CN110438150A - A kind of method and application for formulating rapeseed male sterility new germ plasm by gene editing - Google Patents
A kind of method and application for formulating rapeseed male sterility new germ plasm by gene editing Download PDFInfo
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Abstract
The present invention provides a kind of methods and applications that male sterile line is formulated by gene editing technology.This method carries out gene editing knockout by the conserved domain to MS5 gene, so that MS5 gene function is lost or generates gain-of-function mutation, to form recessive or dominant genic male sterility system.Male sterile line prepared by the present invention is compared with orthodox material, other than fertility difference, without other visible character mutations；If being applied to heterosis utilization or population improvement, breeding efficiency will be greatlyd improve, cost of labor is reduced, has great importance in agricultural production.
Description
Technical field
The invention belongs to field of plant genetic project technology, in particular to utilization CRISPR/Cas9 system to wild cabbage
Type rape MS5 gene is edited, and then obtains different type genie male sterile line.
Background technique
Grease is one of three categories main nutrients for the survival of mankind, and maintenance edible oil supply security is national security
One of the core of strategy.The popularization of cenospecies and the utilization of hybrid vigour are one of the effective way for improving yield of rape (Fu Ting
Status and prospect Anhui the agronomy notification of the production of china rape and breed improvement, 2000,6 (1): 2-8).And it is safe and effective
Pollination control system be cross rape production important prerequisite.
Currently, control system of commonly pollinating in rape mainly has: nuclear male sterility, cytoplasmic male sterility, from
Hand over not affine, the transgenosis Genetic Sterility system of artificially creating and chemical emasculation etc..Wherein, dominant genic male sterility system
Pollen abortion is shown to stablize thoroughly, without cytoplasm negative effect and cytoplasm abundance, sterile character are easy transformation, easy to accomplish
The advantages that " three systems " production of hybrid seeds process of cenospecies, thus obtained the favor of breeder and the product of domestic and international more R&D institutions
Pole research.Yi3A is primary type nuclear male sterility type (Japanese plum woods, the Zhou Xirong dominant genic male sterile of China's discovery, report
The heredity of rape and utilization crop investigations, 1990,4:27-32), once successfully selected in life based on the infertility mutant material
The kind of popularization is commercialized in production.Genetic analysis shows, the fertility of dominant genic male sterile system is multiple etc. by three of same site
Position gene control, respectively MS5a(restoring gene), MS5b(sterile gene) and MS5c(normal fertile or temporary maintainer line gene), and
Aobvious recessive relationship between three is MS5a> MS5b> MS5c(Lu W, Liu J, Xin Q, Wan L, Hong D, Yang G.A
Triallelic genetic male sterility locus in Brassica napus:an integrative
strategy for its physical mapping and possible local chromosome evolution
Around it.Annals of botdny, 2013,111 (2): 305-315).Therefore, in MS5bIn homozygotic state or base
Because type is MS5bMS5cHeterozygous state when, single plant shows as male sterility；And MS5aIn the presence of genotype or MS5cIt is in
When homozygous, single plant then shows as fertile.By map based cloning and transgenosis functional verification, it is successfully separated acquisition restoring gene
MS5a(BnaA08g25920D) (Xin, Q., Shen, Y., Li, X., Lu, W., Wang, X., Han, X., Dong, F., Wan, L.,
Yang, G., and Hong, D. (2016) .MS5 mediates early meiotic progression and its
natural variants may have applications for hybrid production in Brassica
Napus.The Plant Cell 28:1263-1278.).
We have obtained MS5 in early-stage studybThe sequence of section, but its cause Abnormal Circumstances In Meiosis function section and
Molecule mechanism is not parsed always, therefore, parses MS5bThe function of sterile gene, by editing MS5 different structure territory, according to
The hybrid for needing to formulate recessive cytoblast sterile and dominant genic male sterility for Brassica Crops such as cabbage type rape, Chinese cabbage and wild cabbages is excellent
Snobbish use and population improvement will greatly improve sterile line breeding efficiency, provide innovation efficient new way for crop breeding.Through
The domestic and international prior art is retrieved, not yet finds the report for utilizing CRISPR/Cas9 technology creation Brassica napus male sterile line.
Summary of the invention
In view of the shortcomings of the prior art, the present invention causes the function section and molecule of Abnormal Circumstances In Meiosis by parsing
Mechanism is joined to create the CRISPR/Cas9 system of stability and high efficiency in cabbage type rape using MS5 gene and its albumen
The characteristics of with regulation cabbage type rape meiosis, generates new rapeseed plant recessive or dominant by being mutated the albumen specific domain
Genie male sterile line has a very important significance in heterosis utilization and population improvement.
The purpose of the present invention is what is be achieved through the following technical solutions:
In a first aspect, this method uses the present invention provides a kind of method for creating of cabbage type rape male sterility strain
CRISPR/Cas9 or other gene editing systems, fixed point knock out MS5 gene or MS5 gene M S5superfamily structural domain,
So that afunction occurs for the corresponding albumen of the gene.
It is further preferred that the method for creating of cabbage type rape male sterility strain as described above, the specific step of this method
Suddenly include:
Step 1 is chosen positioned at the target spot S1 of coiled coil structural domain on MS5 gene and positioned at MS5 superfamily
The target spot S2 of structural domain, the sequence of the S1 are as follows: 5 '-AAGCGAGAGGCTCGCCGTA-3 ' (SEQ ID NO:1), the S2's
Sequence are as follows: 5 '-GTCCTTGGCATCGAAGAAC-3 ' (SEQ ID NO:2), and primer needed for synthesizing carrier construction；
Step 2 constructs double target spot CRISPR/Cas9 gene editing carriers；
Step 3, extracting plasmid, convert Agrobacterium；
Carrier is converted rape hypocotyls by way of mediated by agriculture bacillus by step 4, and MS5 is screened in transgenic progeny
The plant of gene mutation, to obtain cabbage type rape male sterility strain.
It is further preferred that the method for creating of cabbage type rape male sterility strain as described above, primer described in step 1
Sequence it is as shown in the table:
It is further preferred that the method for creating of cabbage type rape male sterility strain as described above, double target spots in step 2
The construction step of CRISPR/Cas9 gene editing carrier are as follows:
Step 2.1 is with sequence shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6
Primer carries out PCR amplification using high fidelity enzyme, cuts glue after agarose electrophoresis and purify back using pCBC-DT1T2 plasmid as template
Receive PCR product；
Step 2.2, the PCR product for using T4 DNA ligase to recycle step 2.1 simultaneously with BsaI digestion and CRISPR/
Cas9 carrier is assembled into whole carrier；
Whole carrier is converted competent escherichia coli cell, resistance screening, bacterium colony PCR identification, generation sequencing by step 2.3
Determine the accuracy of carrier sequence.
The Agrobacterium it is further preferred that method for creating of cabbage type rape male sterility strain as described above, in step 3
For Agrobacterium GV3101.
It is further preferred that the method for creating of cabbage type rape male sterility strain as described above, in transgenosis in step 4
When screening the plant of MS5 gene mutation in offspring, first with primer S-MS5-L/S-MS5-R to comprising two target sites
Region is expanded, and then the primer SS-MS5-1 and SS-MS5-2 inside amplified fragments is recycled to examine by generation sequencing approach
The sequence for surveying two target sites, determines whether target site is edited；The sequence of the primer S-MS5-L such as SEQ ID
Shown in NO:7, the sequence of the primer S-MS5-R is as shown in SEQ ID NO:8, and the sequence of the primer SS-MS5-1 is such as
Shown in SEQ ID NO:9, the sequence of the primer SS-MS5-2 is as shown in SEQ ID NO:10.
It should be noted that the method for creating of cabbage type rape male sterility strain of the present invention, Wild cabbage type therein
It is MS5 that rape material, which is selected from genotype,cMS5cWestar and/or genotype be MS5aMS5aY127.
Second aspect, the results show of the invention, after CRISPR/Cas9 gene editing, MS5 gene occurs prominent
The heterozygosis single plant of gene delection homozygosis single plant and MS5 superfamily the structural domain missing of change, shows as male not
It educates.Therefore, the present invention provides the applications of fertility-related gene MS5 a kind of, i.e., MS5 gene is in initiative male sterile line of rape
Application, the described application is: being knocked out using CRISPR/Cas9 system and changes MS5 gene, so that MS5 albumen lacks in rape
It loses or truncates, and then obtain male sterile line of rape.The MS5 gene is MS5aOr MS5c。
Compared with prior art, the beneficial effects of the present invention are embodied in terms of following three:
(1) relative to traditional hereditary transfer method, the initiative of provided by the present invention dominant or recessive male sterile line
Method can be quickly obtained the male sterile line that can be used for breeding；
(2) present invention can formulate dominant genic male sterility system for population improvement according to demand, for three systems or
Two be the dominant and genic male sterile system of the production of hybrid seeds, clear mechanism, sterility stabilization；
(3) relative to conventional breeding, the present invention can the preferably original excellent economical character of reserved materials.
Detailed description of the invention
Fig. 1 is sgRNA Expression element group；Wherein, promoter U6-26p, U6-29p；GRNA-Sc is gRNA skeleton；Eventually
Only son is U6-26t.
Fig. 2 is gene editing single plant target site genotype and sterile single plant phenotype: where A MS5aGene editing list
Strain allelotype；B is MS5cGene editing single plant allelotype；C is acceptor material Westar, sterile single plant MS5a-CR-
5, MS5c- CR-36 and sterile line Rs1046A fertility phenotype are shown.From left to right vertical setting of types is followed successively by inflorescence in C, and Pistil And Stamen is male
Chromosome morphology at the end of stamen and meiosis leptotene and meiosis.
Specific embodiment
Further illustrate the content of present invention below with reference to the drawings and specific embodiments, but the present invention can with it is a variety of not
It is same as other methods described herein to implement, therefore, the scope of protection of the invention is subject to claims, is not embodied
The limitation of example.Test method as described below is the conventional method and technology of the art, agents useful for same unless otherwise specified
It is obtained by commercial sources with consumptive material.
Embodiment 1: the acquisition of transgene rape positive plant
(1) building of the selection of gRNA target spot and transgene carrier
Select MS5 gene conserved domain coiled coil structural domain and MS5 superfamily conservative domain section
Carry out the design (sequence as shown in SEQ ID NO:1 and SEQ ID NO:2) of gRNA target spot.Such as according to target sequence design primer
Shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.With high fidelity enzyme Phanta Max
Super-Fidelity DNA Polymerase (promise is only praised, Nanjing) carries out four as template using the pCBC-DT1T2 for diluting 100 times
Primer PCR amplification.- BsF/-BsR is normal primer concentration；- F0/-R0 dilutes 20 times.Establish following digestion-linked system:
5 μ l of system after connection is taken to convert competent escherichia coli cell DH5 α, the LB plate screening of kalamycin resistance is anti-
Property bacterium colony, send one Hui Yuan biotechnology company of day be sequenced determine sequence accuracy.Accurately clone, extraction plasmid convert agriculture for sequencing
Bacillus competent cell GV3101 is used for Agrobacterium-mediated genetic transformation.
(2) rape converts
Seed sterilization, germination.A. it takes Westar and Y127 seed into 50ml sterile centrifugation tube, 75% alcohol, lid is added
It is spun upside down after good, impregnates seed 1min.B. alcohol is sopped up with pipettor, adds suitable 0.1% mercuric chloride, lid covers down
Overturning sterilizes 10-15min.C. thimerosal is sopped up, suitable aseptic water washing is added 3-5 times, lid lid is spun upside down every time,
It keeps being gnotobasis in centrifuge tube.D. M0 culture medium is multicast to the aseptic nipper seed that will sterilize, every ware 20-25.It will culture
Ware is put into sterile culture box, is set and is cultivated 6 days at 24 DEG C of half-light.
Liquid LB training objective bacterium is used after sowing 5 days.20ml vial: 5ml resistance LB+20 μ l target Agrobacterium, in
About 14-16h is cultivated in 28 DEG C of 180-220rpm shaking tables.Get out co-culture medium M1, acetyl is added in about 50 DEG C of culture medium whens
Syringone AS (100 μM of final concentration), infecting buffer, also to add AS (final concentration 100uM) spare.Spectrophotometric measures bacterium OD value,
0.4 or so preferably.Prepare bacterium night, inhale the cultured bacterium solution of 2ml into sterile centrifugation tube, 3000rpm is centrifuged 3min, abandons supernatant；
2ml is added to infect buffer suspension, 3000rpm is centrifuged 3min, abandons supernatant；2ml infects buffer suspension again, and it is spare to put 4 DEG C of refrigerators.
18ml is added in sterilized petri dishes and infects buffer, with sterile scissors clip seedling hypocotyl into plate, each explant is long
Degree is 0.8-1.0cm.A knife is vertically cut as far as possible when cutting explant, every general 150-200 explant of ware.It is outer what is cut
The 2ml bacterium solution prepared just now is poured into implant ware, at this moment liquid volume is 20ml in ware, disseminates 10-15min, and the compartment time shakes
It shakes once, 4~5 times.Start to sop up bacterium solution with pipettor when infecting 8min, clamps explant to sterile with aseptic nipper
A moment is placed on filter paper, it is therefore an objective to siphon away bacterium solution extra on explant.Then explant returns again in M1 culture medium, explant
The lower 24 DEG C of placements of half-light.Co-culture 24-36h after explant is gone in M2, under light normally culture (24 DEG C daytime 16h, at night
8h).28th day, explant is gone in M3, every 2-3 weeks subculture is primary, until there is green bud.There to be the green bud of complete growth point
M4 is transferred to take root.In above-mentioned culture medium, M1 medium component: MS culture medium+30g/L sucrose+mannitol 16~20g/L+2,4-D
0.8~1.2mg/L+Kinetin, 0.25~0.35mg/L+ agar powder 6g/L, pH value are 5.8~6.0；M2 medium component is
MS culture medium+sucrose 28~32g/L+ mannitol 16~20g/L+2,4-D0.8~1.2mg/L+Kinetin 0.25~
0.35mg/L+ agar powder 6g/L, pH value are 5.8~6.0；Temperature is down to Ticarcillin/Clavulanate Acid bacteriostatic agent is added after 50 DEG C after high-temperature sterilization
(TMT) final concentration of 300mg/L, kanamycins (Kan) final concentration 50mg/L, AgNO3Final concentration of 5mg/L；M3 culture medium
Ingredient: the MS culture medium+trans--Zeatin of glucose 8.0~12g/L+, 0.23~0.27g/L+MES of xylose, 0.5~0.7g/L+
1.8~2.2mg/L+IAA, 0.08~0.12mg/L+, 5.8~6.2g/L of agar powder, pH value are 5.8~6.0；It is warm after high-temperature sterilization
Ticarcillin/Clavulanate Acid bacteriostatic agent (TMT) final concentration of 300mg/L, kanamycins (Kan) final concentration 50mg/L are added after spending low 50 DEG C,
AgNO3Final concentration of 5mg/L；M4 medium component: MS culture medium+sucrose 8~12g/L+, 8~12g/L of agar powder, pH value are
5.8~6.0.
Embodiment 2: the extraction and detection of transgene rape plant DNA
(1) extraction of transgene rape plant DNA
T in embodiment 1 is extracted using CTAB method0For transgene rape leaf DNA.Specifically includes the following steps: taking few
Clean steel ball and 400 μ l 2% is added into the 2ml centrifuge tube for finishing writing number in the rape leaf (diameter about 1~2cm) of amount
CTAB solution grinds (28times/s, 30s) with sample grinding machine (Tissulyser II, QIAGEN).Sample after grinding is put into
Water-bath 60min in 58 DEG C of thermostat water bath shakes once every 10min, places and is cooled to room temperature after the completion of water-bath.In draught cupboard
It is middle that 24: 1 isometric (chloroform and isoamyl alcohol are prepared by 24: 1 volume ratio) are added, 10-15min is jiggled, then set
12000r/min is centrifuged 10min in centrifuge.200 μ l supernatants are drawn into the 1.5ml centrifuge tube of identical number with pipettor
Isometric frost (- 20 DEG C) dehydrated alcohol is added in (the NaAc solution for being previously added the 3mol/L of 1/10 volume of supernatant), quiet
Only 8000r/min pours out alcohol after being centrifuged 2min again in centrifuge after 30min.Isometric 76% dehydrated alcohol washing is added
DNA pours out alcohol, and DNA is precipitated as centrifuge tube bottom, is dried at room temperature.The ddH of 100-200 μ l is added2O room-temperature dissolution is good
DNA long-term preservation needs be placed in -20 DEG C of refrigerator.
(2) detection of transgenic positive single plant
Using the transgenic plant DNA of proposition as template, the Plasmid DNA built is positive control, and acceptor material DNA is yin
Property control, with the universal primer 545PKSE-Cas9-1171/2315F+545PKSE-Cas9-1171/ of Cas9 sequence design
2315R detects transgenic positive single plant.
(3) transgenic positive single plant MS5 gene editing site primer
More positive detection primers have the single plant of purpose band to carry out MS5 gene editing site primer.First with primer S-
MS5-L/S-MS5-R expands the region comprising two target sites, then recycles the primer SS- inside amplified fragments
MS5-1 and SS-MS5-2 detects the sequence of two target sites by generation sequencing approach, determines whether target site is edited.
Primer sequence is as follows:
| Primer | Primer sequence (5 ' -3 ') |
| S-MS5c-L (SEQ ID NO:7) | GTGGCATGAAAACGAATCAGCC |
| S-MS5c-R (SEQ ID NO:8) | CGAACAATGGCTATGTGTTTGC |
| SS-MS5C-1 (SEQ ID NO:9) | CATGCCTGTCTCGTATAACATTAC |
| SS-MS5C-2 (SEQ ID NO:10) | GTAATGTTATACGAGACAGGCATG |
According to PCR product sequencing result, target site transgenosis single plant to be edited is selected, is further cloned by TA,
20 clone's sample presentation sequencings are selected, the exact editor's genotype of heterozygosis editor single plant is detected.With Geneious software to sequencing sequence
Column are analyzed, and transgenosis single plant editor situation is as shown in figure 2 a andb.As shown in Figure 2 A, in 8 plants of Y127 of sequencing as receptor
Positive T0For in single plant, 5 plants show as male sterility, wherein the overwhelming majority is that diallele makes a variation.And the site S1 heterozygosis
The MS5 of editora- CR-4 shows as fertile phenotype.At the same time, in 5 plants of Westar positive single plants of sequencing, 4 plants show as hero
Property infertility, wherein MS5c- CR-10 shows as normal fertility in the site S1 heterozygosis, and MS5c- CR-33 shows as the site S2 heterozygosis,
Then as other dialleles or homeotic mutation, sterile phenotype (Fig. 2 B) is shown as.This result shows that, MS5 gene
After the variation of coiled coil structural domain, MS5cGene is knocked, and the allelotype of afunction is shown as relative to wild type
It is recessive.And after MS5 superfamily structural domain is knocked, since there are still competitive can tie coiled coil structural domain
Close MS5 and its interaction albumen, so show as dominant negative effect, and edited gene relative to wild type show as it is dominant not
It educates.
Embodiment 3: sterile strain Phenotypic Observation
In order to further determine the sterile phenotype of gene editing single plant, we look first at the stamen of sterile plant, discovery with
Fertile plant stamen is compared, MS5aAnd MS5cIt is shrivelled that edited sterile plant shows anther, without pollen filling etc. and MS5bIt is pure
Close the similar phenotype of sterile plant.Chromosome opens up piece the results show that visible meiosis prophase is until tetrad is each in fertile plant
The chromosome morphology (Fig. 2 C) in period, and in sterile plant, it is only capable of observing normal chromosome structure in leptotene, subtract later
Number division is stagnated, and chromosome is condensed into one, final degradable (Fig. 2 C), this in MS5bIt is observed in homozygous sterile plant
Chromosome morphology is consistent.
Cabbage type rape nuclear male sterility is formulated by gene editing means in conclusion the present invention provides one kind
The method of system can be quickly obtained recessive and dominant genic male sterility system by the editor to MS5 gene different structure territory,
Population improvement and heterosis utilization for cabbage type rape.
Sequence table
<110>Hua Zhong Agriculture University
<120>a kind of method and application that rapeseed male sterility new germ plasm is formulated by gene editing
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<213>artificial sequence (Artificial Sequence)
<400> 3
atatatggtc tcgattgaag cgagaggctc gccgtagtt 39
<210> 4
<211> 41
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
tgaagcgaga ggctcgccgt agttttagag ctagaaatag c 41
<210> 5
<211> 43
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
aacgttcttc gatgccaagg accaatctct tagtcgactc tac 43
<210> 6
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
attattggtc tcgaaacgtt cttcgatgcc aaggaccaa 39
<210> 7
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
gtggcatgaa aacgaatcag cc 22
<210> 8
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
cgaacaatgg ctatgtgttt gc 22
<210> 9
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
catgcctgtc tcgtataaca ttac 24
<210> 10
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
gtaatgttat acgagacagg catg 24
Claims (9)
1. a kind of method for creating of cabbage type rape male sterility strain, which is characterized in that this method using CRISPR/Cas9 or
Other gene editing systems, fixed point knocks out MS5 gene or MS5 gene M S5 superfamily structural domain, so that described
Afunction occurs for the corresponding albumen of gene.
2. the method for creating of cabbage type rape male sterility strain according to claim 1, which is characterized in that the tool of this method
Body step are as follows:
Step 1, two gRNA target spots for selecting MS5 gene, two gRNA target spots are positioned at coiled coil structural domain
S1, and positioned at the S2 of MS5 superfamily structural domain, the sequence of the target spot S1 is as shown in SEQ ID NO:1, the target
The sequence of point S2 is as shown in SEQ ID NO:2, and primer needed for synthesizing carrier construction；
Step 2 constructs double target spot CRISPR/Cas9 gene editing carriers；
Step 3, extracting plasmid, convert Agrobacterium；
Carrier is converted rape hypocotyls by way of mediated by agriculture bacillus by step 4, and MS5 gene is screened in transgenic progeny
The plant of mutation, to obtain cabbage type rape male sterility strain.
3. the method for creating of cabbage type rape male sterility strain according to claim 2, which is characterized in that institute in step 1
The sequence of primer is stated respectively as shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6.
4. the method for creating of cabbage type rape male sterility strain according to claim 2, which is characterized in that the step 2
In double target spot CRISPR/Cas9 gene editing carriers construction step are as follows:
Step 2.1, with sequence shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 be draw
Object carries out PCR amplification using high fidelity enzyme, glue and purification and recovery is cut after agarose electrophoresis using pCBC-DT1T2 plasmid as template
PCR product；
Step 2.2, the PCR product and CRISPR/Cas9 load for using T4 DNA ligase to recycle step 2.1 simultaneously with BsaI digestion
Body is assembled into whole carrier；
Whole carrier is converted competent escherichia coli cell, resistance screening, bacterium colony PCR identification, generation sequencing determination by step 2.3
The accuracy of carrier sequence.
5. the method for creating of cabbage type rape male sterility strain according to claim 2, which is characterized in that the step 3
In Agrobacterium be Agrobacterium GV3101.
6. the method for creating of cabbage type rape male sterility strain according to claim 2, which is characterized in that the step 4
In when screening the plant of MS5 gene mutation in transgenic progeny, first with primer S-MS5-L/S-MS5-R to including two
The region of target site is expanded, and then primer SS-MS5-1 and SS-MS5-2 inside amplified fragments is recycled to pass through a generation
Sequencing approach detects the sequence of two target sites, determines whether target site is edited；The sequence of the primer S-MS5-L
As shown in SEQ ID NO:7, the sequence of the primer S-MS5-R is as shown in SEQ ID NO:8, the primer SS-MS5-1
Sequence as shown in SEQ ID NO:9, the sequence of the primer SS-MS5-2 is as shown in SEQ ID NO:10.
7. the method for creating of any one of -6 cabbage type rape male sterility strains according to claim 1, which is characterized in that institute
It is MS5 that the brassica napus stated, which is selected from genotype,cMS5cWestar and/or genotype be MS5aMS5aY127.
Application of the 8.MS5 gene in initiative male sterile line of rape, the application is: being struck using CRISPR/Cas9 system
MS5 gene is removed and changed, and then obtains male sterile line of rape.
9. application of the MS5 gene in initiative male sterile line of rape according to claim 8, which is characterized in that described
MS5 gene is MS5aOr MS5c。
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| CN111876440A (en) * | 2020-08-05 | 2020-11-03 | 华中农业大学 | Method for editing BnaARF2 to create high-yield rape germplasm |
| CN112143735A (en) * | 2020-09-01 | 2020-12-29 | 中国农业科学院油料作物研究所 | Male sterile gene, protein, carrier, engineering bacterium of brassica napus and application thereof |
| CN112921051A (en) * | 2021-02-19 | 2021-06-08 | 西北农林科技大学 | Method for creating watermelon male sterile new germplasm by gene editing technology |
| CN113980964A (en) * | 2021-09-14 | 2022-01-28 | 江苏大学 | Site-directed mutagenesis method and application of brassica napus BnHBBD gene |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111876440A (en) * | 2020-08-05 | 2020-11-03 | 华中农业大学 | Method for editing BnaARF2 to create high-yield rape germplasm |
| CN112143735A (en) * | 2020-09-01 | 2020-12-29 | 中国农业科学院油料作物研究所 | Male sterile gene, protein, carrier, engineering bacterium of brassica napus and application thereof |
| CN112143735B (en) * | 2020-09-01 | 2022-09-20 | 中国农业科学院油料作物研究所 | Male sterile gene, protein, carrier, engineering bacterium of brassica napus and application thereof |
| CN112921051A (en) * | 2021-02-19 | 2021-06-08 | 西北农林科技大学 | Method for creating watermelon male sterile new germplasm by gene editing technology |
| CN112921051B (en) * | 2021-02-19 | 2023-07-25 | 西北农林科技大学 | Method for creating male sterile breeding quality of watermelons through gene editing technology |
| CN113980964A (en) * | 2021-09-14 | 2022-01-28 | 江苏大学 | Site-directed mutagenesis method and application of brassica napus BnHBBD gene |
| WO2023040623A1 (en) * | 2021-09-14 | 2023-03-23 | 江苏大学 | Method for and application of site-directed mutation of brassica napus bnhbbd gene |
| CN113980964B (en) * | 2021-09-14 | 2023-10-10 | 江苏大学 | Method for site-directed mutagenesis of BnHBBD gene of brassica napus and application |
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