CN112921013B - Soybean cyst nematode chitin synthetase gene and application thereof - Google Patents

Abstract

The invention provides a chitin synthase gene SCN-CHS cloned from soybean cyst nematode, the DNA sequence of the gene is shown as SEQ ID No.1, the cDNA sequence of the gene SCN-CHS is shown as SEQ ID No.2, and the protein sequence of the gene SCN-CHS is shown as SEQ ID No. 3. A chitin synthesis core structural domain of a soybean cyst nematode chitin synthase gene (SCN-CHS) is selected, and a host plant mediated gene silencing (HIGS) transgenic plant is constructed by taking the structural domain as a target point, so that the influence of the HIGS plant on SCN is independent of physiological race, and the influence on SCN can be stably inherited. The invention is helpful to analyze the biological function of the chitin synthase gene SCN-CHS in the soybean cyst nematode, and determines the application potential of HIGS plants developed based on RNAi in the prevention and control of soybean cyst nematode diseases.

Description

Soybean cyst nematode chitin synthetase gene and application thereof

Technical Field

The invention belongs to the technical field of biology, and relates to a chitin synthase gene derived from soybean cyst nematodes and application of the gene in new nematode-resistant varieties through RNAi development.

Background

From soybean cyst nematodes (soybean cyst nSoybean cyst nematode disease caused by the ematolide Heterodera glycine, SCN) is one of the destructive diseases in the production of soybeans, has the characteristics of wide distribution, serious harm, difficult control and the like, and seriously threatens the production safety of the soybeans. The soybean cyst nematode infection often causes dwarfing and yellowing of plants on the upper part of the soybean, commonly called as 'dragon seedlings', and the lower part of the soybean often shows serious symptoms such as underdeveloped root systems, reduced root nodule quantity, increased fibrous roots and the like. Statistically, SCN damage causes an average soybean reduction of 10-30%, in severe cases 60-70%, and even no grain harvest, resulting in a loss of about 30 billion dollars of soybean production worldwide each year. At present, measures for preventing and treating SCN mainly comprise planting of anti-nematode varieties, crop rotation, use of chemical pesticides and the like, wherein planting of the anti-SCN soybean varieties is the most economic and effective method for preventing and treating soybean cyst nematode.

SCN is commonly generated in northeast and Huang-Huai-Hai soybean production areas of China, the Heilongjiang, Jilin and Liaoning mainly use SCN No.1 and No.3 physiological races, and the SCN No.4, No. 6, No. 9 and No. 14 physiological races are detected in the areas of Yian, Anda, Daqing and the like; Huang-Huai-Hai is mainly SCN4 physiological races, and also has part of SCN 1 and 2 physiological races. Based on the theory of 'gene to gene', the disease resistance of the disease-resistant variety depends on the pathotype or physiological race of pathogens, and the SCN-resistant soybean variety may lose the disease resistance to SCN due to the change of the SCN physiological race in the field. For this reason, there is an urgent need to develop a new species of broad-spectrum resistant soybean that is independent of SCN physiological races.

RNAi (RNA interference) technology specifically targets key sites of pests to control them. The transgenic plant based on RNAi can be used for controlling pests, such as dsRNA for expressing V-ATPase gene in corn, and can obviously reduce the damage of corn rootworm (Diabrotica virgifera). The premise of preventing and controlling the plant diseases and insect pests through an RNAi strategy is to search an ideal action target. Chitin (Chitin) is one of the important components of the cell wall of phytopathogenic fungi, the wall of insects and the peritrophic membrane of the midgut, and the egg shell of phytopathogenic nematodes, but is a linear macromolecule formed by the polymerization of D-N-acetylglucosamine (GlcNAc) through β -1, 4-glycosidic bonds, which is not present in plants and vertebrates, and the biosynthesis process is very complicated, the key enzyme is Chitin synthase (Chs), which is encoded by the Chitin synthase gene CHS. Chitin synthases have been of great interest as drug targets for the design of novel fungicides and insecticides. The function of the chitin synthase gene is systematically researched in pathogenic fungi such as rice blast, maize smut, wheat scab and the like, and a candidate target for designing a novel bactericide aiming at the chitin synthase is disclosed. The gene CHS of chitin synthetase has also been cloned in agricultural pests such as Spodoptera frugiperda, Tripsammophila castanea and the like. In nematodes, 2 CHS genes are cloned from free-living nematodes, namely Caenorhabditis elegans, and researches show that the CHS genes have very important functions in nematode development. Among plant parasitic nematodes, 1 CHS gene was cloned from only peanut root-knot nematode (Meloidogyne artiellia), but studies on chitin synthase of soybean cyst nematode have not been reported. A Host plant mediated Gene Silencing (HIGS) RNAi transgenic wheat (HIGS wheat) is constructed by a Liriopsis graminearum FgChs3b, which is a Chinese scientist, by a Liaoyu research team, by taking Fusarium graminearum FgChs3b as a target, the seedling and the spike of the HIGS wheat can obviously obstruct the colonization and the expansion of the Fusarium graminearum F, the HIGS wheat F shows obvious resistance to the Fusarium graminearum F, and field tests show that the HIGS wheat still shows the characteristic of antagonism F even to the T5 generation, which indicates that the HIGS plant constructed by taking FgChs3b as the target can obtain the lasting resistance to the Fusarium graminearum F.

Based on the CHS gene which is widely existed in agricultural pests such as plant parasitic nematodes, plant pathogenic fungi and the like and has evolutionary conserved regions, and the CHS gene does not exist in crops, human and livestock and the like, the Chs has the potential of becoming a candidate target for developing new varieties for preventing and controlling the soybean cyst nematodes based on the RNAi technology.

Disclosure of Invention

The invention clones the chitin synthase Gene of soybean cyst nematode and constructs Host plant mediated Gene Silencing (HIGS) transgenic soybean plants. The biological function of a chitin synthase gene (CHS) in soybean cyst nematodes is revealed by detecting the influence of HIGS soybean plants on the soybean cyst nematodes, and the application potential of HIGS soybean plants in prevention and control of soybean cyst nematode diseases is determined by developing the HIGS soybean plants through RNAi.

The cloned soybean cyst nematode chitin synthase gene is named as SCN-CHS, and only 1 chitin synthase gene is found in the soybean cyst nematode, and the nucleotide sequence of the chitin synthase gene is shown in SEQ ID No. 1.

The cDNA of the SCN-CHS gene also belongs to the protection scope of the invention, and the cDNA sequence is shown as SEQ ID No. 2.

The protein coded by the SCN-CHS gene also belongs to the protection scope of the invention, and the amino acid sequence is shown as SEQ ID No. 3.

The target sequence of dsRNA is designed to be in the protection scope of the invention, and the sequence is shown as SEQ ID No. 4.

The dsRNA segment produced by the target sequence is also in the protection scope of the invention, and the sequence is shown as SEQ ID No. 5.

Application of the gene of SEQ ID No.1 or 2 in constructing HIGS transgenic plants.

In the application, in order to select the core structural domain of a chitin synthase gene sequence, a target sequence of dsRNA is designed to be shown as SEQ ID No.4, and a dsRNA sequence is shown as SEQ ID No.5, so that a HIGS transgenic soybean plant is constructed, and the soybean plant can obtain the resistance to soybean cyst nematodes.

The application is characterized in that the core structural domain of a chitin synthase gene of soybean cyst nematode is used as a target point, a dsRNA fragment is transferred into a plant by an RNAi method, the sequence of the dsRNA fragment is shown as SEQ ID No.5, when the soybean cyst nematode eats a HIGS transgenic soybean plant, the chitin synthase gene of the soybean cyst nematode is silenced, and the soybean plant has the performance of resisting the soybean cyst nematode.

The plant is soybean.

The invention clones a chitin synthase gene SCN-CHS of soybean cyst nematode, detects the expression spectrums of the SCN-CHS gene in different development stages of Eggs (Eggs), pre-infection second-instar larvae (PreJ2s), post-infection second-instar larvae (PosJ2s), third-instar larvae (J3s), white female worms (J4s) and the like by using qRT-PCR technology, and finds that the expression quantity of the SCN-CHS gene is the highest in the egg (Eggs) stage. A host plant mediated gene silencing (HIGS) vector is constructed by using the core domain of the SCN-CHS chitin synthase gene, a receptor soybean variety is transformed into Jack, and 3 homozygote HIGS plants with single copy insertion are obtained, and the numbers of the homozygote HIGS plants are 48-7-5, 55-8-24 and 57-9-2. Molecular verification proves that the HIGS vector with glyphosate resistance is successfully expressed in transgenic soybeans, and phenotype tests show that main agronomic traits of HIGS plants, such as plant height, root length, fresh weight, hundred grain weight, seed coat color and the like, are not obviously changed. By inoculating soybean cyst nematode SCN4, the HIGS plants of the T2 generation are found to obviously obstruct the growth, development and propagation of SCN4, and the main effects are that the number of SCN in roots of the HIGS plants of the T2 generation is reduced, the development frequency of the HIGS plants in the advanced age stage is reduced, and the number of formed single plant cysts and the number of eggs of the single cysts are obviously reduced. The effect of HIGS plants of the T2 generation on SCN3 is similar to that of SCN4, and the formation amount of cysts is obviously reduced, which indicates that the effect of the HIGS plants on the SCN is independent of physiological races of soybean cyst nematodes and has broad-spectrum resistance on the soybean cyst nematodes.

The effect of HIGS plants of T6 generation on SCN4 infection is tested, the effect of HIGS plants of T6 generation on SCN4 is found to be similar to the effect of HIGS plants of T2 generation on SCN4, and the number of single cysts and the number of eggs of the single cysts formed by the HIGS plants of T6 generation are both obviously reduced, which indicates that the effect of the HIGS plants on SCN can be stably inherited. The expression abundance change condition of the SCN-CHS gene in the cyst formed by the HIGS plant is tested by using a qRT-PCR technology, and the result shows that the SCN-CHS gene of the cyst formed by the HIGS plants of the T2 generation and the T6 generation is obviously reduced.

The invention clones the chitin synthase gene SCN-CHS of the soybean cyst nematode, analyzes the biological function of the chitin synthase gene SCN-CHS in the soybean cyst nematode, determines the application potential of HIGS plants constructed based on RNAi strategy in preventing and controlling soybean cyst nematode diseases, and lays theoretical foundation and technical foundation for effectively controlling the soybean cyst nematode diseases.

Drawings

FIG. 1: PCR amplification of SCN-CHS

And respectively taking genomic gDNA and cDNA of the soybean cyst nematode as templates, and carrying out PCR amplification on gDNA and cDNA fragments.

FIG. 2 shows the developmental expression of SCN-CHS gene in 5-year-old age period detected by qRT-PCR.

The method for relative expression analysis was 2^-ΔΔCtThe method, the reference gene is beta-actin gene. RQ of 2-instar larvae (PreJ2s) before infestation is 1. Egg: eggs; PreJ2 s: infecting the first 2 instar larvae; PosJ2 s: infested 2 instar larvae; j3 s: larvae of 3 years old; j4 s: adult females at age 4.

FIG. 3 is a schematic diagram of the HIGS vector construction strategy.

RB and LB are two sites of recombination of agrobacterium plasmid and genome chromosome, 35S promoter, hairpin structure composed of partial sequence of chitin synthetic core structure domain and iPDK intron, Nos terminator and selection marker gene EPSPS.

FIG. 4 shows HIGS transgenic RT-PCR validation.

Genomic DNA of HIGS plants of T2 generation and T6 generation is amplified by using soybean internal reference Lectin primer and screening marker EPSPS primer.

FIG. 5 shows agronomic traits of HIGS plants.

Wherein A: hundred grain weight of HIGS plants; b: height, root length and fresh weight of HIGS plants.

FIG. 6 shows the effect of HIGS plants at the T2 generation on SCN.

Wherein A: development process of SCN4 in roots of HIGS plants of T2 generation; b: the number of cysts of each individual plant formed by HIGS plants in T2 generation 460 days after SCN inoculation; c: the number of monochiosporadic eggs forming cysts of HIGS plants in T2 generation 460 days after SCN inoculation; d: 360 days after inoculation of SCN, the number of cysts formed in individual plants of HIGS plants at T2 generation.

FIG. 7 shows the effect of HIGS plants at the T6 generation on SCN 4.

Wherein A: development process of SCN4 in roots of HIGS plants of T6 generation; b: the number of single plant cysts formed by HIGS plants in T6 generation 460 days after the SCN is inoculated; c: number of single cyst eggs of cysts formed by HIGS plants in T6 generation 460 days after SCN inoculation; d: expression level of SCN-CHS in cysts formed by HIGS plants of T2 generation and T6 generation.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemicals, unless otherwise specified.

EXAMPLE 1 cloning of the SCN-CHS Gene

1. Collecting 2-instar larvae of soybean cyst nematode (about 5000 heads), washing with DEPC treatment water for 2-3 times, transferring into 1.5ml centrifuge tube, adding 1ml Trizol (Invitrogen), quick freezing in liquid nitrogen for 30s, water bathing at 37 deg.C for 30s, repeatedly freezing and thawing for 4-5 times, standing at room temperature for 5min, extracting total RNA, and extracting with DNA-free^TMThe DNA Removal Kit removes residual DNA from the total RNA and performs reverse transcription to obtain cDNA.

2. Using cDNA as template, adopting primer to make PCR amplification, and its amplification system is 10 XPCR amplification Taq (Mg)²⁺) 5 mul; dNTP (10mM), 4. mu.l; 1. mu.l each of the forward primer (10mM) and the reverse primer (10 mM); advantage Taq, 0.5. mu.l; cDNA template, 1. mu.l; deionized water, 35.5. mu.l in total volume 50. mu.l. The amplification program is denatured at 94 ℃ for 5 min; extension for 4min at 94 ℃, 30sec, 54 ℃, 30sec, 72 ℃ for the next 34 cycles; finally, the extension is carried out for 10min at 72 ℃ and the product is stored at 4 ℃. The PCR product was separated and identified by 1% agarose gel electrophoresis.

3. Using gDNA as template, and adopting primer to make PCR amplification, and its amplification system is 10 XPCR Ex Buffer (Mg)²⁺) 5 mul; dNTP (10mM), 4. mu.l; 1. mu.l each of the forward primer (10mM) and the reverse primer (10 mM); ex Taq, 0.5. mu.l; gDNA template, 1. mu.l; deionized water, 35.5. mu.l in total volume 50. mu.l. The amplification program is denatured at 94 ℃ for 5 min; extension for 5min at 94 ℃, 30sec, 54 ℃, 30sec, 72 ℃ for the next 34 cycles; finally, the extension is carried out for 10min at 72 ℃ and the product is stored at 4 ℃. The PCR product was separated and identified by 1% agarose gel electrophoresis.

4. And (3) respectively connecting the pMD19-T vector with the PCR amplification products in the step 2 and the step 3, then transforming DH5 alpha escherichia coli competent cells, and sequencing. The sequencing result shows that the amplification product has the sequence table SEQ ID N0: 1, having the sequence of SEQ ID N0: 2, as shown in fig. 1, and encodes SEQ ID N0: 3, the protein shown in SEQ ID N0: 1 is named as SCN-CHS gene.

Example 2 analysis of the developmental expression of the SCN-CHS Gene

A large number of soybean cyst nematode 2-instar larvae are inoculated on soybean roots, and 5 soybean cyst nematodes (2-instar larvae before infection, 2-instar larvae after infection, 3-instar larvae, 4-instar females and eggs) at different development stages are obtained by controlling inoculation time and carrying out enzyme cracking treatment separation. Respectively extracting mRNA of 5 ages by adopting a magnetic bead method, carrying out reverse transcription on the mRNA to form first-strand cDNA serving as a template, and designing an SCN-CHS gene specific upstream Primer CHS-QF by adopting Primer 5.0 software: 5'-TTTCAACCAGAGACGGAGG-3', and the reverse primer CHS-QR: 5'-GACGACCATTTGATAGAGAACG-3' are provided. Taking a beta-Actin gene as an internal reference gene, and an upstream primer of Actin-QF: 5'-CGCTGAACCCGAAGGCCAACAGA-3', downstream primer Actin-QR: 5'-TTGATGTCACGGACGATCTCACG-3' are provided. By using

The expression quantity of the SCN-CHS gene at different ages is detected by Real-time RT-PCR with a Select Master Mix kit (Takara) by using an ABI7500 fluorescent quantitative PCR instrument, three times of biological repetition are respectively carried out, and 2 times of biological repetition is adopted^-ΔΔCtAnd analyzing the result by a method, thereby determining the expression condition of the SCN-CHS gene in different developmental stages of the soybean cyst nematode.

Reaction system (20 μ l): 10 μ l SYBR Premix Ex Taq II; 0.4. mu.l ROX reference Dye II; 1. mu.l each of the forward primer (10. mu.M) and the reverse primer (10. mu.M); 1 microliter of template; ddH₂O is added to 20 μ l. Reaction procedure: 50 ℃/2min, 95 ℃/2 min; 40 cycles of 95 ℃/15s, 58 ℃/15s, 72 ℃/30 s; 95 ℃/15s, 60 ℃/1min, 95 ℃/30s, 60 ℃/15 s. As shown in FIG. 2, the SCN-CHS gene was expressed in the highest amount in the egg stage among the 5 developmental stages tested.

Implementation example 3 construction of HIGS transgenic silent plants

Selecting a sequence table SEQ ID N0: 2 (soybean cyst nematode chitin synthase gene sequence), namely a 420bp segment from 1936bp to 2355bp, is used as a target sequence designed by a dsRNA segment (SEQ ID NO: 5), and a HIGS expression vector is composed of a hairpin structure formed by the dsRNA segment and an iPDK intron of soybean, a 35S promoter, a Nos terminator and an anti-glyphosate screening marker EPSPES, as shown in a sequence SEQ ID No.4, and is shown in a figure 3. Transforming the agrobacterium rhizogenes competent cells by an electric shock method, and constructing HIGS transgenic plants by adopting an agrobacterium rhizogenes-mediated soybean cotyledonary node transformation method. The method comprises the following steps: sterilizing soybean seeds with chlorine, inoculating the soybean seeds to a germination culture medium, removing seed coats, cutting hypocotyls under cotyledonary nodes, co-culturing the hypocotyls and hairy root fungus solution carrying a target carrier, and inducing hairy roots. Example 4 detection of root Gene expression in HIGS plants

The young roots of the HIGS plants of the T2 generation and the T6 generation were collected, placed in a sterile mortar, poured into liquid nitrogen and ground thoroughly to a powder. Total RNA of the radicles was extracted using TRIzol (Invitrogen) and PrimeScript was used^TMThe RT reagent Kit with gDNA Eraser (Takara) Kit removed residual DNA in total RNA, and reverse transcribed to obtain cDNA. Taking cDNA as a template, and respectively using soybean internal reference Lectin, EPSPS and CHS specific primers to carry out RT-PCR, wherein the specific sequences are as follows: soybean internal reference Lectin upstream primer Lectin-118F: 5'-GCCCTCTACTCCACCCCCATCC-3', downstream primer Lectin-118R: 5'-GCCCATCTGCAAGCCTTTTTGTGC-3', respectively; EPSPS upstream primer EPSPS-F: 5'-CAAGTCTATCTCCCACAGGTCC-3', downstream primer EPSPS-R: 5'-GCATCAGTCTCAACGGTAAGGT-3', CHS upstream primer CHS-F: 5'-TGACAACACATATATTCTCGCCAT-3', downstream primer CHS-R: 5'-GAAGCAGCGGCGTATTCAAC-3' are provided.

The amplification system was 10 XPCR Mix 12.5. mu.l, 1. mu.l each of the forward (10mM) and reverse (10mM), 1. mu.l of DNA template, 9.5. mu.l of deionized water, and 25. mu.l in total. The internal reference Lectin amplification program is denaturation at 94 ℃ for 5 min; extension at 94 ℃ for 30sec, 60 ℃ for 30sec, 72 ℃ for 1min, 35 cycles; extending for 10min at 72 ℃; storing at 4 ℃. EPSPS and CHS amplification program 94 ℃ denaturation for 5 min; extension at 94 ℃ for 30sec, 55 ℃ for 30sec, 72 ℃ for 1min, 35 cycles; extending for 10min at 72 ℃; storing at 4 ℃. The PCR product was identified by 1% agarose gel electrophoresis. FIG. 4 shows that the internal reference Lectin primers are used for carrying out RT-PCR amplification by respectively using cDNA of HIGS plants and Jack as templates, and specific bands are formed; when the EPSPS and the CHS primers are used for RT-PCR amplification, the cDNA of the HIGS plant is taken as a band of the template, and the cDNA of the control Jack is taken as a band of the template, so that the CHS and the EPSPS genes are successfully expressed at the root of the HIGS plant.

Example 5 Effect of HIGS plants on agronomic traits

Randomly picking 100 HIGS plants and Jack seeds with the same size, measuring the weight of each strain of soybean in hundred, putting the strain into a glass culture dish, and taking a picture. Planting HIGS plants and Jack by adopting a greenhouse PVC tube planting method, preparing sterilized soil and sand, fully and uniformly mixing the soil and the sand according to the ratio of 1: 3, putting the mixture into a PVP tube with the length of 25cm multiplied by the diameter of 3cm, selecting 2 soybeans for each variety, sowing the soybeans in the PVP tube, putting the PVC tube into a plastic box, putting the PVC tube into an incubator, and controlling the temperature to be 25-28 ℃. And (5) measuring the plant height, the root length and the fresh weight 20 days after sowing. The results show that the HIGS plants have no significant difference in the main agronomic traits such as hundred-grain weight, plant height, root length, fresh weight and the like (figure 5).

Example 6 Effect of HIGS plants in T2 generations on Soybean cyst nematodes

The soybean planting method is the same as that in example 3, 2 blue gun heads are respectively inserted at two sides of each soybean plant, inoculation is carried out after the first pair of true leaves of the soybean are completely unfolded (about 14 days after sowing), the blue gun heads are pulled out, small holes are left, SCN 4J 2 suspension is injected into each small hole, and 800 SCN 4J 2 nematodes are inoculated to each plant. At 3d, 8d and 20d after inoculation, soybean plant roots of PVP tubes were washed and magenta-stained. The method comprises the following specific steps: putting soybean roots into a 500mL beaker, adding 20mL of 10% NaC10 and 180mL of distilled water, stirring, and standing for 10 min; then putting the roots on a mesh screen, washing the roots with tap water, putting the roots back into a beaker, adding distilled water to submerge the roots, and replacing the distilled water every 5min to thoroughly remove the residual NaClO; adding 200mL of distilled water and 6mL of acid fuchsin, stirring, and heating in a microwave oven for 5 min; the roots were rinsed with running water, then placed in a petri dish and glycerol was added. Counting was performed with a microscope (OLYMPUS) and photographed, and the result is shown as a in fig. 6. Inoculating for 60d, washing soybean plants with PVP tubes, pouring soybean roots and soil of each PVP tube into a plastic barrel, adding tap water, stirring, standing for 30s, sieving the soil suspension with a sieve (20 meshes sieve is sleeved on an 80 meshes sieve), and repeating for 2 times. The residue on the 80 mesh screen was rinsed with a wash bottle onto the filter paper in the funnel. The number of cysts per plant was counted using a microscope (OLYMPUS). The results of randomly picking 10 full cysts from each variety, placing the cysts on a glass slide, kneading the cysts with forceps, adding a drop of distilled water, and calculating the number of single cyst eggs with a microscope (OLYMPUS) are shown in FIGS. 6B and 6C, and the number of single cysts and the number of single cyst eggs of HIGS plants of T2 generation are reduced, which indicates that the HIGS plants of T2 generation obviously prevent SCN4 infection and development. The method for inoculating the SCN3 is the same as the method for inoculating the SCN4, and the results are shown in FIG. 6D, the number of cysts of each plant and the number of eggs of each cyst of the HIGS plants of the T2 generation are reduced, which indicates that the HIGS plants of the T2 generation obviously block the infection and development of the SCN 3. These results indicate that the effect of T2 generation HIGS plants on SCN is independent of the physiological race of soybean cyst nematode.

Example 7 Effect of T6 generation HIGS plants on Soybean cyst nematode

The specific procedure was the same as in example 4, and the results are shown in FIG. 7, which indicates that the effect of HIGS plants on SCN can be stably inherited. Selecting 20-30 cysts from each variety of HIGS plant, placing in a 1.5mL centrifuge tube, extracting total RNA of cysts by using a magnetic bead method, and adopting DNA-free^TMThe DNA Removal Kit removes residual DNA in the total RNA, and obtains cDNA by reverse transcription. Using cDNA as template, the SCN-CHS gene specific forward primer CHS-QF of example 1: 5'-TTTCAACCAGAGACGGAGG-3', and the reverse primer CHS-QR: 5'-GACGACCATTTGATAGAGAACG-3', an upstream primer of the reference gene beta-Actin, Actin-QF: 5'-CGCTGAACCCGAAGGCCAACAGA-3', downstream primer Actin-QR: 5'-TTGATGTCACGGACGATCTCACG-3' are provided. By using

The Fast Universal qPCR Kit uses ABI7500 fluorescent quantitative PCR instrument to perform Real-time RT-PCR detection on the expression of SCN-CHS gene, performs biological repetition for three times respectively, and adopts 2^-ΔΔCtThe results of the analysis of the method revealed the expression of the SCN-CHS gene in cysts formed by HIGS plants of generations T2 and T6, and the results are shown in FIG. 7, which indicates that HIGS plants of generations T2 and T6 form cysts eggsThe SCN-CHS gene is obviously down-regulated.

Sequence listing

<110> institute of plant protection of Chinese academy of agricultural sciences

<120> soybean cyst nematode chitin synthase gene and application thereof

<141> 2021-04-15

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5121

<212> DNA

<213> Soybean cyst nematodes (Heterodera glycines)

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cgcattccga atgagtactt caagctgttg ttcgaagttc tgatggaatt gactcggttt 2220

tatttaatta gtcaattggt tccgtggtat ttttgctttg tcttgtcaag ttccgacaac 2280

gctgaagcgg acatgtacag ccgtattctt ctgaacactc cgtacggagg tcgacttgtg 2340

ctggcgttga atgggggctc acttcttttt gtgcatctca aagacaaacg tttgatccga 2400

cacaaaaagc gttggtcgca agtgatgtac atgtactttt tgctcgggca tcgaattatg 2460

gactcacatt tgagcgtaga agacaaacaa ttgcaggtct tttgagccta aattttgccc 2520

tgtgcaataa ttcaatgttc gcaccgatgc ttaggctgac aacacatata ttctcgccat 2580

tgatggcgat tccaaattcg aaccagcggt agtgattcgt cttttacatc tgatgaactt 2640

gaaaagcgac gttggctgtg cgtgcggaag aatccatccg attggagaag gtgtgctatc 2700

cttcccatta atggtgaatt tcttaccatt cccaggggtc atggtttggt accaaaagtt 2760

cgagtacgca atcgcccatt ggttccaaaa ggctgctgag catgtgttcg gctgtgtttt 2820

gtgtgccccc ggttgcttct ctctgtttcg tgcttctgct ctcatggatg acaatgcgat 2880

gcacaaatac accaaaactg cctccgaacc acgacatttt gttcaatacg accaagggga 2940

agaccgttgg ctttccactt tgcttcttaa gcaggtccaa ttgcctttct tttttcggaa 3000

taataaccat tttaagggtt atcgggttga atacgccgct gcttctgacg ccgagactta 3060

tgcgcctgaa gggtttgagg aatttttcaa ccagagacgg aggtggacgt cttcttccat 3120

cgcaaatacg ctcgatcttt tagcggatta caaagttttg gatgccattg actttttcat 3180

cattattttt agcttgcatc aagcaacaat gccagcatct ctcggctgta cattctctat 3240

caaatggtcg tcatcgtttt ctctttgctt ggacctgcca tcattttcac tatgcttgtc 3300

tatgcacagg ttattatatg acaaattcta tttaattgct acaaattaat gctcgagcgt 3360

atgaccggtg gtttttcagg tggcggcctt tgccgttgat tcgacgagag tgctcttcta 3420

caatgcagtt ccggtgtgcg gcttcatctt atgctgcttc accatggatt cgtccgtaca 3480

gcttacctac gccaaaattg cttccgtcat ttacgccttc ataatgctgg ccgtccttat 3540

cgccaccaca aaccaaatcg tattgtagac agtcttttcg cccacctcca tgttcgtgtt 3600

gggcatggtg cagatcttct cctttgcctc ctgcatccac ccgaaggaat tcgccaacat 3660

tatttatggt tcgatcttct tcctgatgat cccttccact tatgtcttcc tctcgctgta 3720

ctcattgatc aatttgaacg tgattaattg gggcactcgt gaagccgtag caaaagcgac 3780

agggcaggcg acgttcaggg agaatatagc cgagagagtg ctgcggcgag tggctaatct 3840

caacgacgac aattccttct tgacgcgctt tcttacgaga gttcgcagtg gtgaagaacc 3900

gagcgaaaaa atacggacac ttgaaaggaa attcgaacgc actgaacgat tgttgatgag 3960

catgaaggca tttatggggt gatgggagtt ggatagttta aacatttagg aaatgactga 4020

aaaggcaaga ggagaatccg atgccatgca aatgggcgga cagcattttt gcatgatgga 4080

tgtggtgctt aatgaagagg gtgaacagga attgccttta cgatctggat acgaggtgac 4140

taatgtgttc atcattagag aattttcgcg aaaaactggg aattggtcaa attaaatgtt 4200

tgcttgtact ttaccttcat cgccgcaggc tgttgccatt cgtcggctgg acacttttga 4260

acggcgtgcc attgttactg acccatgcaa acgatccctt tggatggact gtgaatacct 4320

tcagtgttgc gatcgtggga agttgagact tggggaagag actttttggg acgaattgat 4380

cgacaaatat ttgaaggtgg gcttttgggt caaatgcaaa ttgtttcttt tttgttcaat 4440

ggcttttctt tagccagtcg agaccaccac ccaagaacag gcgcaagtcg cgaatggact 4500

cgtcgcattg cgaaaccgaa ttgcattttc catcattttg ctcaattgtt tgcttgtgct 4560

ggcaattttc cttctccaac gacacaaaga tgttctgtcc gtcaaattca ctccttatgg 4620

tgggcattgt tagctctttt tcacttcctt tattttaaag atggatttaa atggacgaaa 4680

atgaatgaaa cgaccgggaa atttgaagac acgactgagg ccctgaaagt tgacccgttg 4740

ggtatcggaa taatcttttt tctgatggga attctcattg tgagaccttt ttcaattgaa 4800

ttcactaatt aattggaaaa ggttcaaacc atcggcatgc tcatccatcg tctaaacact 4860

ctcgtggaag cactgcacga actgagtgag atgaaagacg tacaatacaa caccttcaca 4920

tttactgacc acatcaaagt gctcaatgaa ggtggctttt catcatgtgc catttgcatt 4980

tgtgtctact tttctaattg agctcgtcaa atgattgaca ccgttaatta cgaccgcgcc 5040

cacggggctg atgggtacac tcgccacgga cttggggacc aaaaagtgca aaagaatgtg 5100

ctttacaaac tccaaaagtg a 5121

<210> 2

<211> 3984

<212> DNA

<213> Soybean cyst nematodes (Heterodera glycines)

<400> 2

atgaatttcc agcaacctga tcatagatgg gatgccttca ggtccaatgc tcgttcaaaa 60

aggattttcc agaaatcaga ctcttattat gttcgctggc ttcaatggtt gaagttcggg 120

atttttctga ttgctcattg cacttgtctg ctcgcaacct tcgtatccaa gtcgatcgtc 180

gttattcttg ctacaaatat cggtgcaaat cgtttaatcg agggagttaa attgtacgaa 240

tattgtgcat ccgtgccgtt ggcaattcaa aacttcgaac gcttttcctg cctttgtgct 300

gctctttggc tcatccaagt ggtgccggac ttattcgctc ttttccagtc acttgtcaga 360

tttcggccaa cttctgcttc tgaccattcg ctgtttccga tgttcattct gtttgaatct 420

cttcgtgccg ctggactttc tttcctgact ttgaatgttt ttccgctttt ggacgtgccc 480

cgctgtgttt tgcttggcgc tgctttccat tgcatttctt atcttctccg agtttttcgc 540

tgttttcata gctctgccga ctccaatcgt ccgattggat ggcgcctcat gcgcatcatc 600

gctagcattc cttcagcgct tattttcgct gttcttttca ctggtgccta cctgtggtat 660

cttactcaag cgcatttgga gttccgtttc ggcgttttgc ttccgttatc ctttctgatg 720

tgtgcagttg gacactggga aagttgggtg gacacgaaac acacaagcgg cgtattgcag 780

gaattgtacg agctcaaata tggactgaga aaactgaacg cggagacgcg cgtgtgcgct 840

tgtgtggtcc gttttttgtg tgccgcgctg attttggggt tttcccttcg caatcacaac 900

atcccatttg gtacgctgac ttctctgttg ctcaacgggg gaacgctcca aggccctaaa 960

cttatctttc tgcttgcgct tgttgtcatt tctctcaact tttatctccg cttttgcttc 1020

cgctttttgg ctgcgatgcg acttgaactc attccgcttg gacaccccgt ctttctcacg 1080

actccaatgc ttttcttctt cttctataca ttttgtcatt acgttcctat ttgtcgaatc 1140

aatgctcttt ttcaacattt tgacttgagc atttattgcg taaaaggaat gcaacggcaa 1200

ggcgttttct ccgatttcta cataacattg atttggcttt tctcatatat gaaatggagc 1260

tatcgtttta taagtggtga gaacttcgat cagatggacg aaataattgt gtcgatgtca 1320

ccgatgatga gtgggcttga catttgccaa tctctggttg ttttccgttg ctcaatcgct 1380

aaaaaggaaa ctgattgtga ctccgattac aactaccgtg aaagcatttc aagaatattt 1440

aatggcgaga tggacagaat ccttacactg tatgtctgtg cgacgatgtg gcacgagaca 1500

cggaacgaaa tggcacagat gatcaaatcc attctaaaac ttgatgagga acatgccatt 1560

cgtttgtcgg agaaaaatac ccttgaagga accaaattcc gcttggaagt tcatattttc 1620

ttcgatgact cgtggactga tgacaaagaa tgtggtcgca ttccgaatga gtacttcaag 1680

ctgttgttcg aagttctgat ggaattgact cgttccgaca acgctgaagc ggacatgtac 1740

agccgtattc ttctgaacac tccgtacgga ggtcgacttg tgctggcatt gaatgggggc 1800

tcacttcttt ttgtgcatct caaagacaaa cgtttgatcc gacacagaaa gcgttggtcg 1860

caagtgatat acatgtactt tttgctcggg catcgaatta tggactcaca tttgagcgta 1920

gaagacaaac aattgcaggc tgacaacaca tatattctcg ccattgatgg cgattccaaa 1980

ttcgaaccag cggcagtgat tcgtctttta catctgatga acttgaaaag cgacgttggc 2040

tgtgcgtgcg gaagaatcca tccgattgga gaaggggtca tggtttggta ccaaaagttc 2100

gagtacgcaa tcgcccattg gttccaaaag gctgctgagc atgtgttcgg ctgtgttttg 2160

tgtgcccccg gttgcttctc tctgtttcgt gcttctgctc tcatggatga caatgtgatg 2220

cacaaataca ccaaaactgc ctccgaacca cgacattttg ttcaatacga ccaaggggaa 2280

gaccgttggc tttccacttt gctacttaag cagggttatc gggttgaata cgccgctgct 2340

tctgacgccg agacttatgc gcctgaaggg tttgaggtat ttttcaacca gagacggagg 2400

tggacgcctt cttccatcgc aaatacgctc gatcttttag cggattacaa acttgcatca 2460

agcaacaatg ccagcatctc tcggctgtac gttctctatc aaatggtcgt catcgttttc 2520

tctttgcttg gacctgccat cattttcact atgcttgtct atgcacaggt ggcggccttt 2580

gccgttgatt cgacgagagt gctcttctac aatgcagttc cggtgtgcgg cttcatcttc 2640

tgctgcttca ccatggattc gtccgtacag cttacctacg ccaaaattgc ttccgtcatt 2700

tacgccttca taatgctggc cgtccttatc gccaccacaa accaaatcgt attggaaaca 2760

gtcttttcgc ccacctccat gttcgtgttg ggcatggtgc tgatcttctc ctttgcctcc 2820

tgcatccacc cgaaggaatt cgccaacatt atttatggtt cgatcttctt cctgatgatc 2880

ccttccactt atgtcttcct ctcgctgtac tcattgatca atttgaacgt gattaattgg 2940

ggcactcgtg aagccgtagc aaaagcgaca gggcaggcga cgttcaggga gaatatagcc 3000

gagagagtgc tgcggcgagt ggctaatctc aacgacgaca attccttctt gacgcgcttt 3060

cttacgagag ttcgcagtgg tgaagaaccg agcgaaaaaa tacggacact tgaaaggaaa 3120

ttcgaacgca ctgaacgatt gttgatgagc atgaaggaaa tgactgaaaa ggcaagagga 3180

gaatccgatg ccatgcaaat gggcggacag catttttgca tgatggatgt ggtgcttaat 3240

gaagagggtg aacaggaatt gcctttacga tctggatacg aggctgttgc cattcgtcgg 3300

ctggacactt ttgaacggcg tgccattgtt actgacccat gcaaacgatc cctttggatg 3360

gactgtgaat accttcagtg ttgcgatcgt gggaagttga gacttgggga agagactttt 3420

tgggacgaat tgatcgacaa atatttgaag ccagtcgaga ccaccaccca agaacaggcg 3480

caagtcgcga atggactcgt cgcattgcga aaccgaattg cattttccat cattttgctc 3540

aattgtttgc ttgtgctggc aattttcctt ctccaacgac acaaagatgt tctgtccgtc 3600

aaattcactc cttatgatgg atttaaatgg acgaaaatga atgaaacgac cgggaaattt 3660

gaagacacga ctgaggccct gaaagttgac ccgttgggta tcggaataat cttttttctg 3720

atgggaattc tcattgttca aaccattggc atgctcatcc atcgtctaaa cactctcgtg 3780

gaagcactgc acgaactgag tgagatgaaa gacgtacaat acaacacctt cacatttact 3840

gaccacatca aagtgctcaa tgaagctcgt caaatgattg acaccgttaa ttacgaccgc 3900

gcccacgggg ctgatgggta cactcgccac ggacttgggg accaaaaagt gcaaaagaat 3960

gtgctttaca aactccaaaa gtga 3984

<210> 3

<211> 1327

<212> PRT

<213> Soybean cyst nematodes (Heterodera glycines)

<400> 3

Met Asn Phe Gln Gln Pro Asp His Arg Trp Asp Ala Phe Arg Ser Asn

1 5 10 15

Ala Arg Ser Lys Arg Ile Phe Gln Lys Ser Asp Ser Tyr Tyr Val Arg

20 25 30

Trp Leu Gln Trp Leu Lys Phe Gly Ile Phe Leu Ile Ala His Cys Thr

35 40 45

Cys Leu Leu Ala Thr Phe Val Ser Lys Ser Ile Val Val Ile Leu Ala

50 55 60

Thr Asn Ile Gly Ala Asn Arg Leu Ile Glu Gly Val Lys Leu Tyr Glu

65 70 75 80

Tyr Cys Ala Ser Val Pro Leu Ala Ile Gln Asn Phe Glu Arg Phe Ser

85 90 95

Cys Leu Cys Ala Ala Leu Trp Leu Ile Gln Val Val Pro Asp Leu Phe

100 105 110

Ala Leu Phe Gln Ser Leu Val Arg Phe Arg Pro Thr Ser Ala Ser Asp

115 120 125

His Ser Leu Phe Pro Met Phe Ile Leu Phe Glu Ser Leu Arg Ala Ala

130 135 140

Gly Leu Ser Phe Leu Thr Leu Asn Val Phe Pro Leu Leu Asp Val Pro

145 150 155 160

Arg Cys Val Leu Leu Gly Ala Ala Phe His Cys Ile Ser Tyr Leu Leu

165 170 175

Arg Val Phe Arg Cys Phe His Ser Ser Ala Asp Ser Asn Arg Pro Ile

180 185 190

Gly Trp Arg Leu Met Arg Ile Ile Ala Ser Ile Pro Ser Ala Leu Ile

195 200 205

Phe Ala Val Leu Phe Thr Gly Ala Tyr Leu Trp Tyr Leu Thr Gln Ala

210 215 220

His Leu Glu Phe Arg Phe Gly Val Leu Leu Pro Leu Ser Phe Leu Met

225 230 235 240

Cys Ala Val Gly His Trp Glu Ser Trp Val Asp Thr Lys His Thr Ser

245 250 255

Gly Val Leu Gln Glu Leu Tyr Glu Leu Lys Tyr Gly Leu Arg Lys Leu

260 265 270

Asn Ala Glu Thr Arg Val Cys Ala Cys Val Val Arg Phe Leu Cys Ala

275 280 285

Ala Leu Ile Leu Gly Phe Ser Leu Arg Asn His Asn Ile Pro Phe Gly

290 295 300

Thr Leu Thr Ser Leu Leu Leu Asn Gly Gly Thr Leu Gln Gly Pro Lys

305 310 315 320

Leu Ile Phe Leu Leu Ala Leu Val Val Ile Ser Leu Asn Phe Tyr Leu

325 330 335

Arg Phe Cys Phe Arg Phe Leu Ala Ala Met Arg Leu Glu Leu Ile Pro

340 345 350

Leu Gly His Pro Val Phe Leu Thr Thr Pro Met Leu Phe Phe Phe Phe

355 360 365

Tyr Thr Phe Cys His Tyr Val Pro Ile Cys Arg Ile Asn Ala Leu Phe

370 375 380

Gln His Phe Asp Leu Ser Ile Tyr Cys Val Lys Gly Met Gln Arg Gln

385 390 395 400

Gly Val Phe Ser Asp Phe Tyr Ile Thr Leu Ile Trp Leu Phe Ser Tyr

405 410 415

Met Lys Trp Ser Tyr Arg Phe Ile Ser Gly Glu Asn Phe Asp Gln Met

420 425 430

Asp Glu Ile Ile Val Ser Met Ser Pro Met Met Ser Gly Leu Asp Ile

435 440 445

Cys Gln Ser Leu Val Val Phe Arg Cys Ser Ile Ala Lys Lys Glu Thr

450 455 460

Asp Cys Asp Ser Asp Tyr Asn Tyr Arg Glu Ser Ile Ser Arg Ile Phe

465 470 475 480

Asn Gly Glu Met Asp Arg Ile Leu Thr Leu Tyr Val Cys Ala Thr Met

485 490 495

Trp His Glu Thr Arg Asn Glu Met Ala Gln Met Ile Lys Ser Ile Leu

500 505 510

Lys Leu Asp Glu Glu His Ala Ile Arg Leu Ser Glu Lys Asn Thr Leu

515 520 525

Glu Gly Thr Lys Phe Arg Leu Glu Val His Ile Phe Phe Asp Asp Ser

530 535 540

Trp Thr Asp Asp Lys Glu Cys Gly Arg Ile Pro Asn Glu Tyr Phe Lys

545 550 555 560

Leu Leu Phe Glu Val Leu Met Glu Leu Thr Arg Ser Asp Asn Ala Glu

565 570 575

Ala Asp Met Tyr Ser Arg Ile Leu Leu Asn Thr Pro Tyr Gly Gly Arg

580 585 590

Leu Val Leu Ala Leu Asn Gly Gly Ser Leu Leu Phe Val His Leu Lys

595 600 605

Asp Lys Arg Leu Ile Arg His Arg Lys Arg Trp Ser Gln Val Ile Tyr

610 615 620

Met Tyr Phe Leu Leu Gly His Arg Ile Met Asp Ser His Leu Ser Val

625 630 635 640

Glu Asp Lys Gln Leu Gln Ala Asp Asn Thr Tyr Ile Leu Ala Ile Asp

645 650 655

Gly Asp Ser Lys Phe Glu Pro Ala Ala Val Ile Arg Leu Leu His Leu

660 665 670

Met Asn Leu Lys Ser Asp Val Gly Cys Ala Cys Gly Arg Ile His Pro

675 680 685

Ile Gly Glu Gly Val Met Val Trp Tyr Gln Lys Phe Glu Tyr Ala Ile

690 695 700

Ala His Trp Phe Gln Lys Ala Ala Glu His Val Phe Gly Cys Val Leu

705 710 715 720

Cys Ala Pro Gly Cys Phe Ser Leu Phe Arg Ala Ser Ala Leu Met Asp

725 730 735

Asp Asn Val Met His Lys Tyr Thr Lys Thr Ala Ser Glu Pro Arg His

740 745 750

Phe Val Gln Tyr Asp Gln Gly Glu Asp Arg Trp Leu Ser Thr Leu Leu

755 760 765

Leu Lys Gln Gly Tyr Arg Val Glu Tyr Ala Ala Ala Ser Asp Ala Glu

770 775 780

Thr Tyr Ala Pro Glu Gly Phe Glu Val Phe Phe Asn Gln Arg Arg Arg

785 790 795 800

Trp Thr Pro Ser Ser Ile Ala Asn Thr Leu Asp Leu Leu Ala Asp Tyr

805 810 815

Lys Leu Ala Ser Ser Asn Asn Ala Ser Ile Ser Arg Leu Tyr Val Leu

820 825 830

Tyr Gln Met Val Val Ile Val Phe Ser Leu Leu Gly Pro Ala Ile Ile

835 840 845

Phe Thr Met Leu Val Tyr Ala Gln Val Ala Ala Phe Ala Val Asp Ser

850 855 860

Thr Arg Val Leu Phe Tyr Asn Ala Val Pro Val Cys Gly Phe Ile Phe

865 870 875 880

Cys Cys Phe Thr Met Asp Ser Ser Val Gln Leu Thr Tyr Ala Lys Ile

885 890 895

Ala Ser Val Ile Tyr Ala Phe Ile Met Leu Ala Val Leu Ile Ala Thr

900 905 910

Thr Asn Gln Ile Val Leu Glu Thr Val Phe Ser Pro Thr Ser Met Phe

915 920 925

Val Leu Gly Met Val Leu Ile Phe Ser Phe Ala Ser Cys Ile His Pro

930 935 940

Lys Glu Phe Ala Asn Ile Ile Tyr Gly Ser Ile Phe Phe Leu Met Ile

945 950 955 960

Pro Ser Thr Tyr Val Phe Leu Ser Leu Tyr Ser Leu Ile Asn Leu Asn

965 970 975

Val Ile Asn Trp Gly Thr Arg Glu Ala Val Ala Lys Ala Thr Gly Gln

980 985 990

Ala Thr Phe Arg Glu Asn Ile Ala Glu Arg Val Leu Arg Arg Val Ala

995 1000 1005

Asn Leu Asn Asp Asp Asn Ser Phe Leu Thr Arg Phe Leu Thr Arg Val

1010 1015 1020

Arg Ser Gly Glu Glu Pro Ser Glu Lys Ile Arg Thr Leu Glu Arg Lys

1025 1030 1035 1040

Phe Glu Arg Thr Glu Arg Leu Leu Met Ser Met Lys Glu Met Thr Glu

1045 1050 1055

Lys Ala Arg Gly Glu Ser Asp Ala Met Gln Met Gly Gly Gln His Phe

1060 1065 1070

Cys Met Met Asp Val Val Leu Asn Glu Glu Gly Glu Gln Glu Leu Pro

1075 1080 1085

Leu Arg Ser Gly Tyr Glu Ala Val Ala Ile Arg Arg Leu Asp Thr Phe

1090 1095 1100

Glu Arg Arg Ala Ile Val Thr Asp Pro Cys Lys Arg Ser Leu Trp Met

1105 1110 1115 1120

Asp Cys Glu Tyr Leu Gln Cys Cys Asp Arg Gly Lys Leu Arg Leu Gly

1125 1130 1135

Glu Glu Thr Phe Trp Asp Glu Leu Ile Asp Lys Tyr Leu Lys Pro Val

1140 1145 1150

Glu Thr Thr Thr Gln Glu Gln Ala Gln Val Ala Asn Gly Leu Val Ala

1155 1160 1165

Leu Arg Asn Arg Ile Ala Phe Ser Ile Ile Leu Leu Asn Cys Leu Leu

1170 1175 1180

Val Leu Ala Ile Phe Leu Leu Gln Arg His Lys Asp Val Leu Ser Val

1185 1190 1195 1200

Lys Phe Thr Pro Tyr Asp Gly Phe Lys Trp Thr Lys Met Asn Glu Thr

1205 1210 1215

Thr Gly Lys Phe Glu Asp Thr Thr Glu Ala Leu Lys Val Asp Pro Leu

1220 1225 1230

Gly Ile Gly Ile Ile Phe Phe Leu Met Gly Ile Leu Ile Val Gln Thr

1235 1240 1245

Ile Gly Met Leu Ile His Arg Leu Asn Thr Leu Val Glu Ala Leu His

1250 1255 1260

Glu Leu Ser Glu Met Lys Asp Val Gln Tyr Asn Thr Phe Thr Phe Thr

1265 1270 1275 1280

Asp His Ile Lys Val Leu Asn Glu Ala Arg Gln Met Ile Asp Thr Val

1285 1290 1295

Asn Tyr Asp Arg Ala His Gly Ala Asp Gly Tyr Thr Arg His Gly Leu

1300 1305 1310

Gly Asp Gln Lys Val Gln Lys Asn Val Leu Tyr Lys Leu Gln Lys

1315 1320 1325

<210> 4

<211> 420

<212> DNA

<213> Soybean cyst nematodes (Heterodera glycines)

<400> 4

acatgccatt cgtttgtcgg agaaaaatac ccttgaagga accaaattcc gcttggaagg 60

tctttattaa aattgacaag ctcgccccaa ttgcattttt ctcacctttt ttttgatcta 120

agatttatat tttccagtta atttctacaa aaattgaaac tttccgcgat ttgttcagtt 180

catattttct tcgatgactc gtggactgat gacaaagaat gtggtcgcat tccgaatgag 240

tacttcaagc tgttgttcga agttctgatg gaattgactc ggttttattt aattagtcaa 300

ttggttccgt ggtatttttg ctttgtcttg tcaagttccg acaacgctga agcggacatg 360

tacagccgta ttcttctgaa cactccgtac ggaggtcgac ttgtgctggc gttgaatggg 420

<210> 5

<211> 420

<212> RNA

<213> Soybean cyst nematodes (Heterodera glycines)

<400> 5

acaugccauu cguuugucgg agaaaaauac ccuugaagga accaaauucc gcuuggaagg 60

ucuuuauuaa aauugacaag cucgccccaa uugcauuuuu cucaccuuuu uuuugaucua 120

agauuuauau uuuccaguua auuucuacaa aaauugaaac uuuccgcgau uuguucaguu 180

cauauuuucu ucgaugacuc guggacugau gacaaagaau guggucgcau uccgaaugag 240

uacuucaagc uguuguucga aguucugaug gaauugacuc gguuuuauuu aauuagucaa 300

uugguuccgu gguauuuuug cuuugucuug ucaaguuccg acaacgcuga agcggacaug 360

uacagccgua uucuucugaa cacuccguac ggaggucgac uugugcuggc guugaauggg 420

Claims (7)

1. The amino acid sequence of the chitin synthetase of soybean cyst nematode is shown in SEQ ID No. 3.

2. A gene encoding the chitin synthase of soybean cyst nematode according to claim 1.

3. The gene of claim 2, wherein the nucleotide sequence is as shown in SEQ ID No.1 or SEQ ID No. 2.

4. A target sequence for designing dsRNA, which is the core structure domain of the gene of claim 3, and the nucleotide sequence of the target sequence is shown as SEQ ID No. 4.

5. The dsRNA segment designed according to the target sequence of claim 4, and the sequence of the dsRNA segment is shown as SEQ ID No. 5.

6. The use of the dsRNA segment of claim 5 in the construction of HIGS transgenic plants, said plants being soybean.

7. A method for resisting soybean cyst nematode is characterized in that a core structural domain of a gene of chitin synthase of the soybean cyst nematode is taken as a target, a dsRNA segment is transferred into a plant by an RNAi method, when the soybean cyst nematode eats a HIGS transgenic plant, the gene of the chitin synthase of the soybean cyst nematode is silenced, the plant shows the performance of resisting the soybean cyst nematode, the sequence of the dsRNA segment is shown as SEQ ID No.5, and the plant is soybean.

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