Proteins related to rust resistance as well as coding gene and application of proteins
A coding and rust technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve problems such as the utilization of unfavorable resistance resources and the loss of resistance of disease-resistant resources
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Embodiment 1
[0169] Embodiment 1, the cloning of RppRC1 gene
[0170] Soybean is a paleotetraploid legume, self-pollinated, with a genome size of about 1.1 Gbp. Soybean germplasm with different resistances had obvious differences in response to soybean rust. Soybean (Glycine max (L.) Merrill) ZYRC1 is a rust-resistant resource obtained from Chinese soybean germplasm by the Institute of Oil Crops, Chinese Academy of Agricultural Sciences. It is CGMCC No. 17575, and its reaction to soybean rust is immunity.
[0171] The genome of soybean ZYRC1 (the source of the RppRC1 gene) was used as a template, and primer F and primer R (sequences are as follows) were used for PCR amplification.
[0172] F: 5'-ATGGCAGATAGTGTTGTTGCTTTTCTGC-3' (SEQ ID No.3);
[0173] R: 5'-TCACAGTTCATTAGAGATTTTGAGCTTACAGC-3' (SEQ ID No. 4).
[0174] The resulting amplified product is the sequence shown in SEQ ID No.2. SEQ ID No.2 is the nucleotide sequence of the RppRC1 gene, which encodes the protein shown in SEQ ID ...
Embodiment 2
[0175] Embodiment 2, transform soybean with soybean RppRC1 gene
[0176] 1. Construction of recombinant expression vector
[0177] Take the soybean variety ZYRC1 under normal conditions and cultivate it until the first three primary compound leaves grow out, extract DNA, use the DNA as a template, and under the guidance of primer F and primer R (see Example 1 for the primer sequence), Use conventional PCR method to amplify, after the reaction is over, carry out 1% agarose gel electrophoresis detection on the PCR amplified product, recover and purify the DNA fragment of about 2.5kb; Under the guidance of the general PCR method, the 2525bp promoter pRppRC1 fragment was obtained, which was ligated into the vector pB2GW7 (the vector comes from VIB-plantsystems biology, website: https: / / gateway.psb.ugent.be / search) to obtain the recombinant vector pB2GW7-pRppRC1.
[0178] PF: 5'- GAGCTC AAAGGCTTTTTTGTTAAGGGAAGGT-3' (the underlined part is SacI recognition sequence);
[0179] ...
Embodiment 3
[0219] Embodiment 3, detect the anti-rust effect of transgenic soybean
[0220] This example will use soybean rust physiological race SS4 (recorded in "Shan Zhihui et al. Pathogenic responses of soybean rust on different leguminous hosts. Chinese Journal of Oil Crops, 2008, 30(4): 497-500 " article, the public can obtain from the applicant, can only be used to repeat the experiment of the present invention, not for other use) to inoculate the transgenic plants obtained in Example 2, and score the plant disease symptoms to test the anti-rust effect of the RppRC1 gene.
[0221] First, preliminary tests were performed on T0 transformation events L1 and L2 to assess the effect of the RppRC1 transgene on rust infection. The specific operation is as follows: Take the fully expanded new leaves on the T0 plants, and use 1×10 5 The soybean rust physiological race SS4 bacterial suspension of spores / ml was sprayed and inoculated, and the inoculum size was 10 μl per square centimeter, wi...
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