CN117903267A - Novel soybean cyst nematode resistant gene and application thereof - Google Patents
Novel soybean cyst nematode resistant gene and application thereof Download PDFInfo
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Abstract
The invention discloses a novel soybean cyst nematode resistant gene and application thereof. In particular to a new variant alpha-SNAP lmm3 of soybean alpha-SNAP gene obtained by artificial chemical mutagenesis to enhance the resistance of soybean cyst nematode and application thereof. The invention creates a novel alpha-SNAP variant gene-alpha-SNAP lmm3 which participates in the resistance regulation of soybean cyst nematode by an artificial mutagenesis method. The gene creation, functional analysis and breeding application value exploration provides important gene foundation and theoretical support for research on related mechanisms of soybean cyst nematode resistance, provides valuable gene resources and application guidance for research and application of a plant defense system and cultivation of new soybean varieties with high disease resistance, and has important application value in soybean disease resistance breeding.
Description
Technical Field
The invention relates to the technical field of plant crossbreeding technology and plant genetic engineering, in particular to a novel soybean cyst nematode resistance gene and application thereof.
Background
Soybean cyst nematode disease commonly occurs in 20 provincial soybean main production areas in China, causing economic loss of 1.2 hundred million dollars, and seriously jeopardizing soybean production in China. The excavation of the disease resistance gene and the cultivation of disease-resistant varieties are important measures for preventing and treating the disease. Rhg1 is the major resistance locus for soybean cyst nematode disease consisting of tandem repeats of four genes, providing soybean cyst nematode disease resistance primarily through copy number accumulation, with cultivars of more than 16 copies of Rhg showing resistance to soybean cyst nematode disease, while single copies of Rhg1 are susceptible to soybean cyst nematode disease. The alpha-SNAP gene is the most important gene contributing to soybean cyst nematode resistance in the Rhg1 locus, and in a multi-copy resistant variety of Rhg1, the alpha-SNAP gene achieves nematode resistance by accumulating copies and generating cytotoxicity by impaired conserved vesicle transport functions of its variant atypical rhg1α -SNAP, whereas the multi-copy resistant soybean of Rhg1 can survive and its yield is unaffected because atypical NSF co-evolution releases cytotoxicity of the atypical rhg1α -SNAP gene therein. However, the atypical Rhg1α -SNAP gene which is naturally mutated in soybean varieties which do not contain a single copy of Rhg1 of atypical NSF has not been found yet, whether it has soybean cyst nematode disease resistance and its molecular mechanism of disease resistance are unclear, and the mechanism of maintaining soybean growth survival by this gene has yet to be explored.
Disclosure of Invention
The invention aims to provide a novel soybean cyst nematode resistance gene and application thereof, namely, novel mutation of soybean alpha-SNAP gene obtained by artificial chemical mutagenesis enhances disease and insect resistance and application thereof.
The invention creates a new atypical Rhg1α -SNAP soybean cyst nematode resistant gene α -SNAP lmm3 by artificial mutagenesis in Rhg1 single-copy soybean varieties without atypical NSF. The Gmlmm mutant carrying the cytotoxic alpha-SNAP lmm3 gene shows a spot-like phenotype while improving soybean cyst nematode resistance, and can still complete a complete life cycle through normal fruiting despite a certain decrease in soybean yield. In addition, the heterozygous plant carrying the alpha-SNAP lmm3 gene and the transgenic over-expression plant can not only effectively resist nematodes but also stably produce, which shows that the novel atypical Rhg1alpha-SNAP soybean cyst nematode disease resistance gene alpha-SNAP lmm3 created in the Rhg1 single-copy variety has a disease resistance mechanism and a detoxification mechanism different from the atypical alpha-SNAP in the Rhg1 multi-copy variety. The alpha-SNAP lmm3 gene is created, the functions of the gene are researched and the gene is applied to soybean disease-resistant breeding, and the gene has important scientific significance and application value to soybean industry.
To achieve the object of the present invention, in a first aspect, the present invention provides a novel soybean cyst nematode resistant gene, which is a novel variant α -SNAP lmm3 of soybean α -SNAP gene, which is a gene encoding the following protein (a) or (b):
(a) A protein consisting of the amino acid sequence shown in SEQ ID NO. 1; or (b)
(B) And (b) a protein which is derived from (a) and has equivalent functions and is obtained by substituting, deleting or adding one or more amino acids in the sequence shown in SEQ ID NO. 1.
In a second aspect, the invention provides a biological material comprising the α -SNAP lmm3 gene or an activating factor thereof, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineering bacteria, or transgenic cell lines.
In a third aspect, the invention provides the use of an α -SNAP lmm3 gene or an activator thereof or a biological material containing an α -SNAP lmm3 gene or an activator thereof in genetic breeding of plants or in the preparation of transgenic plants.
In a fourth aspect, the invention provides the use of an α -SNAP lmm3 gene or an activator thereof or a biomaterial containing an α -SNAP lmm3 gene or an activator thereof for modulating resistance to soybean cyst nematode in a plant.
In a fifth aspect, the invention provides the use of an α -SNAP lmm3 gene or an activator thereof or a biological material containing an α -SNAP lmm3 gene or an activator thereof to regulate plant cell death.
In a sixth aspect, the invention provides the use of an α -SNAP lmm3 gene or an activator thereof or a biomaterial comprising an α -SNAP lmm3 gene or an activator thereof for modulating selective autophagy degradation detoxification in a plant.
According to the application of the fourth to sixth aspects, the regulation is positive regulation.
In a seventh aspect, the present invention provides a method of increasing resistance to soybean cyst nematode, enhancing plant cell death, or maintaining plant survival by selective autophagy degradation detoxification in a plant, the method comprising: the alpha-SNAP lmm3 gene is over expressed in plants by using genetic engineering means.
The means of overexpression may be selected from the following 1) to 5), or optionally in combination:
1) By introducing a plasmid having the gene;
2) By increasing the copy number of the gene on the plant chromosome;
3) By altering the promoter sequence of said gene on the plant chromosome;
4) By operably linking a strong promoter to the gene;
5) By introducing enhancers.
Further, an over-expression vector of the gene is constructed and transformed into a plant.
The gene is driven by a soybean cyst nematode inducible promoter HIP, and the promoter HIP is:
i) A nucleotide sequence shown in SEQ ID NO. 3;
ii) the nucleotide sequence shown in SEQ ID NO. 3 is substituted, deleted and/or added with one or more nucleotides and has the same function;
iii) A nucleotide sequence which hybridizes with the sequence shown in SEQ ID No. 3 and has the same function under stringent conditions, i.e., in a 0.1 XSSPE solution containing 0.1% SDS or a 0.1 XSSC solution containing 0.1% SDS, at 65℃and washing the membrane with the solution; or (b)
Iv) a nucleotide sequence which has more than 90% homology with the nucleotide sequence of i), ii) or iii) and has the same function.
The expression vector carrying the gene of interest can be introduced into plant cells by conventional biotechnological methods using Ti plasmids, plant viral vectors, direct DNA transformation, microinjection, electroporation, etc. (Weissbach, 1998,Method for Plant Molecular Biology VIII,Academy Press,New York, pages 411-463; geiserson and Corey,1998,Plant Molecular Biology,2 nd edition).
In the present invention, the plant is a monocotyledonous plant or a dicotyledonous plant. Such plants include, but are not limited to, soybean, arabidopsis, wheat, rice, maize, cotton, peanut, and the like.
By means of the technical scheme, the invention has at least the following advantages and beneficial effects:
The invention creates a novel alpha-SNAP variant gene alpha-SNAP lmm3.α-SNAPlmm3 gene which participates in the regulation and control of soybean cell death, selective autophagy degradation detoxification and soybean cyst nematode resistance in a Rhg1 single copy variety for the first time, and can positively regulate plant cell death and soybean cyst nematode resistance: by increasing the expression level of the alpha-SNAP lmm3 gene, the cytotoxicity of plants can be effectively enhanced, and the resistance of plants to soybean cyst nematode can be improved (the susceptibility and the morbidity of soybean cyst nematode can be reduced). The interaction of alpha-SNAP lmm3 and GmATG8 degrades and detoxifies through a selective autophagy pathway, maintains soybean survival, and achieves the effectiveness of enhancing soybean cyst nematode resistance. The creation and functional analysis of a novel atypical alpha-SNAP gene alpha-SNAP lmm3 coded in the Rhg1 single-copy soybean variety are breakthrough progress in the research of soybean cyst nematode disease resistance mechanism, emphasize the effect of autophagy degradation detoxification in enhancing soybean cyst nematode disease resistance, provide important gene foundation and theoretical support for research of soybean cyst nematode disease resistance related mechanisms, provide valuable gene resources for the research and application of a plant defense system and the cultivation of a novel soybean variety with high disease resistance, and have great application value in soybean disease resistance genetic engineering breeding of the alpha-SNAP lmm3 gene and an activating factor thereof.
Drawings
FIG. 1 is a phenotypic comparison of wild type Williams82 and Gmlmm mutants in example 1 of the present invention. Wherein, A is the whole plant phenotype of Williams82 (left) and Gmlmm mutant (right) planted for 62 days; b is Williams82 (left) and Gmlmm-3 mutant (right) three-leaf compound small She Biaoxing planted for 28 days.
FIG. 2 shows the localization of the α -SNAP lmm3 gene to soybean chromosome 18 in example 2 of the present invention. Wherein A is BSA sequencing positioning target gene candidate interval; b is the map cloning method to locate the target gene.
FIG. 3 shows the resistance phenotype of Gmlmm mutant of soybean cyst nematode inoculated in example 3 of the present invention. Wherein, A is the developmental phenotype of the nematode in Williams 82 (left) and Gmlmm mutant (right) roots 15 days after nematode inoculation; b is the proportion of soybean roots of Williams 82 and Gmlmm mutants that develop over J2 years of nematode growth at 15 days of nematode inoculation; c is the number of soybean cyst nematode cysts per plant 30 days after nematode inoculation. * P <0.05, P <0.001.
FIG. 4 is a graph showing that the defect in the interaction between α -SNAP lmm3 and NSF in example 4 of the present invention resulted in cytotoxicity, and that α -SNAP lmm3 was super-accumulated at the Soybean Cyst Nematode (SCN) infection site. Wherein, A is BiFC experiment to prove that the interaction between alpha-SNAP lmm3 and NSF is damaged; b is Co-IP experiment to prove that the interaction between alpha-SNAP lmm3 and NSF is damaged; transient expression of the alpha-SNAP lmm3 gene in tobacco leaves produces cytotoxicity to induce cell death; d is that the alpha-SNAP lmm3 protein is infected and accumulated by soybean cyst nematode; the E is an immunogold experiment, which shows that the alpha-SNAP lmm3 protein is induced to accumulate at a syncytia position by nematode infection. And (3) injection: different lowercase letters indicate that the differences are significant.
FIG. 5 shows the autophagy process in example 5 of the present invention in which alpha-SNAP lmm3 interacts with GmATG to activate the Gmlmm3 mutant by selective autophagy pathway degradation. Wherein, the accumulation of alpha-SNAP lmm3 protein in the blade of the Gmlmm mutant is obviously reduced; the accumulation of alpha-SNAP lmm3 protein in the Gmlmm mutant root is obviously reduced; c is autophagy activated in the Gmblmm 3 mutant compared to wild-type Williams 82; d is a BiFC experiment demonstrating the interaction of α -SNAP lmm3 and GmATG 8; e is Co-IP experiments demonstrating the interaction of alpha-SNAP lmm3 and GmATG. * P <0.001.
FIG. 6 shows significant breeding potential for the α -SNAP lmm3 heterozygous plants of example 6 of the present invention. Wherein, A is the phenotype of the whole plant after the leaves of the Williams 82 and Gmlmm mutants and the heterozygous plants are removed (upper part) and the corresponding single plant yield graph (lower part), which shows that the heterozygous plants have good yield similar to the wild plants; b is the cyst count of each plant of Williams 82, gmlmm mutant and heterozygous plant after infection by the line, indicating that the heterozygous plant has soybean cyst nematode resistance similar to homozygous mutant Gmlmm 3. And (3) injection: different lowercase letters indicate that the differences are significant.
FIG. 7 shows that the soybean cyst nematode inducible promoter drives expression of the α -SNAP lmm3 gene in transgenic soybean roots of example 7 of the present invention significantly improves soybean cyst nematode resistance. Wherein A is a schematic diagram of a control vector and an overexpression vector of a soybean cyst nematode inducible promoter HIP driven alpha-SNAP lmm3; b is Williams 82 inoculated with nematodes for 12 days through the hair roots after transient overexpression of alpha-SNAP lmm3 by a control vector control and an experimental vector HIP, which shows that the soybean cyst nematode inducible promoter HIP drives the alpha-SNAP lmm3 transgenic hair roots to obviously resist nematodes; c is statistics of nematode susceptibility corresponding to B. And (3) injection: different lowercase letters indicate that the differences are significant.
Detailed Description
The invention aims to provide a soybean anti-soybean cyst nematode related gene alpha-SNAP lmm3 created by artificial mutagenesis and application thereof in regulating and controlling plant cell death, selective autophagy degradation detoxification and soybean cyst nematode resistance.
The invention adopts the following technical scheme:
The invention obtains a soybean mutant library created by artificial chemical mutagenesis, which is a spot-like mutant Gmlmm (Glycine max lesion mimic mutant) related to soybean cyst nematode disease resistance, positions a target gene between 1.57Mb and 1.73Mb of chromosome 18 through map cloning, obtains the target gene through sequencing analysis, and names the target gene as alpha-SNAP lmm3, wherein the amino acid sequence of the encoded protein is shown as SEQ ID NO. 1, and the CDS sequence is shown as SEQ ID NO. 2. The invention discovers that the alpha-SNAP lmm3 can accumulate and regulate the resistance of the soybean cyst nematode at the nematode infection position.
In a first aspect, the invention provides the use of a soybean α -SNAP lmm3 protein or a gene encoding the same or an activator of a gene encoding a soybean α -SNAP lmm3 protein in modulating resistance to soybean cyst nematode in a plant.
In a second aspect, the invention provides the use of a soybean α -SNAP lmm3 protein or a gene encoding it or an activator of a gene encoding a soybean α -SNAP lmm3 protein in modulating plant cell death.
In a third aspect, the invention provides the use of soybean alpha-SNAP lmm3 protein or a gene encoding the same or an activator of a gene encoding soybean alpha-SNAP lmm3 protein in regulating selective autophagy degradation detoxification of plants.
In the application, the soybean cyst nematode resistance of the plant is improved by increasing the expression level of the coding gene of the alpha-SNAP lmm3 protein in the plant.
In a fourth aspect, the invention provides the use of an activator of a soybean α -SNAP lmm3 protein or a gene encoding the same, or a gene encoding a soybean α -SNAP lmm3 protein, in genetic breeding of plants or in the preparation of transgenic plants.
Preferably, the transgenic plant is a disease resistant transgenic plant. More preferably a transgenic plant resistant to soybean cyst nematode.
In the invention, the soybean alpha-SNAP lmm3 protein has any one of the following amino acid sequences:
(1) An amino acid sequence shown as SEQ ID NO. 1;
(2) An amino acid sequence with the same functional protein obtained by replacing, inserting or deleting one or more amino acids in the amino acid sequence shown as SEQ ID NO. 1;
(3) An amino acid sequence having at least 80% homology with the amino acid sequence shown in SEQ ID NO. 1; preferably, the homology is at least 90%; more preferably 95%.
In the invention, the CDS of the soybean alpha-SNAP lmm3 protein has any one of the following nucleotide sequences:
(1) A nucleotide sequence shown as SEQ ID NO. 2;
(2) The nucleotide sequence shown as SEQ ID NO. 2 is obtained by replacing, inserting or deleting one or more nucleotides to obtain the nucleotide sequence encoding the same functional protein.
The amino acid sequence shown in SEQ ID NO. 1 is the amino acid sequence of soybean alpha-SNAP lmm3 protein, and one skilled in the art can substitute, delete and/or add one or more amino acids according to the conventional technical means in the art such as the amino acid sequence disclosed by the invention, conservative substitution of amino acids and the like on the premise of not affecting the activity of the amino acid sequence, so as to obtain the mutant of the alpha-SNAP lmm3 protein with the same activity as the alpha-SNAP lmm3 protein disclosed by the invention.
The nucleotide sequence shown in SEQ ID NO. 2 is the CDS sequence of alpha-SNAP lmm3 protein in soybean. The coding gene of the alpha-SNAP lmm3 protein can be any nucleotide sequence capable of coding the alpha-SNAP lmm3 protein. In view of the degeneracy of codons and the preferences of codons of different species, one skilled in the art can use codons appropriate for expression of a particular species as desired.
Preferably, the activating factor of the encoding gene of the soybean alpha-SNAP lmm3 protein comprises a soybean cyst nematode inducible promoter capable of improving the expression of the encoding gene of the soybean alpha-SNAP lmm3 protein.
The invention provides a soybean cyst nematode inducible promoter for activating the expression of a coding gene of soybean alpha-SNAP lmm3 protein, which comprises a nucleotide sequence shown as SEQ ID NO. 3. The soybean cyst nematode inducible promoter can drive the alpha-SNAP lmm3 protein to induce expression during nematode infection, so that the overexpression of the encoding gene of the soybean alpha-SNAP lmm3 protein is realized.
The above-mentioned alpha-SNAP lmm3 protein or the gene encoding the same or the gene encoding the soybean alpha-SNAP lmm3 protein may be used as the alpha-SNAP lmm3 protein or the gene encoding the same or the gene encoding the soybean alpha-SNAP lmm3 protein, or as an expression cassette, a vector, or a host cell containing the expression cassette or the vector containing the gene encoding the alpha-SNAP lmm3 protein or the activating factor thereof.
In a fifth aspect, the invention provides a method of modulating resistance to soybean cyst nematode in a plant comprising: regulating and controlling the expression quantity of a coding gene of soybean alpha-SNAP lmm3 protein in a plant;
the soybean alpha-SNAP lmm3 protein has any one of the following amino acid sequences:
(1) An amino acid sequence shown as SEQ ID NO. 1;
(2) An amino acid sequence with the same functional protein obtained by replacing, inserting or deleting one or more amino acids in the amino acid sequence shown as SEQ ID NO. 1;
(3) An amino acid sequence having at least 80% homology with the amino acid sequence shown in SEQ ID NO. 1; preferably, the homology is at least 90%; more preferably 95%.
Preferably, the method comprises the steps of: increasing the soybean cyst nematode resistance of said plant by increasing the expression level of the gene encoding the alpha-SNAP lmm3 protein in said plant.
The above-mentioned increase in the expression level of the gene encoding the α -SNAP lmm3 protein in plants can be achieved by conventional means in the art, for example: the coding gene of the alpha-SNAP lmm3 protein in the plant is over expressed by using a transgenic technology.
Preferably, the invention uses the transgenic technology, uses the nucleotide sequence shown as SEQ ID NO. 3 as a soybean cyst nematode inducible promoter to improve the coding gene of the alpha-SNAP lmm3 protein in the plant, and can obviously improve the expression quantity of the coding gene of the alpha-SNAP lmm3 protein in the plant.
The alpha-SNAP lmm3 gene coded protein is characterized in that the C end 24 amino acids of the alpha-SNAP protein are truncated, and a novel atypical alpha-SNAP variant protein is shown. The interaction between the alpha-SNAP variant protein alpha-SNAP lmm3 and NSF protein is damaged, plant cytotoxicity is generated, and the alpha-SNAP lmm3 is induced to accumulate in syncytia by infection of soybean cyst nematodes, so that the soybean cyst nematodes are resistant. Gmlmm3 mutant carrying homozygous α -SNAP lmm3 gene showed increased resistance to soybean cyst nematode. In Gmlmm mutant, the interaction of toxic alpha-SNAP lmm3 protein and GmATG protein is spontaneously degraded through selective autophagy, and plays a role in detoxification, so that Gmlmm mutant maintains the complete life cycle. Based on the soybean cyst nematode resistance of the alpha-SNAP lmm3 and an innovative autophagy spontaneous degradation detoxification mechanism thereof, in order to obtain new soybean varieties which effectively resist nematodes and can be stably produced, hybrid plants carrying the alpha-SNAP lmm3 gene and transgenic soybean roots are obtained through a hybridization technology and a transgenic technology, and the soybean cyst nematode resistance is higher without affecting soybean growth vigor and yield.
The invention provides important gene foundation and theoretical support for research on related mechanisms of soybean cyst nematode resistance, provides valuable gene resources for research and application of plant defense system promotion and cultivation of new soybean varieties with high disease resistance, and has important application value in soybean disease resistance breeding.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or in accordance with the manufacturer's instructions.
Example 1Gmlmm construction of isolated populations of mutants and phenotypic analysis
Mutant Gmlmm was obtained by screening a library of mutants created by artificial EMS mutagenesis Williams 82, whose leaves developed a plaque-like phenotype (FIG. 1), and were subjected to background purification by successive selfing for 4 generations. Obtaining BCF2 segregating populations for BSA sequencing to locate target gene intervals by backcrossing with Williams 82; meanwhile, F2 segregation population for map-based cloning is constructed by hybridization with a remote variety of 'lotus bean 12'; the phenotype observation and statistical analysis of the F2 segregating population show that the mutant is stably inherited and accords with Mendelian's law of inheritance (3:1 segregating ratio), and the plaque-like phenotype of Gmlmm mutant is recessive single-gene control.
Example 2 positioning of soybean anti-soybean cyst nematode related Gene alpha-SNAP lmm3
Obtaining F2 segregating populations for BSA sequencing and positioning target gene intervals by adopting Gmlmm and Williams 82 backcross, wherein the significant peak value of SNP index distribution is positioned in the first 5Mb interval of chromosome 18 (FIG. 2A); gene localization was performed using the F2 segregating population hybridized with Gmlmm and "lotus bean 12", and the coarse localization results indicated that the mutation site was located between chromosome 18 molecular marker MOL4694 (1.19 Mb) and molecular marker MOL4696 (5.20 Mb). To further determine the position of Gmlmm mutant target gene, a new Indel molecular marker was designed in the region between 5.20Mb and 1.19Mb of chromosome 13 to fine-position the mutant individual, and finally the target gene was positioned in the 161kb interval between 1.57Mb and 1.73Mb of chromosome 18 (FIG. 2B). In the 161kb interval, 24 candidate genes are combined, and the BSA sequencing result shows that only mutation of single base G to A exists in the 9 th exon region of Glyma.18G022500 gene in the 161kb interval, the base mutation leads to premature termination of amino acid coding, and the mutated gene is named as an alpha-SNAP lmm3 gene. Thus, it was concluded that the leaf blade appearance of the Gmlmm mutant, the plaque-like phenotype, was caused by the α -SNAP lmm3 gene.
EXAMPLE 3Gmlmm characterization of mutant anti-soybean cyst nematode phenotype
When the Williams 82 and Gmlmm3 mutants were inoculated with soybean cyst nematodes for 15 days, the nematode had developed to the J3 phase in the Williams 82 roots, while the nematodes in the Gmlmm mutant were still in the J2 phase, indicating that Gmlmm inhibited nematode development and had soybean cyst nematode disease resistance (fig. 3A); the ratio of wild type and mutant over J2-age nematodes to all infested nematodes was counted at 15 days of nematode infestation, and the ratio was found to be significantly lower in the mutant than in the wild type (FIG. 3B), and the number of cysts in soybean roots at 30 days of nematode inoculation was further counted, and the number of cysts per plant in Gmlmm mutant was significantly reduced (FIG. 3C), which showed that Gmlmm3 mutant was resistant to soybean cyst nematode disease (FIG. 3).
Example 4 abnormal interaction of alpha-SNAP lmm3 with NSF protein to produce phytocytotoxicity, accumulation of anti-soybean cyst nematode at SCN feeding site
The BiFC experiment was performed using Agrobacterium-injected tobacco to transiently express the α -SNAP lmm3 and NSF protein (NCBI accession number: LOC 100779259), indicating that the two cannot interact (FIG. 4A); likewise, co-IP experiments (Co-immunoprecipitation experiments) again demonstrated impaired alpha-SNAP lmm3 and NSF interactions (FIG. 4B); in addition, transient expression of the α -SNAP lmm3 gene in tobacco leaves found that α -SNAP lmm3 exhibited a pattern similar to that of α -SNAP HC、α-SNAPLC(α-SNAPHC for Rhg1 HCα-SNAP,α-SNAPLC for Rhg1 LC α -SNAP; similar cell death phenomena can be referred to in literature Bayless,A.M.et al.An atypical N-ethylmaleimide sensitive factor enables the viability of nematode-resistant Rhg1 soybeans.Proc.Natl.Acad.Sci.USA 115,E4512-E4521(2018).), demonstrating that α -SNAP lmm3 has cytotoxicity-inducible cell death (fig. 4C); immune hybridization is carried out on wild type Williams 82 varieties, mutants Gmlmm, nematode resistant varieties Forrest and PI88788 which are inoculated with soybean cyst nematodes for 3 days or are not subjected to nematode inoculation by utilizing a synthesized customized antibody anti-alpha-SNAP, and the result shows that alpha-SNAP lmm3 proteins are remarkably accumulated after infection by the nematode (figure 4D); the Gmlmm mutant was inoculated with soybean cyst nematode for 3 days and subjected to immunogold hybridization, and it was found that the α -SNAP lmm3 protein accumulated significantly at the syncytial position (fig. 4E).
EXAMPLE 5 alpha-SNAP lmm3 autophagy degradation detoxification
The synthesized custom antibody anti-alpha-SNAP is utilized to detect the alpha-SNAP protein accumulation in leaves (FIG. 5A) and roots (FIG. 5B) of Williams 82, gmlmm mutant, forrest and PI88788 varieties, and the result shows that the alpha-SNAP lmm3 protein content in the Gmlmm mutant is reduced; to further confirm activation of autophagy in the Gmlmm mutant, root segments from Williams 82 and Gmlmm3 were fixed and analyzed by TEM (transmission electron microscopy). After staining, TEM images (fig. 5C) showed that the presence of the double membrane autophagosome was evident in Gmlmm root cells, whereas it was hardly observed in wild type controls. Quantification of the number of autophagosomes per square micron area indicated that the Gmlmm mutant exhibited nearly 9-fold more autophagosomes than the wild-type control; further by performing BiFC (bimolecular fluorescence complementation) and Co-IP experiments with overexpressed soybean protein in nicotiana benthamiana, it was demonstrated that the α -SNAP lmm3 protein can interact directly with GmATG8 (fig. 5d, e), by which it was speculated that α -SNAP lmm3 protein in Gmlmm mutant was degraded by the selective autophagy pathway to alleviate cytotoxicity so that Gmlmm3 mutant plants could survive.
Example 6α -SNAP lmm3 heterozygous plants showed excellent breeding potential
According to the early analysis of the nematode-resistant mechanism and autophagy detoxification mechanism of the alpha-SNAP lmm3 gene, we used the alpha-SNAP lmm3 gene to search for breeding applications, found that heterozygotes, unlike homozygous Gmlmm3 plants with leaf necrosis and yield loss, did not exhibit leaf damage and maintained normal yield levels (fig. 6A), indicating that the presence of a single copy of the normal alpha-SNAP gene was able to compensate for the cytotoxic effects caused by alpha-SNAP lmm3. Wild-type plants, williams 82, gmlmm mutants, and heterozygous plants were inoculated with J2-age soybean cyst nematodes and the total number of cysts per plant was counted after 45 days, and heterozygotes were found to exhibit significant soybean cyst nematode resistance similar to homozygous mutant Gmlmm (fig. 6B). The result shows that the preparation of the alpha-SNAP lmm3 gene heterozygous plant can have important disease-resistant soybean breeding value.
Example 7α -SNAP lmm3 overexpression of transgenic hairy roots significantly improved soybean cyst nematode resistance
Due to the separation of heterozygous plants and the instability of genetic breeding, the breeding application value of the over-expressed transgene of the alpha-SNAP lmm3 gene is explored. We used root hair transformation, which is both time efficient and provides important insight for phenotypic analysis compared to stable transformation. Firstly, constructing a binary vector for driving alpha-SNAP lmm3 by using pCAMBIA3301 as a starting vector and using a soybean cyst nematode inducible promoter HIP (the promoter HIP sequence is shown as SEQ ID NO: 3) (figure 7A); then we transferred the vector into the Rhg1 single copy variety Williams 82 and the obtained transiently overexpressed roots were inoculated with soybean cyst nematodes, which showed that HIP-driven α -SNAP lmm3 transgenic roots exhibited significant resistance to soybean cyst nematodes compared to control empty transformed roots (fig. 7, b and C). The genetic transformation experimental result further shows the breeding applicability of the alpha-SNAP lmm3, and proves that the novel atypical Rhg1alpha-SNAP soybean cyst nematode disease resistance gene alpha-SNAP lmm3 can be applied to the production of soybean cyst nematode resistance breeding. The novel atypical Rhg1α -SNAP soybean cyst nematode resistant gene α -SNAP lmm3 created by artificial chemical mutagenesis provides valuable gene resources for genetic breeding of plants.
The invention creates a new atypical Rhg1α -SNAP gene- α -SNAP lmm3 which participates in the resistance regulation of soybean cyst nematode in Rhg1 single-copy soybean by artificial mutagenesis. The gene creation, functional analysis and breeding application value exploration provides important gene foundation and theoretical support for research on related mechanisms of soybean cyst nematode resistance, provides valuable gene resources for research and application of a plant defense system and cultivation of new soybean varieties with high disease resistance, and has important application value in soybean disease resistance breeding.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (10)
1. A novel soybean cyst nematode resistant gene, characterized in that it is a novel variant α -SNAP lmm3 of the soybean α -SNAP gene, which is a gene encoding the following protein (a) or (b):
(a) A protein consisting of the amino acid sequence shown in SEQ ID NO. 1; or (b)
(B) And (b) a protein which is derived from (a) and has equivalent functions and is obtained by substituting, deleting or adding one or more amino acids in the sequence shown in SEQ ID NO. 1.
2. A biological material comprising the gene or the activating element thereof according to claim 1, wherein the biological material is a recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector or an engineering bacterium.
3. Use of the gene according to claim 1 or an activator thereof or the biological material according to claim 2 in genetic breeding of plants or in the preparation of transgenic plants.
4. Use of the gene according to claim 1 or an activator thereof or the biomaterial according to claim 2 for modulating resistance to soybean cyst nematode in a plant.
5. Use of the gene of claim 1 or an activator thereof or the biomaterial of claim 2 for regulating plant cell death.
6. Use of the gene of claim 1 or an activator thereof or the biomaterial of claim 2 for regulating selective autophagy degradation detoxification of plants.
7. The use according to any one of claims 4-6, wherein the modulation is positive.
8. A method of increasing resistance to soybean cyst nematode disease in a plant, enhancing plant cell death, or maintaining plant survival by selective autophagy degradation detoxification, the method comprising: overexpressing the gene of claim 1 in plants by genetic engineering means;
The manner of overexpression is selected from the following 1) to 5), or an optional combination:
1) By introducing a plasmid having the gene;
2) By increasing the copy number of the gene on the plant chromosome;
3) By altering the promoter sequence of said gene on the plant chromosome;
4) By operably linking a strong promoter to the gene;
5) By introducing enhancers.
9. The method of claim 8, wherein an over-expression vector for the gene is constructed and transformed into a plant;
the gene is driven by a soybean cyst nematode inducible promoter HIP, and the promoter HIP is:
i) A nucleotide sequence shown in SEQ ID NO. 3;
ii) the nucleotide sequence shown in SEQ ID NO. 3 is substituted, deleted and/or added with one or more nucleotides and has the same function;
iii) A nucleotide sequence which hybridizes with the sequence shown in SEQ ID No. 2 and has the same function under stringent conditions, i.e., in a 0.1 XSSPE solution containing 0.1% SDS or a 0.1 XSSC solution containing 0.1% SDS, at 65℃and washing the membrane with the solution; or (b)
Iv) a nucleotide sequence which has more than 90% homology with the nucleotide sequence of i), ii) or iii) and has the same function.
10. The use according to any one of claims 3 to 7 or the method according to claim 8 or 9, wherein the plant is a monocotyledonous or dicotyledonous plant;
the plant includes soybean, arabidopsis thaliana, wheat, rice, maize, cotton, and peanut.
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