CN107904244B - Application of wild soybean XLOC _023202 gene in improving resistance of soybean plant cyst nematode - Google Patents
Application of wild soybean XLOC _023202 gene in improving resistance of soybean plant cyst nematode Download PDFInfo
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Abstract
The invention relates to the technical field of biology, in particular to application of a wild soybean XLOC _023202 gene in improving the resistance of soybean plant cyst nematodes; the nucleotide sequence of XLOC _023202 gene is shown in SEQ ID NO. 1. Compared with the prior art, the resistance gene of the soybean plant cyst nematode is obviously up-regulated at 9 days and 15 days after the cyst nematode is inoculated, which suggests that the gene has very good application prospect in the aspect of improving the resistance of the soybean plant cyst nematode.
Description
Technical Field
The invention relates to the technical field of biology, in particular to application of a wild soybean XLOC _023202 gene in improving the resistance of soybean plant cyst nematodes.
Background
Soybean cyst nematode disease is an important disease in soybean production. From the interaction angle of soybean-soybean cyst nematode, the soybean resistance related genes are analyzed, the resistance mechanism of soybean to cyst nematode is further analyzed, and the screening of the resistance related genes has important value for soybean anti-cyst nematode breeding.
At present, there are many reports about gene expression analysis of soybean root systems cultivated under soybean cyst nematode infection, and most of the reports aim at early infection to try to analyze the gene expression condition of soybeans in the process of soybean cyst nematode invasion into root systems and syncytia formation. The disease-resistant mechanism of the disease-resistant germplasm is that necrosis occurs around a syncytial, so that the development of soybean cyst nematodes is limited, and even the nematodes die. Therefore, many genes related to plant defense, cellular synthesis, cellular degradation, and metabolic processes have been found in the above reports.
Wild soybeans are ancestors of cultivated soybeans and have more abundant genetic variation than the cultivated soybeans. Thus, there may be a different resistance mechanism than cultivated soybeans. At present, no report about the gene expression of wild soybean root systems under soybean cyst nematode infection is found.
Researches show that soybean has various disease-resistant mechanisms for cyst nematodes, and the disease-resistant germplasm is necrotized around syncytia in the early infection stage. Disease resistance of soybeans to cyst nematodes is present throughout the soybean cyst nematode infestation. Therefore, the soybean cyst nematode resistance genes found in the prior art are not comprehensive enough.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention researches the gene expression conditions of disease-resistant and disease-susceptible wild soybeans at 9,15 and 20 days after inoculation of soybean cyst nematodes, identifies resistance-related genes, analyzes and verifies the genes existing in more than 2 sampling periods, and finds out the gene XLOC _023202 which is specifically and differentially expressed in the disease-resistant wild soybeans and is obviously up-regulated at 9 and 15 days after inoculation and 2 periods.
The invention relates to application of wild soybean XLOC _023202 gene in improving the resistance of soybean plant cyst nematode; the nucleotide sequence of XLOC _023202 gene is shown in SEQ ID NO. 1. The gene is a homologous gene of cultivated soybean Glyma.14G003800, and the protein coded by the gene is predicted to have 6 amino acids more than that coded by Glyma.14G003800.
Preferably, the XLOC _023202 gene is used as a molecular marker to breed new varieties of soybean plants with high cyst nematode resistance.
Preferably, for use as described above, the XLOC _023202 gene is transgenically transferred to plants of the genus Glycine to increase cyst nematode resistance.
Preferably, the transgenic method specifically comprises, as described above for use:
(1) constructing an expression vector containing the XLOC _023202 gene;
(2) directly introducing the expression vector obtained in the step (1) into a soybean plant cell; or by intermediate host cell-mediated introduction into a plant cell of the genus Glycine;
(3) regenerating a transgenic plant; and
(4) selecting a transgenic plant; and is
(5) Optionally, propagating the plant obtained in step (4) to obtain progeny.
Preferably, for the uses described above, the preferred intermediate host cells are bacterial cells, especially bacterial cells for cloning or storing polynucleotides, or for transforming plant cells, such as E.coli, Agrobacterium tumefaciens and Agrobacterium rhizogenes, most preferably Agrobacterium.
The Agrobacterium may comprise a plasmid (e.g., a conventional plasmid such as the Ti or Ri plasmid) carrying the nucleotide sequence of XLOC _023202, which is transferred to the plant after transfection with the Agrobacterium, and the sequence of interest carried by the plasmid is integrated into the genome of the plant cell. The nucleotide sequences of the invention are particularly suitable for use in plant cells of soybean, but do not exclude the role of cyst nematode resistance also in other species (e.g. oilseed rape, wheat, oats, potato, etc.).
Preferably, the method for directly introducing a plant cell of the genus glycine comprises: gene gun mediated transformation, pollen tube channel method or liposome transformation.
The nucleotide sequence of XLOC _023202 can be used to transform plants and obtain transformed plants. Plant transformation is widely described in the prior art. It is well known that various systems such as plasmid vectors, liposomes, electroporation, microinjection, diffusion, gene guns, calcium phosphate coprecipitation, the use of viral vectors, etc. can be employed. In the present invention, all of which form part of the present invention.
Preferably, in the use as described above, when a transgenic plant is selected in step (4), the expression level of said XLOC _023202 gene transferred is identified, and a plant with a high expression level of the expressed gene is selected.
Preferably, the method for selecting a plant having a high expression level of an expressed gene according to the above-described application comprises:
extracting total RNA of the transgenic plant to be detected, and carrying out qRT-PCR detection on XLOC _023202 by using upstream and downstream primers shown in SEQ ID No. 2 and SEQ ID No. 3.
Preferably, for the above applications, the annealing temperature of qRT-PCR is 55-65 ℃ in the cyclic amplification.
Preferably, for use as described above, the plants of the genus glycine comprise: soybean (L.) merr, short-staple soybean (giycomentella Hayata, wild soybean (giycoja sieb. et zucc.), Penghu soybean (giycoandundina wendl.), broad-leaf vine (giycomycin) skv, tobacco bean (giycobacteria benth), hyacinth bean (leguminous soybean) and broad-leaf soybean (giycocarpa Tateishi & Ohashi).
Compared with the prior art, the resistance gene of the soybean plant cyst nematode is obviously up-regulated at 9 days and 15 days after the cyst nematode is inoculated, which suggests that the gene has very good application prospect in the aspect of improving the resistance of the soybean plant cyst nematode.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 shows that soybean cyst nematode successfully infested the root system of the wild soybeans tested; after 9 days of inoculation, 2-instar larvae already invade the root system (figures 1A and E), 15 days after inoculation, part of 2-instar larvae develop into 3-instar larvae (figures 1B and F), and 20 days after inoculation, 2-instar, 3-instar and 4-instar soybean cyst nematodes simultaneously exist in the root system (figures 1C, D, G and H);
FIG. 2 shows the RNA-seq expression amounts (Log) of 10 differentially expressed genes2(FC)) and qRT-PCR expression level (Log)2(FC)) D9: 9 days after inoculation, D15: 15 days after inoculation; d20: 20 days after inoculation.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Examples
Materials and methods
1. Preparation of soybean cyst nematode egg solution
The rhizosphere soil containing soybean cyst nematode was obtained from Jilin province: 279500, Jilin province, Nanmunicipality, academy of agricultural sciences: 27950and Douda of southern Integrated laboratory. The soybean cyst nematode HG type is HG 0. The soybean cyst nematode in the diseased soil is propagated on the soybean variety Suhong 14. Planting the seed of the seinhong 14 in the diseased soil, pulling out the root from the soil after 35 days, placing the root on a layer sieve (20, 40 and 60 meshes from top to bottom respectively), washing the cyst on the root system with water flow with larger pressure, and collecting the cyst on the 60 meshes. The cysts on the 60-mesh screen were collected in a 1L beaker, and the cyst solution was further purified using the difference in sedimentation rate of cysts, root fragments, soil, and other substances. Pouring the purified cyst solution onto a 60-mesh screen, crushing the cyst solution onto a screen by using a rubber rod to release eggs and 2-instar larvae, washing the eggs and the 2-instar larvae with a washing bottle, washing the eggs and the 2-instar larvae onto 2 layers of sleeved screens (200 meshes and 500 meshes from top to bottom in sequence), washing the 200-mesh screen for multiple times by using tap water, and collecting the eggs on the 500-mesh screen. Eggs on a 500 mesh screen were transferred to a 50mL beaker, and 23000 eggs/mL-1 were prepared as an egg suspension for inoculation.
2. Disease and infection resistant wild soybean germplasm
The wild soybean germplasm ZYD03685 is high-resistance soybean cyst nematode HG 0 population has a cyst index of 1.4, and the ZYD02688 high-susceptibility soybean cyst nematode HG 0 population has a cyst index of 101.6
3. Inoculation treatment method
60 wild soybean seeds to be tested were sterilized with 0.5% NaClO solution for 3min and then rinsed 4 times with running tap water. The disinfected seeds are carved with skin and planted in a plastic bowl filled with high-temperature sterilized perlite. After culturing for 7d at 27 ℃ under the condition of 9h light/15 h dark, the first three-leaf complex is unfolded. The seedlings were carefully pulled out and washed clean. Plants with consistent growth were divided into 6 groups of 5 plants each, 3 groups were treated with inoculated eggs and 3 groups were treated with control (inoculated seed was tap water). Each group of plants was transferred to a plastic pot containing sterilized perlite at high temperature (perlite accounts for 2% of the volume of the plastic pot), 5mL of fresh egg suspension (23000 eggs. mL-1) (inoculated egg group) or 5mL of tap water (control group) was inoculated on the root surface, and then 2cm thick sterilized perlite was coated. The cells were incubated at 27 ℃ for 9h light/15 h dark.
At 9d after inoculation, every time 1 control group and 1 treatment group are randomly selected, plants in each plastic pot are gently pulled out, and the root systems are washed clean. And randomly taking root systems of 3 plants from root systems of 5 plants, quickly placing the root systems in liquid nitrogen, and observing the infection condition of the soybean cyst nematodes by the remaining 2 plants. And pulling out the plants of the remaining 4 groups slightly, washing away the eggs and the nematodes on the surfaces of the roots, transplanting the plants into a plastic pot filled with high-temperature sterilized perlite, and placing the plastic pot at the temperature of 27 ℃ under the condition of 9h light/15 h dark for continuous culture. At 15d and 20d after inoculation, 1 control group and 1 treatment group were randomly selected, plants in each plastic pot were gently pulled out, and the roots were washed clean. And randomly taking root systems of 3 plants from root systems of 5 plants, quickly placing the root systems in liquid nitrogen, and observing the infection condition of the soybean cyst nematodes by the remaining 2 plants.
4. Extraction and sequencing of RNA
After grinding root samples stored in liquid nitrogen into powder in liquid nitrogen, total RNA was extracted using Trizol reagent ((Invitrogen, Shanghai) according to the instructions, RNA concentration and quality were determined using Agilent2100 Analyzer and Nanodrop Spectrophotometer, library construction and sequencing (Illumina HiSeq)TM2000) Completed by BGI (Shenzhen).
The integrity of the RNA was checked by electrophoresis on a 1.5% agarose gel and the concentration and purity of the RNA was checked by NanoDrop 2000. cDNA synthesis was performed using TaKaRa PrimeScript RT reagent Kit with gDNA Eraser in 40. mu.L containing 14. mu.L of RNA solution.
5. Data processing
The original sequencing sequence was subjected to the treatment of deletion of the linker and low quality sequences (the number of bases having a quality value Q. ltoreq.30 DE is 10% or more) using FastQC and FASTX toolkits. The sequence was spliced using Tophat2(v 2.0.8) with the wild soybean genome (GenBank/EMBL/DDBJ database, accession number AZNC00000000.1) as the reference genome. (Qi et al.2014.) its parameters are set to: mismatches of 1 base, 30-100,000 introns in length (Lin et al.2014) were allowed. Gene alignments and gene annotations were performed using Cufflinks v.2.2.1, using the cultivated soybean genome (g.max wm82a2.v1., downloaded from Phytozome10) as the reference genome. (Trapnell et al 2012) gene expression statistical analysis was performed using HTSeq v.0.6.1 (Anders et al 2015), edgeR v.3.2.3 differential expression analysis (Robinson et al 2010; Anders et al 2013) deviation estimation of the generalized linear model was performed using homologous sequences of 28 housekeeping genes, and the threshold value of differential significance of vaccination treatment vs control was FDR 0.05.
6. GO analysis
GO annotation was performed using the (biolnfo. cau. edu. cn) gene ontology analysis program of the AgriGO webpage (Du et al 2010; Zhou et al 2010), using Yekutieli corrected P with reference to genome g.max wm82a2.v1 model. fisher test, and the significance level was set to α 0.05.
7、qRT-PCR
The root samples tested were identical to those of the RNA-seq experiment. After grinding the root samples stored in liquid nitrogen into powder in liquid nitrogen, RNA extraction was performed using TaKaRa MiniBEST Plant RNAextraction Kit according to the instructions, RNA integrity was checked by 1.5% agarose gel electrophoresis, and RNA concentration and purity were checked by NanoDrop 2000. cDNA synthesis was performed using TaKaRa PrimeScript RT reagent Kit with gDNA Eraser in 40. mu.L containing 14. mu.L of RNA solution.
As a control, Cons6/SKIP16(Glyma12G05510) was used to verify the expression level of 10 genes by qRT-PCR. qRT-PCR primer sequences (Table 1) were designed using Oligo 6.0 software and synthesized from the Huada gene (Beijing). qRT-PCR was performed on an EcoRM fluorescent quantitative PCR instrument, a Gene Co., Ltd, with reference to the kit instructions of TaKaRa SYBR Premix Ex TaqTM II (Tli RNAaseH Plus). A20. mu.L qRT-PCR reaction system contained 10. mu.L SYBR Premix Ex TaqTM solution, 8. mu.L cDNA solution, 0.38. mu.M upstream primer and 0.38. mu.M downstream primer. The reaction procedure is as follows: 2min at 50 ℃; 30S at 95 ℃; (95 ℃ 5S, 60 ℃ 30S). times.40 cycles; 15S at 95 ℃; 15S at 55 ℃; 15S at 95 ℃.2 replicates were set up for each sample.
TABLE 1 primer sequences
8. DNA extraction and Gene sequence determination
Taking 35-day-old seedling leaves of the disease-resistant germplasm ZYD03685, and grinding the leaves in liquid nitrogen to form powder. The leaf genome DNA was extracted according to the instructions attached to the DNA extraction kit (the limited liability company for Changsheng biotechnology, Beijing ancient cooking) (CTAB method). XLOC-023202 PCR amplification was performed using primers SEQ ID No. 16 and SEQ ID No. 17, and the XLOC-023202 sequence was obtained by direct sequencing of the PCR products.
Second, result in
1. Infection detection of soybean cyst nematode
FIG. 1 shows that soybean cyst nematode successfully infested the root system of the wild soybean that was tested against. At 9 days after inoculation, 2-instar larvae have invaded the root system (fig. 1A, E), 15 days after inoculation, part of 2-instar larvae have developed into 3-instar larvae (fig. 1B, F), and 20 days after inoculation, 2-, 3-and 4-instar soybean cyst nematodes are present in the root system at the same time (fig. 1C, D, G, H).
2. Differentially expressed genes
RNA-seq yielded a total of 81466062 original reads, and 6788839 original reads were obtained on average per library. After the impurities are removed, 73152136 Clean reads are obtained in total, and 6096011 Clean reads are averagely obtained in each library. 58.4% -91.3% of Clean reads in the library can be aligned to the wild soybean genome. 619, 65 and 8 differentially expressed genes were found in disease-resistant wild soybeans at 3 sampling periods 9,15 and 20 days after inoculation, respectively; 327, 460 and 115 differentially expressed genes were found in infected wild soybeans, respectively.
TABLE 2 parameters of the RNA-Seq library
aD9: 9 days after inoculation; d15: 15 days after inoculation; d20: 20 days after inoculation; c: control treatment (inoculation water); t: inoculating soybean cyst nematode eggs; r: disease-resistant wild soybean; s: susceptible wild soybean.
3. Differentially expressed genes found in more than 2 sampling periods in disease-resistant wild soybeans
Compared and analyzed, only the differentially expressed genes in the disease-resistant wild soybeans exist, and 24 differentially expressed genes are found to exist in more than 2 sampling periods, wherein 18 differentially expressed genes are found to be related to soybean sclerotinia rot, SDS (sodium dodecyl sulfate), rust disease, soybean cyst nematode and soybean defense by predecessors, and the other 6 differentially expressed genes are not reported to be related to disease resistance.
4. qRT-PCR validation
Aiming at the 6 differential expression genes which are not reported to be related to disease resistance, qRT-PCR verification of the expression is carried out. The research shows that the gene expression level of RNA-seq is extremely obviously related to the gene expression level of qRT-PCR (P is 0.01), and the correlation coefficient is 0.69. The correlation between the gene expression quantity of RNA-seq of the differential expression gene XLOC _023202 and the gene expression quantity of qRT-PCR is very high, reaching 0.992, and the expression quantity is obviously up-regulated compared with the photographic ratio at 9 days and 15 days after inoculation. XLOC-023202 is a homologous gene of cultivated soybean Glyma.14G003800 (unknown gene), and its expression level was increased by 5-fold or more at 9 days and 15 days after inoculation as compared with that without inoculation.
5. XLOC-023202 gene sequence
The XLOC _023202 gene sequence of the disease-resistant wild soybean germplasm ZYD03685 is obtained by a PCR product direct sequencing method. The nucleotide sequence of XLOC _023202 gene is shown in SEQ ID NO. 1. Is a homologous gene of the cultivated soybean Glyma.14G003800, but the protein coded by the gene is predicted to have 6 amino acids more than that coded by Glyma.14G003800.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
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Claims (1)
1. The application of wild soybean XLOC _023202 gene in improving soybean cyst nematode resistance; the XLOC-023202 gene is characterized in that the nucleotide sequence of the XLOC-023202 gene is shown as SEQ ID NO. 1.
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