Soybean cyst nematode resistance gene and application thereof

Abstract

The invention relates to a soybean cyst nematode resistance gene and application thereof. The cDNA (complementary deoxyribonucleic acid) fragment (SRC-J-6) provided by the invention is derived from soybean (Glycine max (L.) Merrill.) variety L-10, and has a nucleotide sequence shown in 1) a nucleotide sequence shown in SEQ ID NO. 1; 2) a DNA (deoxyribonucleic acid) molecule capable of hybridizingwith the nucleotide sequence shown in SEQ ID NO. 1 and being expressed under strict conditions; 3) an mRNA (messenger ribonucleic acid) molecule, which has homology of more than 90% with the nucleotide sequence shown in 1) and can be expressed; or 4) a DNA (deoxyribonucleic acid) molecule, which has homology of more than 90% with the nucleotide sequence shown in 1) and can be expressed. After theplant tissues are transformed by the gene disclosed by the invention, the resistance to cyst nematode of the transgenic soybean is significantly improved. The soybean cyst nematode resistance gene provided by the invention has important significance, not only can effectively guide conventional breeding, can also provide excellent genetic resources for the transgenic breeding of soybean and has broad application prospects in the soybean production.

Description

Technical field

[0001] The invention relates to a soybean cyst nematode resistance gene and its application, and belongs to the field of compound libraries containing RNA or DNA encoding proteins. Background technique

[0002] In my country, especially in Northeast China, due to the increasing phenomenon of indiscriminate cultivation, indiscriminate grazing, and indiscriminate logging, the area of ​​arable land desertification, drought and saline-alkali land is increasing year by year. According to reports, the area of ​​saline-alkali land in the country is over 500 million mu, and the area of ​​heavy saline-alkali land in Heilongjiang Province is 16 million mu ("Saline-alkali Land Reform and Organic Industry Development Forum", 2010). Because soybean cyst nematode likes arid and alkaline soil environment (humidity 60%, pH: 8.0), the land desertification, drought and the expansion of saline-alkali land area have caused the soybean cyst nematode to be more and more serious. [0003...

Examples

Embodiment 1

[0054] Example 1 Nucleotide sequence of soybean anti-cyst nematode gene

[0055] The nucleotide sequence of soybean cyst nematode resistance is shown in SEQ ID NO. 1, or shown in any of the following 1)-3),

[0056] 1) DNA molecules that can hybridize and express under stringent conditions with the nucleotide sequence defined in SEQ ID NO. 1;

[0057] 2) An mRNA molecule that has more than 90% homology with the nucleotide sequence described in SEQ ID NO. 1 and can be expressed;

[0058] 3) DNA molecules that have more than 90% homology with the nucleotide sequence described in SEQ ID NO. 1 and can be expressed.

[0059] The stringent conditions can be hybridization in a solution of 6×SSC, 0.5% SDS at 65° C., and then wash the membrane once with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS.

[0060] The primer pair for amplifying any of the above-mentioned DNA fragments also belongs to the protection scope of the present invention, for example: SEQ ID NO.3 and SEQ ID NO.4.

[0061] SEQ ID NO. 2 bel...

Embodiment 2

[0062] Example 2 Obtaining soybean anti-cyst nematode gene

[0063] 1. Gene cloning

[0064] 1) Extraction and reverse transcription of total plant RNA: Take the root and leaf tissues of L-10 and Heinong 37 12 hours after inoculation with nematodes, and extract RNA. The total RNA is detected by agarose gel electrophoresis, 18S and 28S RNA The band type is clearly distinguishable, UV spectrophotometer can detect OD 260 / OD 280 The ratio is between 1.9 and 2.0, indicating that the RNA has good integrity and high purity. Reverse transcription reaction refer to promega ImProm-II TM (Promega) reverse transcription kit for operation.

[0065] 2) Cloning of the full-length sequence of disease resistance candidate genes: using the above reverse transcribed L-10 and Heinong 37 root and leaf cDNA as templates, using the designed gene-specific primers (SEQ ID NO. 3 and SEQ ID NO. ID NO.4) and use the drop-down PCR method to clone the target gene. The reaction system and procedures are show...

Embodiment 3

[0074] Example 3 Functional verification of soybean resistance to cyst nematode gene

[0075] 1. Construction of plant expression vector

[0076] Clone the SRC-J-6 gene sequence into the vector pGFPGUS to construct an overexpression vector pGFPGUS-SRC-J-6. The specific primer sequences are shown in Table 2:

[0077] Table 2. SRC-J-6 amplification primer sequence

[0078]

[0079] The target gene amplification, PCR product purification, ligation, transformation and identification and sequencing of positive clones are the same as above. The pMD18-T-SRC-J-6 recombinant plasmid with the target gene and the empty pGFPGUS vector linked to it were digested with Xba I and Sac I, respectively, and identified by electrophoresis, and recovered about 1.6kb SRC-J with a gel recovery kit -6 fragments and a large fragment of about 13kb of the vector. The obtained small fragment is ligated with the large fragment of pGFPGUS vector to obtain the ligation product. The ligation product was transforme...

Technical field

[0001] The invention relates to a soybean cyst nematode resistance gene and its application, and belongs to the field of compound libraries containing RNA or DNA encoding proteins. Background technique

[0002] In my country, especially in Northeast China, due to the increasing phenomenon of indiscriminate cultivation, indiscriminate grazing, and indiscriminate logging, the area of ​​arable land desertification, drought and saline-alkali land is increasing year by year. According to reports, the area of ​​saline-alkali land in the country is over 500 million mu, and the area of ​​heavy saline-alkali land in Heilongjiang Province is 16 million mu ("Saline-alkali Land Reform and Organic Industry Development Forum", 2010). Because soybean cyst nematode likes arid and alkaline soil environment (humidity 60%, pH: 8.0), the land desertification, drought and the expansion of saline-alkali land area have caused the soybean cyst nematode to be more and more serious. [0003...

Examples

Embodiment 1

[0054] Example 1 Nucleotide sequence of soybean anti-cyst nematode gene

[0055] The nucleotide sequence of soybean cyst nematode resistance is shown in SEQ ID NO. 1, or shown in any of the following 1)-3),

[0056] 1) DNA molecules that can hybridize and express under stringent conditions with the nucleotide sequence defined in SEQ ID NO. 1;

[0057] 2) An mRNA molecule that has more than 90% homology with the nucleotide sequence described in SEQ ID NO. 1 and can be expressed;

[0058] 3) DNA molecules that have more than 90% homology with the nucleotide sequence described in SEQ ID NO. 1 and can be expressed.

[0059] The stringent conditions can be hybridization in a solution of 6×SSC, 0.5% SDS at 65° C., and then wash the membrane once with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS.

[0060] The primer pair for amplifying any of the above-mentioned DNA fragments also belongs to the protection scope of the present invention, for example: SEQ ID NO.3 and SEQ ID NO.4.

[0061] SEQ ID NO. 2 bel...

Embodiment 2

[0062] Example 2 Obtaining soybean anti-cyst nematode gene

[0063] 1. Gene cloning

[0064] 1) Extraction and reverse transcription of total plant RNA: Take the root and leaf tissues of L-10 and Heinong 37 12 hours after inoculation with nematodes, and extract RNA. The total RNA is detected by agarose gel electrophoresis, 18S and 28S RNA The band type is clearly distinguishable, UV spectrophotometer can detect OD 260 / OD 280 The ratio is between 1.9 and 2.0, indicating that the RNA has good integrity and high purity. Reverse transcription reaction refer to promega ImProm-II TM (Promega) reverse transcription kit for operation.

[0065] 2) Cloning of the full-length sequence of disease resistance candidate genes: using the above reverse transcribed L-10 and Heinong 37 root and leaf cDNA as templates, using the designed gene-specific primers (SEQ ID NO. 3 and SEQ ID NO. ID NO.4) and use the drop-down PCR method to clone the target gene. The reaction system and procedures are show...

Embodiment 3

[0074] Example 3 Functional verification of soybean resistance to cyst nematode gene

[0075] 1. Construction of plant expression vector

[0076] Clone the SRC-J-6 gene sequence into the vector pGFPGUS to construct an overexpression vector pGFPGUS-SRC-J-6. The specific primer sequences are shown in Table 2:

[0077] Table 2. SRC-J-6 amplification primer sequence

[0078]

[0079] The target gene amplification, PCR product purification, ligation, transformation and identification and sequencing of positive clones are the same as above. The pMD18-T-SRC-J-6 recombinant plasmid with the target gene and the empty pGFPGUS vector linked to it were digested with Xba I and Sac I, respectively, and identified by electrophoresis, and recovered about 1.6kb SRC-J with a gel recovery kit -6 fragments and a large fragment of about 13kb of the vector. The obtained small fragment is ligated with the large fragment of pGFPGUS vector to obtain the ligation product. The ligation product was transforme...

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