CN101054586A - Rice chlorine ion passage gene OsCLC, coding protein and clone method thereof - Google Patents
Rice chlorine ion passage gene OsCLC, coding protein and clone method thereof Download PDFInfo
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- CN101054586A CN101054586A CN 200710038698 CN200710038698A CN101054586A CN 101054586 A CN101054586 A CN 101054586A CN 200710038698 CN200710038698 CN 200710038698 CN 200710038698 A CN200710038698 A CN 200710038698A CN 101054586 A CN101054586 A CN 101054586A
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Abstract
The present invention relates to rice chlorine ion passage gene OsCLC, its coded protein and clone method. The gene has sequence indicated by SEQ NO 3. The gene of 2429bp is cloned from differentiated rice callus. The opening reading frame of cloned gene encodes protein of 808 amino acid (molecular weight 88KD)which has 11 trans-membrane region indicated by trans-membrane region analysis. Cluster analysis indicates the OsCLC gene is new member of CLC family. The OsCLC gene expression level has positive correlation with salt concentration rise in a certain time range and respond to the salt stress and other environmental stress. The gene can be induced to produce partial complementary GEF1 function in yeast mutant and partially inhibit the sensitivity to NaCl and recover growth function. The gene is sensitive to pH value variety and not sensitive to different cation.
Description
Technical field
The invention belongs to and relate to a kind of rice chlorine ion passage gene OsCLC, its proteins encoded and cloning process thereof.
Background technology
Paddy rice is the staple food crop that surpasses half population in the world, and gene order-checking work is tentatively finished, and is a kind of good model of molecular biosciences research.In the injury of major cause one abiotic stress that causes the crop yield potentiality not give full play to, salinization is limiting growth and the important factor that improves output, yet salt stress causes, and plant photosynthesis descends, energy consumption increases, old and feeblely quicken, output descends or finally hungry dead because of carbon, about the physiology and the molecular biology research of plant salt tolerance mechanism mostly concentrates on Na
+On, relate to Cl
-Research then seldom.
Anion channel extensively is present in animal and plant and the microorganism, has the important physical effect, but its research is lagged far behind cationic channel.As one of important anion channel, chloride channel (ChlorideChannel, CLC) generally be considered to cell turgor and osmoregulation, ion stable state, born of the same parents in pH, stomatal movement, nutritive substance transportation, metal patience, signal identification and transduction etc. all multi-functional relevant.Nineteen ninety, Hechenberger etc. utilize Africa xenopus ovocyte gene expression technique to be separated to chloride channel protein gene C LC-O first in electric ray belongs to the electric organ of bag fish (Torpedo marmorata), have found the newcomer of CLC gene afterwards in bacterium, yeast, Mammals and plant successively.Topological studies show that, CLC passage generally are made up of 10～12 membrane spaning domains (domains), and N-end and C-end are located in the kytoplasm, are different from single hole passage in traditional cationic channel, and the CLC passage exists with dimer diplopore form usually.On Mammals (people and mouse), find 9 kinds of CLC channel proteins altogether at present,, can be divided into 3 subfamilies, studies show that some human inheritance's disease is the variation that is caused by the CLC channel gene according to the size of its homology degree.And simultaneously, be very limited about the reporting quantities of plant chloride ion channel.
Summary of the invention
One of purpose of the present invention is to provide a kind of rice chlorine ion passage gene OsCLC.
Two of purpose of the present invention is to provide the proteins encoded of this gene.
Three of purpose of the present invention is to provide the cloning process of this gene.
For achieving the above object, the present invention adopts following technical scheme:
A kind of rice chlorine ion passage gene OsCLC is characterized in that this gene has the base sequence shown in the SEQ NO 7.
Above-mentioned rice chlorine ion passage gene OsCLC proteins encoded is characterized in that having the aminoacid sequence shown in the SEQ NO 8.
The cloning process of above-mentioned rice chlorine ion passage gene OsCLC is characterized in that this method has following steps:
A. the cultivation of the differentiation callus of paddy rice;
B. the clone of rice chlorine ion passage gene OsCLC: adopting total RNA of the paddy rice differentiation callus of gained among the Trizol method extraction step a, is template with this total RNA, adopts Oligo (dT)
18Be primer, cDNA first chain is synthesized in reverse transcription; Through pcr amplification, the response procedures of this PCR is again: 94 ℃ of 5min → (94 ℃ of 30s, 62 ℃ of 40s, 72 ℃ of 150s) * 35 circulations are extended 10min for → 72 ℃, are obtained pcr amplification product; Then this pcr amplification product is connected on the pMD-19-T carrier, transformed into escherichia coli DH5 α obtains positive colony by the screening of basket hickie method again, and this positive colony is rice chlorine ion passage gene OsCLC; The Oligo that is adopted (dT)
18Primer is respectively:
5 ' end primer is: 5 '-CGATGGCGCCAAGGGACCAGTCAT-3 ',
3 ' end primer is: 5 '-TACTAGCGAGCGTCAGAAGACGAG-3 '.
The concrete steps of the cultural method of the differentiation callus of above-mentioned paddy rice are: ripe rice paddy seed is peelled off clever shell, 70% ethanol rinsing 1min, and 0.1% mercuric chloride soaks 20min; Be seeded in MS+2 behind aseptic water washing 4-5 time, on the inducing culture of 4-D2mg/L, place dark place to cultivate evoked callus down for 26 ℃; After two weeks callus is placed on the division culture medium MS+6-BA2mg/L+KT2mg/L+NAA0.2mg/L, collect the callus of differentiation, behind liquid nitrogen flash freezer, be stored in-80 ℃.
The rice chlorine ion passage protein gene OsCLC that the total length that the present invention is cloned into from the callus of paddy rice differentiation is 2429bp.Open reading frame coding length is the protein of 808 amino acid (molecular weight is 88kD), strides film and distinguishes to analyse and show that having 11 strides diaphragm area, and cluster analysis demonstration OsCLC gene is the newcomer of CLC family.This OsCLC expression of gene level in the certain hour scope with salt concn variations that be proportionate that raise, can present to express to the salt stress environmental stress and respond.And this gene can be induced in yeast mutant and be produced the complementary GEF1 function of part, partly suppresses the sensitivity to NaCl, and recovers the growth function; The pH value is changed responsive; Different positively charged ions there is not responsive phenomenon.
Description of drawings
Fig. 1 strides the film analysis result for rice chlorine ion passage gene OsCLC amino acid sequence coded.
Fig. 2 is the cluster analysis result of rice chlorine ion passage gene OsCLC amino acid sequence coded.
At shows Arabidopis thaliana; Os shows paddy rice; Nt shows tobacco; St shows potato; Sc shows yeast saccharomyces cerevisiae.The sequence that is used for analyzing is as follows at the sequence number of GenBank: CLC-0 (CAA40078); CLC-1 (CAA44683); CLC-2 (CAA45500); CLC-3 (BAA04471); CLC-4 (CAA54417); CLC-5 (CAA91216); CLC-6 (CAA58292); CLC-7 (CAA91556); AtCLC-a (CAA96057); AtCLC-b (CAA96058); AtCLC-c (CAA96059); AtCLC-d (CAA96065); StCLC (CAA71369); ScCLC (CAA80663); OsCLC-1 (BAB97267); OsCLC-2 (BAB97268); CLC-Nt1 (CAA64829)
Fig. 3 is that rice chlorine ion passage gene OsCLC real-time fluorescence quantitative PCR detects the expression amount result of variations.
Fig. 4 is the function compensation experimental result of rice chlorine ion passage gene OsCLC in the yeast saccharomyces cerevisiae mutant strain.
1:WT:BY4741; 2:Mutant:YJR040W; The 3:pGAL1::OsCLC transformant; The 4:pYES2 transformant
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these examples only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted concrete experiment condition in the following example, usually according to normal condition, molecular cloning (Molecular Cloning:A Laboratory Manual, 3rd ed.) or molecular biology of plants-laboratory manual (Plant Molecular Biology-A Laboratory Manual, Melody S.Clark compiles, Springer-verlag Berlin Heidelberg, 1997) condition described in, or the condition of advising according to manufacturer.
Embodiment one
The clone of OsCLC full length coding region and analysis
(1) callus of differentiation preparation
Ripe rice paddy seed is peelled off clever shell, 70% ethanol rinsing 1min, and 0.1% mercuric chloride soaks 20min; Be seeded in MS+2 behind aseptic water washing 4-5 time, on the inducing culture of 4-D2mg/L, place dark place to cultivate evoked callus down for 26 ℃; After two weeks callus is placed on the division culture medium MS+6-BA2mg/L+KT2mg/L+NAA0.2mg/L, collect the callus of differentiation, behind liquid nitrogen flash freezer, be stored in-80 ℃.
(2) total RNA extracts
Adopting the Trizol method to extract total RNA of paddy rice differentiation callus, is template with total RNA, adopts Oligo (dT)
18Be primer, cDNA first chain, Oligo (dT) are synthesized in reverse transcription
18Be respectively:
5 ' end primer is 5 '-CGATGGCGCCAAGGGACCAGTCAT-3 ',
3 ' end primer is 5 '-TACTAGCGAGCGTCAGAAGACGAG-3 ',
Through pcr amplification, the PCR response procedures is 94 ℃ of 5min → (94 ℃ of 30s, 62 ℃ of 40s, 72 ℃ of 150s) * 35 circulation is extended 10min for → 72 ℃, again pcr amplification product is connected to the pMD-19-T carrier, transformed into escherichia coli DH5 α obtains positive colony by the screening of basket hickie method, hands over the order-checking of Sangon company; Carry out cluster analysis with MEGA3.0, stride the diaphragm area analysis with TMHMM2.0 software.
The result shows the rice chlorine ion passage protein gene (GenBank:DQ272692) that total length of this positive colony is 2429bp, and called after OsCLC is referring to Fig. 1.Open reading frame coding length is the protein of 808 amino acid (molecular weight is 88kD), and iso-electric point is 8.1.Through the comparison of BLAST homology, the result shows that the homology of OsCLC amino acid sequence coded and CLC-Nt1, AtCLC-a, AtCLC-b, AtCLC-c, AtCLC-d reaches 59%, 54%, 52%, 58% and 47% respectively.OsCLC has 11 hydrophobic film districts of striding, stride between the film district and the 3rd stride aminoacid sequence GSGIPE (182aa), GKAGPLVH (224aa) and the PVGGVLF (283aa) that has 3 conservative regions in the film district the 2nd and the 3rd, consistent with the aminoacid sequence (GxGxPE, GKxGPxxH and PxxGxLF) that is considered to 3 high conservatives relevant of report existence in other species before with the anion-selective of passage.With the systematic evolution tree analysis of the CLC gene of other species of report, the OsCLC gene is the newcomer among the CLC protein family.
Embodiment two
The real-time fluorescence quantitative PCR of salt inductive OsCLC gene expression amount detects
(1) specimen preparation
According to a pair of Auele Specific Primer of rice Os Actin gene (Genbank:X15865) sequences Design: 5 ' end primer (5 '-TTG TGA GGG ACA TGA AGG AGA A-3 '); 3 ' end primer (5 '-TAT GAA GGA AGG CTGGAA GAG G-3 ') fragment length is 193bp, as the internal reference of real-time fluorescence quantitative PCR.Design a pair of Auele Specific Primer at goal gene OsCLC, 5 ' end primer: 5 '-ACA TCG TAT TTC CTG GGC ATT T-3 '; 3 ' end primer: 5 '-CAG TCA TTC TCA TTG ATC CTC C-3 ', fragment length is 179bp.With synthetic cDNA first chain among the embodiment one is template, and the PCR response procedures is 94 ℃, and 5min circulates through following 30 times: 94 ℃, and 20s, 57 ℃, 20s, 72 ℃, 20s; Last 72 ℃ are extended 5min.Then respectively two kinds of products are connected into the pMD19-T carrier and transform DH5 α, obtain positive colony by the screening of basket hickie method, alkaline process extracts plasmid and identifies definite positive recombinant plasmid that obtains after enzyme is cut.
The Trizol method is extracted the total RNA that tolerates growth 0hr, 1hr, 8hr, 16hr, 24hr blade in 0mM NaCl, 50mM NaCl, 100mM NaCl, 4 concentration of salt solution environment of 200mM NaCl respectively.With Oligo (dT)
18Be primer, cDNA first chain is synthesized in reverse transcription, ℃ deposits stand-by as real-time fluorescence quantitative PCR reaction template-20.
(2) standard curve making
Be diluted to 10 with ultrapure water by 10 times of gradeds
2, 10
3, 10
4, 10
5, 10
6, 10
7, 10
8, 10
9Individual copy number carries out the PCR reaction.It is quantitative respectively internal reference and goal gene plasmid to be carried out copy number, is X-coordinate with the natural logarithm value of copy number, is ordinate zou with the Ct value, the drawing standard graphic representation, and find the solution regression equation.
(3) real-time fluorescence quantitative PCR reaction system
Add SYBR Green Realtime PCR Master Mix 10 μ l, every 0.4 μ l of primer (20 μ M), testing sample template 1 μ l in the 20 μ l reaction systems respectively, mend ddH
2O to 20 μ l.
(4) real-time fluorescence quantitative PCR reaction conditions
Pre-94 ℃ of 5min of sex change; 94 ℃ of 30s of sex change, the 57 ℃ of 20s that anneal extend 72 ℃ of 20s, fuse 83 ℃ of 1s; Read plate after the fusion; Cycle number is 40; 72 ℃ are extended 5min; The melting curve temperature range is set to 65.0 ℃ to 95.0 ℃, the 0.3 ℃ of reading in every interval once, each 1s.Be reflected at Opticon
TMCarry out in the 2 type real-time fluorescence quantitative PCR instrument (MJ Research company).
By the PCR quantitative amplification, according to twice replicate(determination), 3 reactions of the parallel repetition of each every duplicate samples, extrapolate OsActin gene and the pairing DNA copy number of OsCLC respectively by two typical curves, the OsCLC copy number of each sample and the ratio of OsActin gene copy number can compare tolerance growth 0hr, 1hr, 8hr, 16hr, 5 time point OsCLC of 24hr expression variation in blade respectively in 50mM NaCl, 100mM NaCl, 200mM NaCl concentration of salt solution environment.
The expression level real-time fluorescence PCR detected result of OsCLC under the salt stress environment shows, gradient increase along with NaCl concentration, the expression amount of OsCLC transfers to the climax on growth under the salt stress environment is after 1 hour, wherein the expression amount under 50mM NaCl, 100mM NaCl, the 200mM NaCl environment reaches 1.46 times, 1.23 times and 1.42 times of initial amount respectively, expression amount after 8 hours under the 50mM environment is kept 1.23 times, and 100mM and 200mM then drop to 0.98 and 0.97 times.Expression amount after 16 hours under the 50mM environment also falls after rise to 0.92 times, and after 24 hours, expression amount does not then have considerable change under each concentration environment.
Therefore, illustrate under condition of salt stress, OsCLC expression of gene level in the certain hour scope with salt concn variations that be proportionate that raise, can present to express to the salt stress environmental stress and respond.
Embodiment three
The mutant function complementation experiment
(1) construction of recombinant plasmid
With synthetic cDNA first chain among the embodiment one is template, the design Auele Specific Primer: 5 ' the end primer (5 '-AGC TGA GCT CGA TGG CGC CAA GGG ACC A-3 ') that contains Sac I site; 3 ' the end primer (5 '-TAG CTC TAG AGA AGA CGA GGT TGG TGA A-3 ') that contains Xbal I.Products therefrom is inserted among the shuttle vectors pYES2 after Sac I and Xbal I enzyme are cut, earlier express recombinant expression plasmid pGAL1::OsCLC (Apr in bacillus coli DH 5 alpha
+), by the positive colony that the Apr screening obtains, plasmid is cut the positive back Lithium Acetate method that is accredited as through Sac I and Xbal I enzyme
[12]Be transformed among the yeast saccharomyces cerevisiae mutant strain YJR040W (gef1::kam), save composition substratum (Ura at CM
-) on, cultivate after 3-5 days for 30 ℃ and grow pGAL1::OsCLC (Ura
+) and pYES2 empty carrier transformant yeast colony, extract cerevisiae dna respectively as template, utilize the OsCLC gene-specific primer that designs in the 1.2.2 trifle to carry out pcr amplification and identify positive bacterium colony.
(2) yeast culture
Wild-type Wine brewing yeast strain BY4741, yeast saccharomyces cerevisiae mutant strain YJR040W, recombinant plasmid pGAL1::OsCLC (Ura
+) transformant and pYES2 empty carrier transformant be respectively at pH=5, pH=6, pH=7 and add respectively on the YPG substratum of NaCl (250mM), NaCl (500mM), NaCl (750mM) and NaCl (1M), cultivated 3-7 days for 30 ℃.
Function complementation experiment result shows, functional complementation experimental result demonstration in the YPG of different pH values substratum, and 30 ℃ of constant temperature culture are after 3 days, pGAL1::OsCLC (Ura under the environment of pH=6
+) can the complementary GEF1 function of part, it is the most obvious to recover the growth functional effect.
In the YPG substratum, add the growth of NaCl (250mM), NaCl (500mM) and NaCl (750mM) back after 4 days; Add NaCl (1M) growth after 7 days, pGAL1::OsCLC can the complementary GEF1 function of part, partly suppresses the sensitivity to NaCl, recovers the growth function.Along with salt concn in the substratum increases, the degree that has complementary functions is obvious more.
In substratum, add identical Cl
-The salt of concentration, different cation type comprises NaCl, CaCl
2, KCl and MgCl
2The result shows that dissimilar positively charged ions recovers growing state did not influence (data do not show) for pGAL1::OsCLC after cultivation, a couple of days.
Therefore, the OsCLC gene can be induced in yeast mutant and be produced the complementary GEF1 function of part, partly suppresses the sensitivity to NaCl, and recovers the growth function; The pH value is changed responsive; Different positively charged ions there is not responsive phenomenon.
Sequence table
＜110〉Shanghai University
＜120〉rice chlorine ion passage gene OsCLC, its proteins encoded and cloning process thereof
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＜213〉artificial sequence (Artificial)
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＜213〉artificial sequence (Artificial)
<220>
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＜223〉primer
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＜213〉artificial sequence (Artificial)
<220>
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＜223〉primer
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＜213〉artificial sequence (Artificial)
<220>
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＜223〉primer
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<210>7
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＜213〉paddy rice (Oryza sativa)
<220>
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121?gcgatggcga?cgaggaggcg?cgacggcggc?ggcggaggtt?cctcctcgcc?ggccgctccc
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481?tttcgaattt?cgcccttacg?atgttagcaa?cggtgctgac?ggtgtatgtg?gcgccggcgg
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781?atgataggga?ccgaagggac?cttgttactt?gtggtgctgg?tgctggtatt?gctgctgctt
841?tccgggcacc?agttggtgga?gtcctgtttg?ctcttgaagc?agtgtcttca?tggtggagga
901?gtgccctact?ctggcgagca?tttttcacaa?cagcaatggt?tgctgttgtg?cttagggcat
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1261?gtccaagtga?caccgaagaa?gcctgtccat?ccataggaag?atctggtaat?tttaagaagt
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2101?gctcaggaaa?gcatgacaga?atagaggaaa?tagaattcag?tgcagaagaa?ctggaaatgt
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2281?aatcgtctaa?gagagcgcct?gttgtaggca?tactgacaag?gcacgacttc?atgcctgagc
2341?atatattggg?tcttcatcct?ttcctcttca?agactagatg?gaagaaggtg?cggtttggca
2401?agtcagcatt?caccaacctc?gtcttctga
<210>8
<211>808
<212>PRT
＜213〉paddy rice (Oryza sativa)
<400>8
1?maprdqscgd?ggevdpeggi?eapllssgss?ffqdpahedg?dgdeearrrr?rrfllagrsq
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121?aaagfvanlg?venvagakfv?vtsnlmlagr?ygtafavflv?snfaltmlat?vltvyvapaa
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301?allwrafftt?amvavvlral?idfcksdkcg?lfgkgglimf?dvtsdyityh?lvdlppvitl
361?gvlggvlgsl?hnffldkvlr?lynfinekgq?kyklllaavv?tictscclfg?lpwiasckpc
421?psdteeacps?igrsgnfkky?qcamneyndl?aslffntndd?tirnlysagt?ddefhissil
481?vffftsyflg?ifsyglalps?glfvpviltg?atygrlvgml?igsqstldhg?lfavlgsaal
541?lggsmrmtvs?vcvvileltn?nllmlplvml?vllisktvad?afnaniydll?vklkgfpyle
601?ghvepymrql?svsdvvtgpl?qafngiekvg?hivhvlrttg?hngfpvvdep?pfsdspvlfg
661?lvlrahllvl?lrkkdfipnc?sasaldaskq?flphdfakpg?sgkhdrieei?efsaeelemf
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Claims (6)
1. a rice chlorine ion passage gene OsCLC is characterized in that this gene has the base sequence shown in the SEQ NO 7.
2. a rice chlorine ion passage gene OsCLC proteins encoded according to claim 1 is characterized in that having the aminoacid sequence shown in the SEQ NO 8.
3. the cloning process of a rice chlorine ion passage gene OsCLC according to claim 1 is characterized in that this method has following steps:
A. the cultivation of the differentiation callus of paddy rice;
B. the clone of rice chlorine ion passage gene OsCLC: adopting total RNA of the paddy rice differentiation callus of gained among the Trizol method extraction step a, is template with this total RNA, adopts Oligo (dT)
18Be primer, cDNA first chain is synthesized in reverse transcription; Through pcr amplification, the response procedures of this PCR is again: 94 ℃ of 5min → (94 ℃ of 30s, 62 ℃ of 40s, 72 ℃ of 150s) * 35 circulations are extended 10min for → 72 ℃, are obtained pcr amplification product; Then this pcr amplification product is connected on the pMD-19-T carrier, transformed into escherichia coli DH5 α obtains positive colony by the screening of basket hickie method again, and this positive colony is rice chlorine ion passage gene OsCLC; The Oligo that is adopted (dT)
18Primer is respectively:
5 ' end primer is: 5 '-CGATGGCGCCAAGGGACCAGTCAT-3 ',
3 ' end primer is: 5 '-TACTAGCGAGCGTCAGAAGACGAG-3 '.
4. the cloning process of rice chlorine ion passage gene OsCLC according to claim 3, the concrete steps of cultural method that it is characterized in that the differentiation callus of described paddy rice are: ripe rice paddy seed is peelled off clever shell, 70% ethanol rinsing 1min, 0.1% mercuric chloride soaks 20min; Be seeded in MS+2 behind aseptic water washing 4-5 time, on the inducing culture of 4-D2mg/L, place dark place to cultivate evoked callus down for 26 ℃; After two weeks callus is placed on the division culture medium MS+6-BA 2mg/L+KT 2mg/L+NAA 0.2mg/L, collect the callus of differentiation, behind liquid nitrogen flash freezer, be stored in-80 ℃.
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| CN 200710038698 CN101054586A (en) | 2007-03-29 | 2007-03-29 | Rice chlorine ion passage gene OsCLC, coding protein and clone method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104203974A (en) * | 2012-03-20 | 2014-12-10 | 英美烟草(投资)有限公司 | Transgenic plants having lower nitrate content in leaves |
| CN108522279A (en) * | 2018-03-21 | 2018-09-14 | 浙江师范大学 | The direct seedling tissue culture method of wild rice stem seed embryo and used medium |
| CN108841834A (en) * | 2018-06-27 | 2018-11-20 | 中国烟草总公司郑州烟草研究院 | One tobacco chloride channel protein NtCLC2 and its application |
| CN113136389A (en) * | 2021-04-16 | 2021-07-20 | 河南农业大学 | Genetic engineering application of gene GhCLcg-1A and/or gene GhCLcg-1D |
-
2007
- 2007-03-29 CN CN 200710038698 patent/CN101054586A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104203974A (en) * | 2012-03-20 | 2014-12-10 | 英美烟草(投资)有限公司 | Transgenic plants having lower nitrate content in leaves |
| CN108522279A (en) * | 2018-03-21 | 2018-09-14 | 浙江师范大学 | The direct seedling tissue culture method of wild rice stem seed embryo and used medium |
| CN108522279B (en) * | 2018-03-21 | 2021-06-18 | 浙江师范大学 | Tissue culture method for direct seedling formation of wild rice stem seed embryo and culture medium used by same |
| CN108841834A (en) * | 2018-06-27 | 2018-11-20 | 中国烟草总公司郑州烟草研究院 | One tobacco chloride channel protein NtCLC2 and its application |
| CN113136389A (en) * | 2021-04-16 | 2021-07-20 | 河南农业大学 | Genetic engineering application of gene GhCLcg-1A and/or gene GhCLcg-1D |
| CN113136389B (en) * | 2021-04-16 | 2023-02-14 | 河南农业大学 | Genetic engineering application of gene GhCLcg-1A and/or GhCLcg-1D |
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