Disease resistance and heat tolerance related protein tarhp1 and related biological materials and method for cultivating disease resistance and heat tolerance plants
A biomaterial and protein technology, applied in the fields of disease resistance and heat resistance related protein TaRHP1 and its related biomaterials and cultivation of disease resistance and heat resistance plants, can solve the problems of lack and slow progress.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0089] Embodiment 1, the cloning of the gene encoding TaRHP1 of disease-resistant and heat-tolerant wheat
[0090] 1. Cloning of TaRHP1 gene
[0091] The inventors of the present invention isolated and cloned a wheat gene TaRHP1 related to wheat disease resistance and high temperature resistance from sheath blight resistant wheat germplasm CI12633, as shown in sequence 1 of the sequence listing, and named its encoded protein as TaRHP1 protein, as shown in Shown in sequence 2 of the sequence listing. The specific cloning method is as follows:
[0092] Extract the total RNA from the stems of wheat CI12633 inoculated with Rhizoctonia solani R0301, and reverse-transcribe the extracted RNA samples to synthesize the first-strand cDNA according to the procedure of the first-strand cDNA synthesis kit of Tiangen Biochemical Company, as a template for gene cloning. TaRHP1-OF1: 5'-CAGCAGCAGCCCACCTCTCTCT-3' and TaRHP1-OR1: 5'-TTAGAAATCGAGCTGCGAGC-3' were used as primers for PCR amplific...
Embodiment 2
[0110] Example 2. Acquisition and identification of disease-resistant and heat-resistant transgenic Arabidopsis thaliana
[0111] 1. Construction of recombinant expression vector
[0112] 1. Construct the complete ORF sequence of the TaRHP1 gene on the dicotyledonous plant expression vector pCAMBIA1300, the specific operation is as follows: using pEASY-TaRHP1 in Example 1 as a template, using A1300-TaRHP1-inF: GCTGTACAAGGTCG AC ATGGCCGCCGCCGCCG (the underlined sequence is the linker sequence on the vector) and A1300-TaRHP1-inR: TACCG GATCCACTAGT The primer pair composed of GAAATCGAGCTGCGAG (the underlined sequence is the linker sequence on the vector), under the action of the high-fidelity amplification enzyme KOD-FX (TOYOBO company), PCR amplifies the 62-1216th core of sequence 1 in the sequence list Nucleotides were obtained to obtain PCR amplification products and recovered.
[0113] PCR reaction program: 98°C pre-denaturation for 1min; 35 cycles of 98°C for 10s, 58...
Embodiment 3
[0138] Embodiment 3, cultivating the transgenic wheat resistant to sheath blight
[0139] 1. Construction of recombinant expression vector
[0140] Construct the complete ORF sequence of TaRHP1 gene into the monocot transformation vector pWMB132, the specific operation is as follows:
[0141] Using pCAMBIA1300-TaRHP1 as a template and using a primer pair composed of TaRHP1-O-F and TaRHP1-O-R, PCR amplification was performed under the action of the high-fidelity amplification enzyme PRIMERSTAR (TAKARA Company), and the PCR amplification product was obtained and recovered.
[0142] TaRHP1-O-F:5'-cg GGATCC ATGGCCGCCGCCGCCG-3' (the sequence indicated by the underline is the BamH I restriction site sequence);
[0143] (The sequence indicated by the underline is the Sac I restriction site sequence, and the sequence indicated by the box is the 6×HIS tag coding sequence).
[0144] PCR reaction program: pre-denaturation at 98°C for 1min; 35 cycles of 98°C for 10s, 56°C for 15s, a...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More