CN107904244A - Application of the wild soybean XLOC_023202 genes in soybean plants cyst nematode resistance is improved - Google Patents
Application of the wild soybean XLOC_023202 genes in soybean plants cyst nematode resistance is improved Download PDFInfo
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- CN107904244A CN107904244A CN201711217564.0A CN201711217564A CN107904244A CN 107904244 A CN107904244 A CN 107904244A CN 201711217564 A CN201711217564 A CN 201711217564A CN 107904244 A CN107904244 A CN 107904244A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8285—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for nematode resistance
Abstract
The present invention relates to biological technical field, specifically, application of the wild soybean XLOC_023202 genes in soybean plants cyst nematode resistance is improved；The nucleotide sequence of XLOC_023202 genes such as SEQ ID NO:Shown in 1.Compared with prior art, soybean plants cyst nematode resistance gene provided by the present invention was substantially raised at 9 days and 15 days after being inoculated with cyst nematode, this prompts the gene to have extraordinary application prospect in terms of soybean plants cyst nematode resistance is improved.
Description
Technical field
The present invention relates to biological technical field, and soybean is being improved in particular to wild soybean XLOC_023202 genes
Application in platymiscium cyst nematode resistance.
Background technology
Soybean cyst nematode is the important disease on Soybean production.From soybean-soybean cyst nematode Heterodera glycines interaction angle, analysis
Soybean resistance related gene, and then resistance mechanism of the soybean to cyst nematode is parsed, screening resistance related gene is for Soybean Resistance
Cyst nematode breeding has important value.
At present, related soybean cyst nematode Heterodera glycines infect lower the cultivated soybean root system gene expression analysis report it is more, Er Qieqi
Middle most reports are to be directed to infect early stage, it is intended to parse soybean cyst nematode Heterodera glycines intrusion root system and Syncytium formation process
In, the expression conditions of soybean.The disease resistance mechanisms of Resistant gerplasm are necrosed around plasomidum, and then limit soybean born of the same parents
Capsule elegans development, even results in nematode death.Therefore, it is found that many related plant defenses, cell synthesize, are thin in above-mentioned report
Born of the same parents' degraded, the relevant gene of metabolic process.
Wild soybean is the ancestors of the cultivated soybean, has the hereditary variation than the cultivated soybean more horn of plenty.Therefore may deposit
In the resistance mechanism different from the cultivated soybean.At present, there is not yet on soybean cyst nematode Heterodera glycines infect lower wild soybean root system basis because
The report of expression.
Research shows, soybean has the disease resistance mechanisms of cyst nematode a variety of, and infects early stage, Resistant gerplasm is in plasomidum week
Enclose the Just One Of Those Things that necroses.All there is the disease resistance of cyst nematode in whole soybean cyst nematode Heterodera glycines infection processs in soybean.
Therefore, the soybean cyst nematode resistance gene found in the prior art is not comprehensive enough.
In view of this, it is special to propose the present invention.
The content of the invention
The present invention have studied inoculation soybean cyst nematode Heterodera glycines after 9,15 and 20 days when disease-resistant and susceptible wild soybean gene table
Up to situation, identify resistance related gene, and to more than 2 all existing gene of Proper Sampling Period parsed and verified,
So as to have found special differential expression in disease-resistant wild soybean, and the gene that 9 and 15 days 2 periods substantially raised after inoculation
XLOC_023202。
The present invention relates to wild soybean XLOC_023202 genes answering in soybean plants cyst nematode resistance is improved
With；The nucleotide sequence of XLOC_023202 genes such as SEQ ID NO:Shown in 1.The gene is the cultivated soybean
The homologous gene of Glyma.14G003800,6 amino more than the albumen that the albumen of predictive coding is encoded than Glyma.14G003800
Acid.
Preferably, application as described above, using the XLOC_023202 genes as molecular labeling, cultivates high cyst line
The soybean plants new varieties of worm resistance.
Preferably, application as described above, is transferred to Glycine by transgenic method by the XLOC_023202 genes and plants
Thing is to improve its cyst nematode resistance.
Preferably, application as described above, the transgenic method specifically include：
(1) expression vector of the structure containing the XLOC_023202 genes；
(2) expression vector that step (1) obtains is introduced directly into soybean plants cell；Or it is situated between by intermediate host's cell
Import soybean plants cell；
(3) genetically modified plants are regenerated；With
(4) genetically modified plants are selected；And
(5) optionally, the plant that amplification step (4) obtains is to obtain offspring.
Preferably, application as described above, preferable intermediate host's cell is bacterial cell, in particular for cloning or storing up
Deposit polynucleotides or the bacterial cell for converting plant cell, such as Escherichia coli, Agrobacterium, Agrobacterium tumdfaciens and hair
Root agrobacterium, most preferably Agrobacterium.
Agrobacterium can include plasmid (such as the conventional plasmid such as Ti or Ri plasmids), and the XLOC_ is loaded with the plasmid
023202 nucleotide sequence, the plasmid are transferred to plant after with Agrobacterium infection, and the purpose sequence that plasmid is loaded with
Row are integrated into the genome of plant cell.The nucleotide sequence of the present invention is particularly suitable for the plant cell of soybean, but not
Exclusion also has the function that cyst nematode resistance in other species (such as rape, wheat, oat, potato etc.).
Preferably, application as described above, the method for being introduced directly into soybean plants cell include：Particle gun mediates
Conversion method, pollen tube passage method or liposome transformation.
The nucleotide sequence of the XLOC_023202 can be used for conversion plant and obtain the plant of conversion.In the prior art
In extensively description Plant Transformation.It is well known that multiple systems such as plasmid vector, liposome, electroporation, micro note can be used
Penetrate, spread, particle gun, coprecipitation of calcium phosphate, uses viral vector etc..In the present invention, its all portion for all forming the present invention
Point.
Preferably, application as described above, in step (4), when selecting genetically modified plants, identification is transferred to described
The expression of XLOC_023202 genes, selects the high plant of expressing gene expression quantity.
Preferably, application as described above, the method for the high plant of the selection expressing gene expression quantity for：
The total serum IgE of genetically modified plants to be detected is extracted, uses SEQ ID No:2 and SEQ ID No:Upstream and downstream shown in 3
Primer pair XLOC_023202 carries out qRT-PCR detections.
Preferably, application as described above, annealing temperatures of the qRT-PCR when carrying out cyclic amplification be 55 DEG C~
65℃。
Preferably, application as described above, the soybean plants include：It is soybean Glycine max (L.) Merr., short
Suede Wild soybean Glycine tomentella Hayata, wild soybean Glycine soja Sieb.et Zucc., Penghu soybean
Glycine clandestina Wendl., climing beans Glycine gracilis Skv. of wide leaf, cigarette beans Glycine tabacina
Benth., hyacinth bean pod soybean variety Glycine dolichocarpa Tateishi＆Ohashi and broad-leaved soybean Glycine
The first in tomentella Hayata is a variety of.
Compared with prior art, soybean plants cyst nematode resistance gene provided by the present invention is in inoculation cyst nematode
Afterwards 9 days and 15 days when substantially raise, this prompt the gene in terms of soybean plants cyst nematode resistance is improved have it is very good
Application prospect.
Brief description of the drawings
, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution of the prior art
Embodiment or attached drawing needed to be used in the description of the prior art are briefly described, it should be apparent that, in describing below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
Put, other attached drawings can also be obtained according to these attached drawings.
Fig. 1 shows that soybean cyst nematode Heterodera glycines have successfully infected the root system for examination wild soybean；9 days after inoculation, 2 instar larvaes are
Root system (Figure 1A, E) is invaded, 15 days after inoculation, existing 2 instar larvae of part develops into 3 instar larvaes (Figure 1B, F), 20 days after inoculation,
There are 2 ages, 3 ages and 4 age soybean cyst nematode Heterodera glycines (Fig. 1 C, D, G, H) at the same time in root system；
Fig. 2 is the RNA-seq expression quantity (Log of 10 difference expression genes2And qRT-PCR expression quantity (Log (FC))2
(FC))D9：9 days after inoculation, D15：15 days after inoculation；D20：20 days after inoculation.
Embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person, the condition suggested according to normal condition or manufacturer carry out.Reagents or instruments used without specified manufacturer, is
Can be with conventional products that are commercially available.
Embodiment
First, materials and methods
1st, the preparation of soybean cyst nematode Heterodera glycines ovum solution
Rhizosphere soil containing soybean cyst nematode Heterodera glycines is derived from the synthesis examination of Jilin Province Taonan City Jilin Academy of Agricultural Science Taonan
Test station Soybean Field.Its soybean cyst nematode Heterodera glycines HG types are HG 0.Soybean cyst nematode Heterodera glycines in sick soil are pacified in agriculture 14 in soybean varieties
Expand numerous.The seed of peaceful agriculture 14 is planted in sick soil, is extracted root from soil after 35 days, it is upper (from top to bottom to be placed in layer sieve
The mesh mesh screen of respectively 20,40 and 60), the cyst on root system is rinsed out with the current of larger pressure, is collected in 60 mesh sieve nets
On.The online cyst of 60 mesh sieves is collected into the beaker of 1L, and utilizes the sedimentation speed of the materials such as cyst, root system fragment, soil
Degree is different, and cyst solution is further purified.Cyst solution after purification is poured on 60 mesh mesh screens, is pulverized with rubber bar
On sieve, it is allowed to discharge ovum and 2 instar larvaes, is rinsed with wash bottle, ovum and 2 instar larvaes is poured on 2 layers of bushing screen (from top to bottom
It is followed successively by 200 mesh and 500 mesh), 200 mesh mesh screens are repeatedly rinsed with tap water, by ovum collecting on 500 mesh mesh screens.By 500 mesh nets
Ovum on sieve is transferred in 50mL beakers, is configured to the ovum suspension of 23000 ovum mL-1, and standby inoculation is used.
2nd, disease-resistant and susceptible wild soybean germplasm
0 populations of wild soybean germplasm ZYD03685 highly resistance soybean cyst nematode Heterodera glycines HG, its cyst index are 1.4, ZYD02688
0 populations of height sense soybean cyst nematode Heterodera glycines HG, its cyst index is 101.6
3rd, it is inoculated with processing method
After examination 60 0.5%NaClO solution disinfections 3min of wild soybean seed, rinsed 4 times with flowing tap water.Will
Sterilized seed is planted in the polypots for filling high-temperature sterilization perlite after carving skin.At 27 DEG C, 9h illumination/15h dark
Under the conditions of cultivate 7d after, the expansion of first trifoliolate leaf.Seedling is carefully extracted, and is rinsed well.By the consistent plant of growing way
It is divided into 6 groups, every group of 5 plant, 3 groups are inoculation worm's ovum processing, and 3 groups are control treatment (inoculation liquid is tap water).Every group is planted
Strain moves on in the polypots for filling high-temperature sterilization perlite (perlite accounts for the 2% of polypots volume), new in root surface inoculation 5mL
Fresh worm egg suspension (23000 ovum mL-1) (inoculation worm's ovum group) or the tap water (control group) of 5mL, then cover 2cm thickness
High-temperature sterilization perlite.27 DEG C are positioned over, is cultivated under conditions of 9h illumination/15h dark.
9d after inoculation, often randomly selects 1 control group and 1 treatment group, the plant of each polypots is gently extracted, and
Root system is rinsed well.The root system of 3 plant is taken quickly to be positioned in liquid nitrogen at random from the root system of 5 plant, remaining 2 plants of use
In the observation of soybean cyst nematode Heterodera glycines colonization status.Plant 4 groups remaining is gently extracted respectively, and the worm's ovum of root surface and nematode are rushed
It is transplanted to again after washing off in the polypots for filling high-temperature sterilization perlite, is placed on 27 DEG C, the condition of 9h illumination/15h dark
Under continue to cultivate.Distinguish 15d and 20d after inoculation, 1 control group and 1 treatment group are randomly selected, by the plant of each polypots
Strain is gently extracted, and root system is rinsed well.The root system of 3 plant is taken quickly to be positioned over liquid at random from the root system of 5 plant
In nitrogen, remaining 2 plants of observations for being used for soybean cyst nematode Heterodera glycines colonization status.
4th, the extraction and sequencing of RNA
The root system sample that will be preserved in liquid nitrogen, pulverizes last in liquid nitrogen, utilizes Trizol reagents
((Invitrogen, Shanghai) carries out the extraction of total serum IgE according to its specification, with Agilent2100 analyzers and Nanodrop points
Light photometer carries out RNA concentration and quality determination.The structure in library and sequencing (Illumina HiSeqTM2000) it is (deep by BGI
Ditch between fields) complete.
With 1.5% agarose gel electrophoresis detection RNA integrality, with NanoDrop 2000 detect RNA concentration and
Purity.CDNA synthesis is carried out using TaKaRa PrimeScriptTM RT reagent Kit with gDNA Eraser, instead
Transcription system is 40 μ L, wherein containing 14 μ L RNA solutions.
5th, data processing
Connector and low quality sequence (quality are carried out to primitive sequencer sequence using FastQC and FASTX toolkits
Value Q≤30DE base numbers account for more than 10%) delete processing.With wild soybean genome (GenBank/EMBL/DDBJ data
Storehouse, registration number AZNC00000000.1) it is reference gene group, the splicing of sequence is carried out using Tophat2 (v 2.0.8).(Qi
Et al.2014) its parameter is arranged to：Allow the mispairing of 1 base, 30-100, length (the Lin et of 000 introne
al.2014).It is reference gene group with the cultivated soybean genome (G.max Wm82a2.v1., under be downloaded from Phytozome10), profit
Gene comparison and gene annotation are v.2.2.1 carried out with Cufflinks.(Trapnell et al.2012) utilizes HTSeq
V.0.6.1 gene expression amount statistical analysis (Anders et al.2015) is carried out, v.3.2.3 differential expression divides using edgeR
Analyse (Robinson et al.2010；Anders et al.2013) selects the homologous sequence of 28 house-keeping genes to carry out broad sense
The estimation of deviation of linear model, the significance of difference critical value of inoculation processing vs controls is FDR=0.05.
6th, GO is analyzed
GO is carried out using (bioinfo.cau.edu.cn) gene ontology analysis programs of AgriGO webpages
Annotate (Du et al.2010；Zhou et al.2010), reference gene group is G.max Wm82a2.v1 model.Fisher
Test uses Yekutieli corrected P, and significance is set to α=0.05.
7、qRT-PCR
Tested for examination root system sample with RNA-seq identical.The root system sample that will be preserved in liquid nitrogen, is ground into liquid nitrogen
After powder, the extraction of RNA is carried out according to its specification using TaKaRa MiniBEST Plant RNAExtraction Kit,
The integrality of RNA is detected with 1.5% agarose gel electrophoresis, the concentration and purity of RNA are detected with NanoDrop 2000.Make
CDNA synthesis, reverse transcription system are carried out with TaKaRa PrimeScriptTM RT reagent Kit with gDNA Eraser
For 40 μ L, wherein containing 14 μ L RNA solutions.
With Cons6/SKIP16 (Glyma12G05510) for control, the qRT-PCR for having carried out 10 gene expression amounts is tested
Card.Using 6.0 Software for Design of Oligo qRT-PCR primer sequences (table 1), synthesized by Hua Da gene (Beijing).QRT-PCR exists
Carried out on gene Co., Ltd EcoTM fluorescence quantitative PCR instruments, operation is with reference to TaKaRa SYBR Premix Ex TaqTM II
The kit specification of (Tli RNAaseH Plus) carries out.20 μ L qRT-PCR reaction systems include the SYBR of 10 μ L
Premix Ex TaqTM solution, the cDNA solution of 8 μ L, 0.38 μM of sense primer and 0.38 μM of anti-sense primer.Response procedures
For：50℃2min；95℃30S；(95 DEG C of 5S, 60 DEG C of 30S) × 40 circulations；95℃15S；55℃15S；95℃15S.Each sample
Product set 2 repetitions.
1 primer sequence of table
8th, DNA extractions and gene sequencing
Take 35 day age seedling leaves of Resistant gerplasm ZYD03685, the grind into powder in liquid nitrogen.According to DNA extracts reagents
Specification extraction leaves genomic DNA (CTAB methods) appended by box (Beijing DingGuo ChangSheng Biology Technology Co., Ltd).Profit
With primer SEQ ID No:16 and SEQ ID No:17 carry out the PCR amplification of XLOC_023202, using PCR product direct Sequencing
Method obtains XLOC_023202 sequences.
2nd, result
1st, soybean cyst nematode Heterodera glycines infect detection
Fig. 1 shows that soybean cyst nematode Heterodera glycines have successfully infected the root system for resisting, feeling wild soybean for examination.9 days after inoculation, 2 ages children
Worm has invaded root system (Figure 1A, E), and 15 days after inoculation, existing 2 instar larvae of part develops into 3 instar larvaes (Figure 1B, F), after inoculation
20 days, there are 2 ages, 3 ages and 4 age soybean cyst nematode Heterodera glycines (Fig. 1 C, D, G, H) at the same time in root system.
2nd, difference expression gene
RNA-seq obtains 81466062 original reads altogether, and average each library acquisition 6788839 is original
reads.Obtain 73152136 Clean reads altogether after impurity elimination, average each 6096011 Clean reads in library.
The Clean reads for having 58.4%-91.3% in library can be compared on wild soybean genome.After inoculation 9,15, and
20 days 3 Proper Sampling Periods, find 619,65 and 8 difference expression genes in disease-resistant wild soybean respectively；In susceptible wild soybean
327,460 and 115 difference expression genes are found respectively.
The parameter in 2 RNA-Seq libraries of table
aD9：9 days after inoculation；D15：15 days after inoculation；D20：20 days after inoculation；C：Control treatment (inoculation water)；T：Connect
Kind soybean cyst nematode Heterodera glycines worm's ovum；R：Disease-resistant wild soybean；S：Susceptible wild soybean.
3rd, in disease-resistant wild soybean, the difference expression gene of Proper Sampling Period discovery more than 2
Comparative analysis only exists the difference expression gene in disease-resistant wild soybean, and discovery shares 24 difference expression genes and exists
More than 2 Proper Sampling Periods all exist, wherein 18 are found and Soybean Resistance sclerotiniose, SDS, rust, soybean cyst nematode Heterodera glycines by forefathers
Disease and soybean defence it is related, remaining 6 there is not yet with disease-resistant relevant report.
4th, qRT-PCR is verified
For above-mentioned 6 there is not yet difference expression gene with disease-resistant relevant report, the qRT-PCR for having carried out its expression are tested
Card.Research finds that gene expression amount and the qRT-PCR gene expression amounts of RNA-seq are in extremely significantly correlated (P=0.01), related
Coefficient is 0.69.The gene expression amount of the RNA-seq of difference expression gene XLOC_023202 and qRT-PCR gene expression amount phases
Close property it is very high, up to 0.992, and after inoculation 9 days and 15 days when expression quantity compare photograph than substantially raise.XLOC_
023202 when not being the homologous gene of the cultivated soybean Glyma.14G003800 (unknown gene), after inoculation 9 days and 15 days, and not
Inoculation is compared, its expression quantity adds more than 5 times.
5th, XLOC_023202 gene orders
The XLOC_023202 genes of disease-resistant wild soybean germplasm ZYD03685 are obtained using PCR product direct Sequencing method
Sequence.The nucleotide sequence of XLOC_023202 genes such as SEQ ID NO:Shown in 1.It is the cultivated soybean Glyma.14G003800
Homologous gene, but 6 amino acid more than the albumen that is encoded than Glyma.14G003800 of the albumen of predictive coding.
Finally it should be noted that：The above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe is described in detail the present invention with reference to foregoing embodiments, but it will be understood by those of ordinary skill in the art that：Its
It can still modify to the technical solution described in foregoing embodiments, either to which part or all technical characteristic
Carry out equivalent substitution；And these modifications or replacement, the essence of appropriate technical solution is departed from various embodiments of the present invention skill
The scope of art scheme.
SEQUENCE LISTING
<110>Jilin Academy of Agricultural Science
<120>Application of the wild soybean XLOC_023202 genes in soybean plants cyst nematode resistance is improved
<160> 17
<170> PatentIn version 3.3
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Claims (10)
1. application of the wild soybean XLOC_023202 genes in soybean plants cyst nematode resistance is improved；XLOC_023202
The nucleotide sequence of gene such as SEQ ID NO:Shown in 1.
2. application according to claim 1, it is characterised in that using the XLOC_023202 genes as molecular labeling, training
Educate the soybean plants new varieties of high cyst nematode resistance.
3. application according to claim 1, it is characterised in that the XLOC_023202 genes are passed through into transgenic method
Soybean plants are transferred to improve its cyst nematode resistance.
4. application according to claim 3, it is characterised in that the transgenic method specifically includes：
(1) expression vector of the structure containing the XLOC_023202 genes；
(2) expression vector that step (1) obtains is introduced directly into soybean plants cell；Or led by the way that intermediate host is cell-mediated
Enter soybean plants cell；
(3) genetically modified plants are regenerated；With
(4) genetically modified plants are selected；And
(5) optionally, the plant that amplification step (4) obtains is to obtain offspring.
5. application according to claim 4, it is characterised in that the host cell includes：Agrobacterium.
6. application according to claim 4, it is characterised in that the method bag for being introduced directly into soybean plants cell
Include：Biolistic mediated transformation method, pollen tube passage method or liposome transformation.
7. application according to claim 4, it is characterised in that in step (4), when selecting genetically modified plants, identification
The expression for the XLOC_023202 genes being transferred to, selects the high plant of expressing gene expression quantity.
8. application according to claim 7, it is characterised in that the method for the high plant of the selection expressing gene expression quantity
For：
The total serum IgE of genetically modified plants to be detected is extracted, uses SEQ ID No:2 and SEQ ID No:Upstream and downstream primer shown in 3
QRT-PCR detections are carried out to XLOC_023202.
9. application according to claim 8, it is characterised in that annealing temperature of the qRT-PCR when carrying out cyclic amplification
Degree is 55 DEG C~65 DEG C.
10. according to claim 1-9 any one of them applications, it is characterised in that the soybean plants include：Soybean
Glycine max (L.) Merr., glycine tomentella Glycine tomentella Hayata, wild soybean Glycine soja
Sieb.et Zucc., Penghu soybean Glycine clandestina Wendl., the climing beans Glycine gracilis of wide leaf
Skv., cigarette beans Glycine tabacina Benth., hyacinth bean pod soybean variety Glycine dolichocarpa Tateishi＆
The first in Ohashi and broad-leaved soybean Glycine tomentella Hayata is a variety of.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711217564.0A CN107904244B (en) | 2017-11-28 | 2017-11-28 | Application of wild soybean XLOC _023202 gene in improving resistance of soybean plant cyst nematode |
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| CN108374020A (en) * | 2017-12-28 | 2018-08-07 | 南京农业大学 | A kind of agriculture bacillus mediated soybean transformation in planta method of improvement |
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| CN108374020A (en) * | 2017-12-28 | 2018-08-07 | 南京农业大学 | A kind of agriculture bacillus mediated soybean transformation in planta method of improvement |
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