, relate to a kind of method preparing male sterile pepper.

Background technology

Capsicum ( capsicumannum) belonging to Solanaceae (Solanaceae) Capsicum 1 year or per nnial herb, its fruit is a kind of global vegetables and processing and seasoning product.Capsicum is Constantly allogamous plant, and clearly, a superior hybrid generation can increase production 30% ~ 50% than conventional variety to hybrid vigour.Heterotic utilization, has become capsicum and has produced the upper important means improving output and increase economic benefit.But at present China's hybrid seeding many employing flower bud phases artificial emasculation, pollination, the not only high but also very difficult guarantee seed purity of breeding cost.Utilize male sterile line to prepare half-blood, not only can simplify production of hybrid seeds program, reduce seed produces cost, and the purity of hybrid can be improved.Therefore, the seed selection of male sterile line and applied research thereof are more and more subject to people's attention.

Pollen abortion is that the phenotype that plants male sterility occurs embodies, and understands fully that the whole process of pollen development and molecule mechanism study basis and the key point of plants male sterility.Pollen development relates to many polygenic expression regulations, not only can understand the molecular mechanism of pollen development, can also provide fundamental basis for artificially creating male sterile to the research of pollen development genes involved.Knocking out of these genes can cause plant part or completely sterile, but very few to the research of pollen development genes involved in capsicum.The developmental genes involved of capsicum pollens can be knocked out by RNA perturbation technique, formulate male sterile capsicum material.

Summary of the invention

The present invention is directed to the deficiencies in the prior art, provide a kind of method preparing male sterile pepper, the method is by building the recombinant vectors containing flower pesticide specific gene, prepare the material that Pollen Activity reduces, by to this material improvement with optimize can prepare hot pepper male sterile material further, have important value to the seed selection of capsicum new variety.

Above-mentioned purpose of the present invention is achieved by the following technical programs.

Prepare a method for male sterile pepper, comprise the steps:

S1. build containing, for example shown in SEQIDNO:1 ~ 2 caMS1the recombinant RNA i interference carrier of gene;

S2. the recombinant RNA i interference carrier transformation Agrobacterium obtained by S1, obtains recombinational agrobacterium;

S3. use recombinational agrobacterium transfection capsicum explant, obtain transformed plant through cultivating, screening obtains male sterile pepper.

Described in S1 caMS1the cDNA sequence of gene as shown in SEQIDNO:1, long 1919bp; Gene caMS1open reading frame sequence as shown in SEQIDNO:2, long 1758bp.

Preferably, described in S1, the construction process of RNAi interference carrier is:

S11. utilize sense primer shown in SEQIDNO:3 ~ 4 to increase to obtain caMS1the sense fragment of gene, utilizes antisense primer shown in SEQIDNO:5 ~ 6 to increase and obtains caMS1the antisense fragments of gene;

S12. sense fragment and antisense fragments are successively connected to same RNAi interference carrier, obtained caMS1the recombinant RNA i interference carrier of gene.

Step S11 clones the primer pair of this gene sense fragment as shown in SEQIDNO:3 ~ 4, clones the primer pair of this gene antisense fragment as shown in SEQIDNO:5 ~ 6.The primer pair of positive and negative adopted sequence is respectively:

UP-SP4-1：5′-CGG ATTTAAATAGTCAGTGGAGCAAGCAA-3′，

UP-SP4-2：5′-CGC GGATCCAGTCAGTGGAGCAAGCAA-3′；

DW-SP4-1：5′-CATG CCATGGTTAGCCCTGGAATGTGGA-3′，

DW-SP4-2：5′-TCC CCCGGGTTAGCCCTGGAATGTGGA-3′。

Preferably, described RNAi interference carrier is pFGC5941 carrier.

Preferably, introduce at the two ends of sense fragment swai and ncoi restriction enzyme cuts site, introduces at the two ends of antisense fragments smai and bamhI restriction enzyme cuts site, cuts ligation sense fragment and antisense fragments are successively connected in pFGC5941 carrier by enzyme.

Preferably, described in S3, capsicum explant is: all cut together with terminal bud and peripheral meristem from petiole base by the side cotyledon of the aseptic seedling of cultivation, only stay side cotyledon.

Preferably, the OD described in S3 during recombinational agrobacterium transfection Agrobacterium capsicum explant ₆₀₀be 0.4 ~ 0.8, transfection time is 12min.

Preferably, described in S3, cultivation comprises the steps:

S31. explant and bacterium liquid shake up and are seeded in division culture medium, constant temperature dark culturing 2 ~ 3d under 25 ± 1 DEG C of conditions;

S32. micro-organisms: be inoculated in 27 DEG C of cultivation 4 ~ 5d in micro-organisms base;

S33. screening and culturing: be inoculated in PPT screening culture medium, every two weeks subcultures once, subculture 2 ~ 3 times; Until only have a little outer planting physical efficiency to grow resistant buds in screening culture medium, and most of explant chlorisis in the screening culture medium containing PPT, till flavescence coking;

S34. root culture: as Elongation of adventitious bud to the 1.5 ~ 2.5cm broken up, it cut from base portion, be inoculated in root media and induce it to take root, then hardening 2 ~ 3d, transplant and obtain male sterile pepper.

The substratum that above-mentioned division culture medium, micro-organisms base, screening culture medium, root media adopt capsicum group training field common.

Preferably, in S33, the PPT concentration of screening culture medium is 2mg/L.

Preferably, before bacterium immersion dye, described capsicum explant is put into described division culture medium, in 27 DEG C of dark preculture 2 ~ 3d.

Preferably, the method obtaining capsicum aseptic seedling is: by pepper seed after 75% alcohol, 2% hypochlorite disinfectant, soak with sterilized water, remove sterilized water, suck the moisture of seed-coat, under 25 ~ 28 DEG C of dark conditions, cultivate 5 ~ 6d, then seed taken out and be put into 27 DEG C, cultivate under illumination 2000lx14h/d culture condition, when capsicum seedling grows two panels cotyledon and vegetative point is about to expose, obtain capsicum aseptic seedling.

Compared with prior art, beneficial effect of the present invention is: provide a kind of method preparing male sterile pepper, the method is by building the recombinant vectors containing flower pesticide specific gene, prepare the material that Pollen Activity reduces, by to this material improvement with optimize can prepare hot pepper male sterile material further, have important value to the seed selection of capsicum new variety.The present invention screened through 2 ~ 3 generations, obtained resistance capsicum, T ₀39.6%, T is reached for transgenic positive rate ₁29.2% is reached for transgenic positive rate; T ₀generation caMS1the pollen germination rate of interference plant is 21.84%, and wild type control is 52.63%, illustrates that the transfer-gen plant prepared has certain male infertility.

Accompanying drawing explanation

Fig. 1 is the capsicum fertile plant or sterile strain eight grade mixing bud RNA plain agar sugar gel electrophoresis figures extracted; Wherein, F is fertile plant, and S is sterile strain.

Fig. 2 is caMS1cDNA total length and the aminoacid sequence of derivation; Initiator codon and terminator codon black matrix represent; Two setting-out part is that FAR-N-SDR-e guards territory; Single underscore part is that STERILE guards territory.

Fig. 3 is caMS1in the expression of capsicum bud different development stage; A.RT-PCR analyzes, actinfor reference gene; B.qRT-PCR analyzes; F1 ~ 8, S1 ~ 8 are respectively fertile plant and sterile strain 1 ~ 8 grade of bud.

Fig. 4 is caMS1at the expression analysis at capsicum different tissues position; A.RT-PCR analyzes, actinfor reference gene; B.qRT-PCR analyzes; F1 ~ 8, S1 ~ 8 are respectively fertile plant and sterile strain root, stem, leaf, open flower, calyx, petal, flower pesticide and gynoecium.

Fig. 5 is caMS1positive and negative adopted fragment PCR electrophorogram; M:DL2000Marker; 1-sense fragment; 2-antisense fragments.

Fig. 6 is caMS1sense fragment sequencing result.

Fig. 7 is caMS1antisense fragments sequencing result.

Fig. 8 is caMS1object sequencing fragment blastn comparison result.

Fig. 9 is caMS1the restriction enzyme digestion and electrophoresis figure of pFGC5941-B plasmid ( bamh I, smai); M:DL2000; 1 ~ 5 is recombinant plasmid.

Figure 10 is caMS1the bacterium liquid PCR electrophorogram of RNAi carrier transformation Agrobacterium; M:DL2000; CK: negative control: 1 ~ 13 is caMS1pFGC5941-B bacterium liquid.

Figure 11 is caMS1t ₀pCR for transformed plant detects (Bar gene primer); M:DL2000Marker; 1-positive control; 2-blank; 3-negative control; 4 ~ 24 are caMS1t ₀for resistant plant.

Figure 12 is caMS1t ₀pCR for transformed plant detects (35S promoter primer); M:DL2000Marker; 1-positive control; 2-blank; 3-negative control; 4 ~ 24 are caMS1t ₀for resistant plant

Figure 13 is transfer-gen plant amplified fragments sequence alignment result.

Figure 14 is transfer-gen plant and contrast upgrowth situation.

Figure 15 is caMS1disturb plant and contrast flower organ morphology.

Figure 16 is in-vitro pollen germination microscopical view; A- caMS1reticent plant; B-wild-type.

Figure 17 is caMS1t0 detects (35S promoter primer) for the PCR of transformed plant; M:DL2000; 1-negative control; 2-positive control; 3 ~ 9 are caMS1t ₁for transfer-gen plant.

Embodiment

Below in conjunction with Figure of description and specific embodiment, the present invention is described in further details, but embodiment does not limit in any form the present invention.Unless stated otherwise, the present invention adopts reagent, method and apparatus are the art conventional reagent, method and apparatus.

the extraction of embodiment 1 chilli kernel male sterile dual purpose lines RNA and the synthesis of cDNA

With chilli kernel male sterile dual purpose lines AB114 for material, totally 8 the grade buds adopting Trizol method to extract this material fertile plant and sterile strain respectively (are respectively: 1 lobus cardiacus is curled into strip, and calyx wraps tightly corolla; 2 lobus cardiacuses half exhibition, calyx wraps tightly corolla; 3 lobus cardiacuses launch, and calyx wraps tightly corolla, and anthocaulus is upright; 4 calyx split a little, micro-dew corolla, and anthocaulus bends; 5 calyx flush with corolla; 6 corollas stretch out calyx part and are about the long half of calyx; 7 corollas stretch out calyx part and calyx isometric; The flower that 8 next day will open) and root, stem, leaf, open flower, calyx, petal, flower pesticide and gynoecium total serum IgE, concrete steps are as follows:

S1. in 2.0mL centrifuge tube, add 1mLTrizol extracting solution, take to transfer to after 0.1g material is ground into powder rapidly in liquid nitrogen and be equipped with in the centrifuge tube of extracting solution, vibration mixing, leaves standstill 5min on ice;

S2. add 0.25mL chloroform, thermal agitation 30s, leave standstill 5min on ice;

At S3.4 DEG C, the centrifugal 15min of 13000rpm;

S4. repeating step S2, S3 once;

S5. supernatant liquor is transferred to another new centrifuge tube, add 2/3 volume isopropanol, vibration mixing, leaves standstill 10min on ice, at 4 DEG C, and the centrifugal 10min of 12000rpm;

S6. abandon supernatant, precipitate 2 ~ 3 times by 75% washing with alcohol;

The centrifugal 5min collecting precipitation of S7.10000rpm, thoroughly removes 75% ethanol, slightly after drying with appropriate RNase-free water dissolution RNA ,-75 DEG C save backup.

Learnt from else's experience DNaseI(RNaseFree) process and the capsicum fertile plant of purifying or sterile strain eight grades mix bud total serum IgE samples, and carry out agarose gel electrophoresis detection, 28S and 18S band is clear, sees Fig. 1.

After suitably being diluted by capsicum bud total serum IgE sample TE solution, record A260/A280 ratio and be greater than 2.0 at about 2.0, A260/A230 ratio, show that RNA purity is high with nucleic acid-protein instrument, integrity is good, and concrete outcome is in table 1.

The RNA absorbance of table 1 capsicum fertile plant or sterile strain eight grade mixing buds measures

Then, with the RNA extracted for template, adopt commercial reagent box to complete the synthesis of each tissue site cDNA of capsicum, obtain the cDNA of sterile strain and fertile plant 8 grade buds and root, stem, leaf, open flower, calyx, petal, flower pesticide and gynoecium.

embodiment 2 caMS1the synthesis of gene and order-checking

Eight the grade mixing buds of RNA-Seq method to capsicum fertile plant and sterile strain are utilized to carry out rna expression variance analysis, find that the est sequence of a 983bp is only expressed in fertile plant, with this sequence for probe compare of analysis in NCBI shows, this EST and many male sterile associated protein 2(malesterility2) there is higher similarity (about 75%), these malesterility2 play an important role in pollen development process, infer that this gene may be relevant to pollen development.This gene is increased by electronic cloning, 3 ' RACE and 5 ' RACE, obtains cDNA and splice sequence, called after caMS1, its nucleotide sequence is as shown in SEQIDNO:1.

In order to verify supposition, devise a pair special primer:

Upstream (5 '-3 '): CTATAATCTTTCCTTCCATTCCCTTTG

Downstream (5 '-3 '): AATCACAAGTCCTTCGGAAAGAAAA

With the cDNA of fertile plant capsicum eight grade mixing mixing buds for template, above-mentioned primer is utilized to carry out specific PCR.The reaction system of amplification is: cDNA template 1 μ L, 10 × cDNAPCRbuffer2.5 μ L, dNTPMix(2.5mmol/L) 2 μ L, upstream primer 1 μ L, downstream primer (10 μm of ol/L) 1.0 μ L, taqenzyme 0.25 μ L, ddH ₂o17.25 μ L, cumulative volume 25.0 μ L.Pcr amplification program is: 94 DEG C of denaturation 3min; 94 DEG C of sex change 50s, 50 ~ 54 DEG C annealing 40s, 72 DEG C extend 2min, 35 circulation after again 72 DEG C extend 5min.Reclaim PCR primer, and cloning and sequencing.

Above-mentioned PCR obtains the band of a treaty 2000bp, reclaims this band, and cloning and sequencing, obtain caMS1cDNA sequence total length 1919bp, comprise the maximum open reading frame of a 1758bp, as shown in Figure 2, its coding aminoacid sequence as shown in SEQIDNO:2.By analysis, find that this sequence is correct, and demonstrate splicing result.

embodiment 3RT-PCR and qRT-PCR analyzes caMS1the spatial and temporal expression characteristic of gene

rT-PCR analyzes:

The sterile strain obtained with embodiment 1 respectively and fertile plant 8 grade buds and root, stem, leaf, open flower, calyx, petal, flower pesticide and gynoecium one chain cDNA for template, with actinfor internal reference carries out semi-quantitative RT-PCR analysis, detect the expression of each pollen development genes involved.

CaMS1 gene specific primer:

Upstream: 5 '-AAATCTTCCCTCAGGAGTCAATCAG-3 ';

Downstream: 5 '-AATCACAAGTCCTTCGGAAAGAAAA-3 '

And actinspecial primer:

Upstream: 5 '-CCTCTTCACTCTCTGCTCTCTCCTCA-3 ';

Downstream: 5 '-GTCATTTTCTCTCTATTTGCCTTGGG-3 '

The reaction system of RT-PCR is: cDNA template 1.0 μ L, 10 × PCRbuffer2.0 μ L, upstream and downstream special primer (10 μMs) each 0.6 μ L, dNTPs (2.5mM) 1.6 μ L, taqdNApolymerase(5U/ μ L) 0.2 μ L, supplies ddH ₂o to 20 μ L.RT-PCR program thereby is: 94 DEG C of denaturation 3min; 94 DEG C of sex change 30s, 50 ~ 55 DEG C annealing 30s, 72 DEG C extend 60s, 26 ~ 30 circulation after again 72 DEG C extend 5min, at 10 DEG C preserve.PCR primer 1.5% agarose gel electrophoresis detects, and takes pictures.

With actinfor reference gene, use caMS1gene specific primer pair caMS1carried out the RT-PCR expression analysis at flower bud development different times and different tissues position, result shows, caMS1different expression levels is had at the different development stage of bud.The expression of this gene is almost can not see in 1st ~ 4 grades of buds of fertile plant.Suddenly express when the 5th grade of bud strong, and can't detect its expression at the bud of the 6th grade of bud and later grade thereof.In the buds at different levels of sterile strain, all can't detect its expression, (Fig. 3 a).This explanation caMS1express in the mid-term of capsicum flower bud development, infer caMS1the growth of tapetum may be take part in.In addition, find after the expression of Different Organs is analyzed, caMS1only express in the flower pesticide of fertile plant, and at other position, as root, stem, leaf, calyx, petal, gynoecium are not all expressed.This genes of position of all detections in sterile strain does not express that (Fig. 4 a), also illustrates that this gene may be relevant with anther development.

analyze:

In order to verify the result of RT-PCR further, this research has carried out qRT-PCR analysis to this gene.First with the cDNA of capsicum bud at different levels for template carries out qRT-PCR, and then with the cDNA of the root of capsicum, stem, leaf, open flower, calyx, petal, flower pesticide, gynoecium for template, carry out qRT-PCR reaction, 3 repetitions are established in each reaction.

CaMS1 gene specific primer:

Upstream: 5 '-ATGATGGCGAAAGAGGTTGACG-3 ';

Downstream: 5 '-CCATTTACATACGCTGTGGATACTTG-3 '

And actinspecial primer:

Upstream: 5 '-AATCAATCCCTCCACCTCTTCACTC-3 ';

Downstream: 5 '-CATCACCAGCAAATCCAGCCTT-3 '

QRT-PCR reaction system is as follows: cDNA template 1.0 μ L, 10 μ L2 × SYBRGreenIMIX, 15 μm of olL ^-1forward primer 0.2 μ L, 15 μm of olL ^-1reverse primer 0.2 μ L, adds water to cumulative volume and reaches 20 μ L.PCR program: 94 DEG C of 2min; 94 DEG C of 10s, 56 DEG C of 20s, 72 DEG C of 35s, 40 circulations; Product melt curve analysis analysis, and verify with agarose gel electrophoresis.Data collected by the IQ5 software of BIO-RAD company.2 of the relative expression quantity CT value of each gene between differing materials ^{-△ △ CT}method process obtains (Livaketal, 2001).

Result shows, in fertile plant, caMS1expression first in the bud of the 4th grade, faint expression detected, reach maximum value at the 5th grade of expression amount, and also only have faint expression in the 6th grade of bud.Fertile line the 7th, 8 grades, and 1st ~ 8 grades of sterile strain all can't detect the expression (Fig. 3 b) of this gene.This result is similar with RT-PCR result above, demonstrates result above.Right caMS1when expression in Different Organs is analyzed, qRT-PCR demonstrates the result (Fig. 4 b) of RT-PCR above equally, namely caMS1only expressing in the flower pesticide of fertile plant, is the special gene of a flower pesticide.

embodiment 4 builds caMS1the RNAi interference carrier of gene

1. caMS1the clone of the positive and negative adopted fragment of gene RNAi

According in embodiment 2 caMS1the sequencing result of full length gene, by Blast comparison, chooses one section of sequences Design 2 pairs of special primer containing intron in the middle of gene respectively, introduces at the two ends of sense fragment swai and ncoi restriction enzyme cuts site, introduces at the two ends of antisense fragments smai and bamhI restriction enzyme cuts site, and with capsicum eight grade mixing mixing bud cDNA for template, pcr amplification goes out justice, inverted defined gene fragment, and its object clip size is about 200bp.

The primer of positive and negative adopted sequence is:

UP-SP4-1：5′-CGG ATTTAAATAGTCAGTGGAGCAAGCAA-3′，

UP-SP4-2：5′-CGC GGATCCAGTCAGTGGAGCAAGCAA-3′；

DW-SP4-1：5′-CATG CCATGGTTAGCCCTGGAATGTGGA-3′，

DW-SP4-2：5′-TCC CCCGGGTTAGCCCTGGAATGTGGA-3′。

(1) amplification system:

(2) reaction conditions

94 DEG C, 5min; 94 DEG C, 1min, 54 DEG C, 1min, 72 DEG C, 2min, 30 circulations; 72 DEG C, 10min; 16 DEG C of preservations.

PCR primer reclaims test kit with DNA gel and reclaims.Getting 20 μ LPCR recovery products send order-checking company to check order.

Above-mentioned caMS1the pcr amplification of object fragment, amplifies the positive and negative adopted fragment (Fig. 5) that size is about 210bp, is connected on pMD19-T carrier, and sequencing result is be 215bp, 212bp(Fig. 6, Fig. 7 respectively), analyze through Blast and show amplified fragments correct (Fig. 8).

2. the insertion of sense fragment

(1) double digestion of pFGC5941 empty plasmid and sense fragment

Adopt the pFGC5941 empty plasmid in alkaline lysis method of extracting intestinal bacteria, then all use respectively swai and ncothe sense fragment that I double digestion pFGC5941 empty plasmid and recovery obtain, 37 DEG C of enzymes cut through night.Double digestion system is as follows: 10 × buffer(K) 4 μ L, BSA4 μ L, plasmid DNA 10 μ L, SwaI1 μ L, NcoI1 μ L, adds ddH ₂o to 40 μ L, mixing.

(2) connection of pFGC5941 empty plasmid and sense fragment

First get double digestion product and reclaim large fragment and sense fragment, then use the T of Takara ₄dNA ligase 16 DEG C of connections are spent the night.Ligation system is as follows: T ₄buffer2.5 μ L, sense fragment 10 μ L, plasmid vector 2 μ L, T ₄dNA ligase 1 μ L, adds ddH ₂o to 25 μ L, mixing.

(3) product conversion intestinal bacteria are connected

Preparation dH5 α competent cells, will connect product conversion dH5 α competence, the dull and stereotyped 37 DEG C of overnight incubation of Km100mg/LLB.Within second day, picking list bacterium colony spends the night with shaking bacterium containing Km100mg/LLB liquid 37 DEG C, and bacterium liquid extracts plasmid.Plasmid is used ncoi and swai double digestion detects.Insert the carrier called after pFGC5941-A of sense fragment.

3. the insertion of antisense fragments

(1) double digestion of pFGC5941-A plasmid and antisense fragments

With the pFGC5941-A plasmid in alkaline lysis method of extracting intestinal bacteria, then all use respectively bamhI and smathe antisense fragments of I double digestion pFGC5941-A plasmid and recovery, 30 DEG C of enzymes cut through night.Double digestion system is as follows: 10 × buffer(T) 2 μ L, BSA4 μ L, plasmid DNA 10 μ L, bamhI1 μ L, smai1 μ L, adds ddH ₂o to 40 μ L, mixing.

(2) connection of pFGC5941-A plasmid and antisense fragments

First get double digestion product and reclaim large fragment and antisense fragments, then use the T of Takara ₄dNA ligase 16 DEG C of connections are spent the night.Ligation system is with the ligation system of aforementioned pFGC5941 empty plasmid and sense fragment.

(3) product conversion intestinal bacteria are connected

The connection product conversion intestinal bacteria of pFGC5941-A plasmid and antisense fragments and the extraction of plasmid are with reference to the colibacillary method of aforementioned connection product conversion.Same plasmid is used respectively bamhI, smai and swai, ncoi two overlaps double digestion and detects, and result cuts out the object band (Fig. 9) that expection size is 200bp, and illustration purpose gene is successfully inserted in plant expression vector pFGC5941, by its called after pFGC5941-B.

4. recombinant plasmid transformed Agrobacterium

(1) transformation Agrobacterium competent cell

1) get fresh preparation or-70 DEG C of 200 μ L Agrobacterium competent cells preserved, put on ice, gently by cell suspension after thawing completely;

2) 5 μ L are added caMS130min is placed on ice after the mixing of pFGC5941-B plasmid;

3) centrifuge tube is put into liquid nitrogen quick-frozen 5min, heat shock 1min in 37 DEG C of water-baths, will go on ice rapidly, ice bath 2min;

4) 1mLYEP liquid nutrient medium is added, 28 DEG C of shaking culture 1h;

5) the centrifugal 5min of 4000rpm, stays 100 μ LYEP nutrient solution suspension cells;

6) bacterium liquid is coated on the YEP flat board containing Km100mg/L+Str100mg/L, cultivate 24-48h for 28 DEG C.

(2) recon qualification

The YEP liquid 28 DEG C of the Agrobacterium bacterium colony Km100mg/L+Str100mg/L that picking grows shakes bacterium 24h, extract plasmid, PCR detection is carried out again with plasmid, object fragment (Figure 10) can be obtained, illustrate that plant expression vector successfully proceeds in Agrobacterium, expression vector establishment success, for later genetic transformation creates condition.

the pepper transformation of the PFGC5941-CaMS1 that embodiment 5 is agriculture bacillus mediated

(1) acquisition of capsicum aseptic seedling

First full, that grain is large pepper seed aseptic water washing 2-3 time is selected, remove impurity, then use 75% alcohol disinfecting 1min, aseptic water washing 2 ~ 3 times, then sterilize during 12min(with 2% chlorine bleach liquor and constantly shake), with aseptic washing 3 times, remove sterilized water, then soak 10min ~ 15min with sterilized water, finally remove sterilized water, suck seed-coat residual moisture with sterilizing filter paper, sow in 1/2MS substratum, be placed in artificial culture room and cultivate.Cultivate under being placed in 25 ~ 28 DEG C of dark conditions, 5 ~ 6d both can observe hypocotyl and emerge, then cultivate under seed taking-up being put into temperature about 27 DEG C, illumination 2000lx14h/d culture condition, can explant be produced when capsicum seedling grows two panels cotyledon and vegetative point is about to expose, approximately need about 14d from planting seed to explant can be prepared.

(2) preculture

Fleming height Bill (Flamingo-bill) outer explant is put into division culture medium, 27 DEG C of dark preculture 2 ~ 3d in artificial culture room.Described Flamingo-bill explant is namely: all cut together with terminal bud and peripheral meristem from petiole base by the side cotyledon of the aseptic seedling of cultivation, only stay side cotyledon.

(3) Agrobacterium is infected and Dual culture

Infect front 4d and take out the Agrobacterium containing expression vector be stored in the Ultralow Temperature Freezer of-80 DEG C, on YEP solid medium, (Str100mg/L, Km100mg/L) draws dull and stereotyped, 28 DEG C of constant temperature culture 24 ~ 48h.Picking list bacterium colony is placed in the YEP liquid nutrient medium of 10mL containing Str100mg/L, Km100mg/L and AS200mg/L, and 200rpm shaken overnight at 28 DEG C, goes in 50mLYEP liquid nutrient medium next day, continues to be cultured to OD ₆₀₀be 0.4 ~ 0.8.

After capsicum explant preculture 2d, with ready Agrobacterium bacterium immersion dye 12min on Bechtop, period shakes frequently, then be put on the newspaper of autoclave sterilization to half-dried, inoculate in same division culture medium, the explant of every bottle graft kind about 15, and under (25 ± 1) DEG C condition constant temperature dark culturing 2 ~ 3d.

(4) micro-organisms

The Flamingo-bill explant of Dual culture 2d is inoculated in micro-organisms base, is placed on 27 DEG C, artificial culture room and cultivates 4 ~ 5d.

(5) screening and culturing

After explant micro-organisms, be inoculated in screening culture medium, every two weeks subcultures once, subculture 2 ~ 3 times.Until only have a little outer planting physical efficiency to grow resistant buds in screening culture medium, and most of explant chlorisis in the screening culture medium containing PPT, till flavescence coking.

This experiment PPT screens, and because PPT is comparatively large to plant poisoning effect, finds the screening pressure of applicable capsicum very important.Divide two groups in experiment and be provided with 11 concentration gradients (table 3) altogether, result shows: when PPT concentration is greater than 3mg/L, and the differentiation of capsicum Flamingo-bill explant indefinite bud is subject to strongly inhibited, and most of brownization is withered; When PPT concentration is less than 1.5mg/L, the good differentiation of capsicum Flamingo-bill explant indefinite bud, but false positive is more, and the screening concentration therefore adopted is 2mg/L, and obtains resistant plant smoothly.

Table 3Flamingo-bill explant is to the sensitivity experiments of PPT

(6) root culture

As Elongation of adventitious bud to the 1.5 ~ 2.5cm broken up, it is cut from base portion, is inoculated in root media and induces it to take root.When root grows to about 1.5cm, quantity reaches after more than 5, about about 30d, uncork lid hardening 2 ~ 3d, then moves to and is equipped with in autoclaved perlite and peat soil mixotrophism alms bowl or flowerpot, finally obtains 48 strain resistance seedlings.30 strains are capsicum 612 kinds, and 18 strains are capsicum 59 kinds.

embodiment 6 caMS1the molecule of Gene interfere plant and phytology are identified

1.T ₀pCR for transfer-gen plant detects

(1) primer sequence

1) basis bargene primer sequence (Zang Ning etc., 2008)

bar-UP：5′-ATGAGCCCAGAACGACGC-3′

bar-DW：5′-TCTCAAATCTCGGTGACG-3′

2) according to CaMV35S promoter primer sequence

S1-UP：5′-GAGGACCTAACAGAACTCG-3′

S2-DW：5′-GTCTTGCGAAGGATAGTGG-3′

(2) pcr amplification reaction system

DNA profiling 1 μ L, 10 × DNAPCRbuffer2.5 μ L, dNTPMix(2.5mmol/L) 2 μ L, upstream primer (10 μm of ol/L) 1 μ L, downstream primer (10 μm of ol/L) 1 μ L, rTaq enzyme 0.25 μ L, ddH ₂o17.25 μ L, cumulative volume 25.0 μ L.

(3) pcr amplification reaction program

94 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C extend 1min, 32 circulations; 72 DEG C extend 5min; 12 DEG C of preservations.

(4) PCR positive rate and conversion data statistical method

Extract the genomic dna of embodiment 5 transformed plant, use respectively bartwo pairs of primer pairs 48 strain of gene and CaMV35S promotor turns caMS1(30 strains are capsicum 612 kinds to the resistance capsicum of gene, 18 strains are capsicum 59 kinds) carry out PCR detection, result has 19 strains to amplify to be about 550bp object band (Figure 11, Figure 12), PCR positive rate is respectively 39.6%, and preliminary proof RNAi carrier proceeds to capsicum.

Choose and turn caMS1gene 3 strain, carries out pcr amplification, the bright band obtained is cut glue and reclaims (CaMV35S promoter primer), be connected on pMD19-T carrier, through conversion, the screening of blue hickie, chooses hickie and carries out shaking bacterium and plasmid extraction.Recon is sent to the order-checking of order-checking company, has measured full gene sequence, with expection identical (Figure 13), be defined as transfer-gen plant.

2.T ₀pollen Activity for transfer-gen plant detects (the in vitro rudiment assay method of agar-agar)

The preparation of substratum: 10% sucrose, 100mg/kg boric acid (HB0 ₃), 5mg/kg Plant hormones regulators,gibberellins, 100mg/kg nitrocalcite (Ca (NO ₃) ₂4H ₂o), the agar of 0.8%.

The collection of pollen: points 3 days collect flower, repeat as 3 times, acquisition time the morning 10 time about.Pollen, for having bloomed but not yet loose powder, is shaken off on template after picking flowers, and is fully mixed by the standard gathering flower.

Concrete operations: by the culture dish of falling people after substratum heat fused, the thin uniform layer that the about 0.5cm that congeals into is thick, is made into solid medium.With dissecting needle, pollen is evenly applied on substratum, builds and put into incubator and cultivate.Cultivate 3 hours under substratum being placed on dark, constant temperature (25 scholar l) DEG C condition, then observe germination rate under an optical microscope.Substratum all establishes 3 repetitions, the visual field of each repeated measures 3 non-overlapping copies, statistics more than 200 pollen granules sprouting situation, using pollen tube length more than l/2 pollen diameter as sprouting standard.

Pollen germination rate=(the pollen granule number sprouted/total pollen granule number) × 100%.

All transgenic peppers that makes discovery from observation all show nourishes and grows and reproductive growth (Figure 14) normally.Relative to wild-type and contrast, caMS1the anther dehiscence of repressed transfer-gen plant is comparatively slow, and pollen tube is shorter than wild-type, and the pollen less (Figure 15) in flower pesticide.Finally choose brighter and much the same 3 strains of brightness of PCR band caMS1rNAi transfer-gen plant carries out isolated culture and detects its Pollen Activity, and result is the pollen germination rate of interference plant is 21.84%, and wild-type is 52.63%(Figure 16).

3, T ₁pCR for transfer-gen plant detects

Extract test kit according to Ezup pillar Plant Genome and extract T ₁for the DNA of transgenic pepper, carry out PCR detection with the primer pair 24 strain capsicum of CaMV35S promotor, result has 7 strain T ₁can amplify for transgenic pepper and be about 550bp object band (Figure 17), segregation ratio is 17:7, and positive rate is 29.2%.

SEQUENCELISTING

<110> Agricultural University Of South China

<120> mono-kind prepares the method for male sterile pepper

<130>

<160>6

<170>PatentInversion3.3

<210>1

<211>1919

<212>DNA

The cDNA sequence of <213> gene C aMS1

<400>1

ctataatctttccttccattccctttgaacttttttctcccttgtttctctttgcatcta60

tggcggctatgggtagtctatgttcttcctcttgtatatcaaaaactgtgatgaaattgt120

ctaagaattggagatggtgccctcccaagaaggtatattgtcaaactagtggtacaaagt180

ctggtaatgtttcttctgttgtaacagagagatcatcggtgattagctcggaaactttag240

gaagtttggttttgagtccaaatgccgaaatcaaagtcaaggatttggtgccttatggtc300

agtcaaggcatgatgatggtataggcattaccaagtttctgagagggaaagcatttctca360

ttactggtgcaactggttttctgggaaaagttctaattgagaagatcttaaggacagcac420

ctgatgtgaacaaaatattcatcttgatcaaggcaaagaacaaagaagttgctatgcaga480

gattgaagaatgaaatcctcaatgctgatatattcaactgcctcaaacaagttcatggga540

aatcctatcagactttcatgttgagcaagttggtacctctgttaggaaatgtttgtgaag600

ctaaccttggaattgatgaagacacagccaacatgatggcgaaagaggttgacgtaattg660

taaattctgctgcgaataccactttcgatgaaaggtacgatatttcacttgatataaata720

ctgaaggacctagccgccttatgaactttgcaaaacaatgtcgcaaccttaaactctttc780

ttcaagtatccacagcgtatgtaaatggacagcgacaaggtagaattatggaaaaggcat840

tcggcattggagacagtatagcaagggaaaatcttccctcaggagtcaatcagagctcct900

ccccctctttgaatgttgaagatgagataaagttagttttggagtccaaacaaggtttag960

aagataattcagtggctcagaaaatgaaagagattggtttaaggagagctaacaaatttg1020

gatggcaagacacttacgtattcacaaaggcaatgggagagatgatgatagacagcatga1080

gaggtgatattccggtagtaattattcgaccaagtgttattgagagcacctacaaggaac1140

catttcctggatggatggaagggagcaggatgatggatccaatcatcttgtactatggta1200

aaggacagctcacagggtttctcgtagaccctaatggagttcttgatgtggttccagctg1260

acatggttgtgaatgcaaccttggcagccatggcaaaacatgggacagaaggaaaaccag1320

gaagtagcagtgtttaccaggttgcttcatctgctgtaaatccattagtcttcaaggacc1380

tggccagaatgctatttgagcacttcaatcgttcaccctatattgattccaaaggaagac1440

caattcatgttccaaaaatgtcgctgctcagatccatggaggacttatcatcccatctat1500

ggcgagacgccattaacagaagtggcctaacagatttgacagatccaaatgggaagttgt1560

ccaggaaacttgagaatatctgtaggaagtcagtggagcaagcaaagtaccttgctaata1620

tttatgaaccgtacactttttatggaggaagatttgacaacagcaatacccagaggttga1680

tggaatgcatgtctaaagaagaaagatggcaatttggatttgatgtagaaagcatagatt1740

ggaaagattacatctctaatgtccacattccagggctaaggaagcatgtgatgaaaggaa1800

gaggatcatgcagttaatgccagtctgtcctttatgtttctgcatcttatcttaactgta1860

atacaagtatacgaccgctggttttatttatactttttctttccgaaggacttgtgatt1919

<210>2

<211>1758

<212>DNA

The open reading frame sequence of <213> gene C aMS1

<400>2

atggcggctatgggtagtctatgttcttcctcttgtatatcaaaaactgtgatgaaattg60

tctaagaattggagatggtgccctcccaagaaggtatattgtcaaactagtggtacaaag120

tctggtaatgtttcttctgttgtaacagagagatcatcggtgattagctcggaaacttta180

ggaagtttggttttgagtccaaatgccgaaatcaaagtcaaggatttggtgccttatggt240

cagtcaaggcatgatgatggtataggcattaccaagtttctgagagggaaagcatttctc300

attactggtgcaactggttttctgggaaaagttctaattgagaagatcttaaggacagca360

cctgatgtgaacaaaatattcatcttgatcaaggcaaagaacaaagaagttgctatgcag420

agattgaagaatgaaatcctcaatgctgatatattcaactgcctcaaacaagttcatggg480

aaatcctatcagactttcatgttgagcaagttggtacctctgttaggaaatgtttgtgaa540

gctaaccttggaattgatgaagacacagccaacatgatggcgaaagaggttgacgtaatt600

gtaaattctgctgcgaataccactttcgatgaaaggtacgatatttcacttgatataaat660

actgaaggacctagccgccttatgaactttgcaaaacaatgtcgcaaccttaaactcttt720

cttcaagtatccacagcgtatgtaaatggacagcgacaaggtagaattatggaaaaggca780

ttcggcattggagacagtatagcaagggaaaatcttccctcaggagtcaatcagagctcc840

tccccctctttgaatgttgaagatgagataaagttagttttggagtccaaacaaggttta900

gaagataattcagtggctcagaaaatgaaagagattggtttaaggagagctaacaaattt960

ggatggcaagacacttacgtattcacaaaggcaatgggagagatgatgatagacagcatg1020

agaggtgatattccggtagtaattattcgaccaagtgttattgagagcacctacaaggaa1080

ccatttcctggatggatggaagggagcaggatgatggatccaatcatcttgtactatggt1140

aaaggacagctcacagggtttctcgtagaccctaatggagttcttgatgtggttccagct1200

gacatggttgtgaatgcaaccttggcagccatggcaaaacatgggacagaaggaaaacca1260

ggaagtagcagtgtttaccaggttgcttcatctgctgtaaatccattagtcttcaaggac1320

ctggccagaatgctatttgagcacttcaatcgttcaccctatattgattccaaaggaaga1380

ccaattcatgttccaaaaatgtcgctgctcagatccatggaggacttatcatcccatcta1440

tggcgagacgccattaacagaagtggcctaacagatttgacagatccaaatgggaagttg1500

tccaggaaacttgagaatatctgtaggaagtcagtggagcaagcaaagtaccttgctaat1560

atttatgaaccgtacactttttatggaggaagatttgacaacagcaatacccagaggttg1620

atggaatgcatgtctaaagaagaaagatggcaatttggatttgatgtagaaagcatagat1680

tggaaagattacatctctaatgtccacattccagggctaaggaagcatgtgatgaaagga1740

agaggatcatgcagttaa1758

<210>3

<211>29

<212>DNA

<213>UP-SP4-1

<400>3

cggatttaaatagtcagtggagcaagcaa29

<210>4

<211>27

<212>DNA

<213>UP-SP4-2

<400>4

cgcggatccagtcagtggagcaagcaa27

<210>5

<211>28

<212>DNA

<213>DW-SP4-1

<400>5

catgccatggttagccctggaatgtgga28

<210>6

<211>27

<212>DNA

<213>DW-SP4-2

<400>6

tcccccgggttagccctggaatgtgga27

Claims (10)

1. prepare a method for male sterile pepper, it is characterized in that, comprise the steps:

S1. build containing, for example shown in SEQIDNO:1 ~ 2 caMS1the recombinant RNA i interference carrier of gene;

S2. the recombinant RNA i interference carrier transformation Agrobacterium obtained by S1, obtains recombinational agrobacterium;

S3. use recombinational agrobacterium transfection capsicum explant, obtain transformed plant through cultivating, screening obtains male sterile pepper.

2. prepare the method for male sterile pepper according to claim 1, it is characterized in that, described in S1, the construction process of RNAi interference carrier is:

S11. utilize sense primer shown in SEQIDNO:3 ~ 4 to increase to obtain caMS1the sense fragment of gene, utilizes antisense primer shown in SEQIDNO:5 ~ 6 to increase and obtains caMS1the antisense fragments of gene;

S12. sense fragment and antisense fragments are successively connected to same RNAi interference carrier, obtained caMS1the recombinant RNA i interference carrier of gene.

3. want according to right the method preparing male sterile pepper described in 2, it is characterized in that, described RNAi interference carrier is pFGC5941 carrier.

4. prepare the method for male sterile pepper according to claim 3, it is characterized in that, introduce at the two ends of sense fragment swai and ncoi restriction enzyme cuts site, introduces at the two ends of antisense fragments smai and bamhI restriction enzyme cuts site, cuts ligation sense fragment and antisense fragments are successively connected in pFGC5941 carrier by enzyme.

5. prepare the method for male sterile pepper according to claim 1, it is characterized in that, described in S3, capsicum explant is: all cut together with terminal bud and peripheral meristem from petiole base by the side cotyledon of the aseptic seedling of cultivation, only stay side cotyledon.

6. prepare the method for male sterile pepper according to claim 1, it is characterized in that, the OD described in S3 during recombinational agrobacterium transfection Agrobacterium capsicum explant ₆₀₀be 0.4 ~ 0.8, transfection time is 12min.

7. prepare the method for male sterile pepper according to claim 1, it is characterized in that, cultivate described in S3 and comprise the steps:

S31. explant and bacterium liquid shake up and are seeded in division culture medium, constant temperature dark culturing 2 ~ 3d under 25 ± 1 DEG C of conditions;

S32. micro-organisms: be inoculated in 27 DEG C of cultivation 4 ~ 5d in micro-organisms base;

S33. screening and culturing: be inoculated in PPT screening culture medium, every two weeks subcultures once, subculture 2 ~ 3 times; Until only have a little outer planting physical efficiency to grow resistant buds in screening culture medium, and most of explant chlorisis in the screening culture medium containing PPT, till flavescence coking;

S34. root culture: as Elongation of adventitious bud to the 1.5 ~ 2.5cm broken up, it cut from base portion, be inoculated in root media and induce it to take root, then hardening 2 ~ 3d, transplant and obtain male sterile pepper.

8. prepare the method for male sterile pepper according to claim 7, it is characterized in that, in S33, the PPT concentration of screening culture medium is 2mg/L.

9. prepare the method for male sterile pepper according to claim 5, it is characterized in that, before bacterium immersion dye, described capsicum explant is put into described division culture medium, in 27 DEG C of dark preculture 2 ~ 3d.

10. prepare the method for male sterile pepper according to claim 5, it is characterized in that, the method obtaining capsicum aseptic seedling is: by pepper seed after 75% alcohol, 2% hypochlorite disinfectant, soak with sterilized water, remove sterilized water, suck the moisture of seed-coat, 5 ~ 6d is cultivated under 25 ~ 28 DEG C of dark conditions, then seed taken out and be put into 27 DEG C, cultivate under illumination 2000lx14h/d culture condition, when capsicum seedling grows two panels cotyledon and vegetative point is about to expose, obtain capsicum aseptic seedling.