Rice thioredoxinase gene osndu, protein, vector, host cell, molecular marker method and application
A thioredoxase, rice technology, applied in the field of plant genetic engineering
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Embodiment 1
[0051] [Example 1] Obtaining of rice OsNDU gene
[0052] A thioredoxinase OsNDU was found by screening the interacting protein in the rice 9311 library against the brown planthopper gene Bph6, and the ORF sequence and genome sequence of the gene were amplified in the cDNA of Nipponbare by designing primers.
Embodiment 2
[0053] [Example 2] Subcellular localization of rice OsNDU gene
[0054] Primers were designed at both ends of the full-length ORF of the OsNDU gene, and BamHI restriction sites and protective bases were added. After the amplified fragment was recovered, it was digested with BamHI enzyme and connected to the vector PCXUN::GFP digested with the same enzyme. , The positive clones were sent for sequencing, and it was confirmed that there was no mutation in the forward connection to extract the plasmid, and the plasmid was transformed into protoplasts. The specific process is as follows:
[0055] The rice seeds were sown in 1 / 2 MS medium, and cultured in a dark incubator at 28°C for 10 to 12 days. Take about 100 seedlings, cut the stems into small pieces of about 0.5mm with a blade, equilibrate in 0.6M mannitol for 10 minutes, transfer them to the enzymatic hydrolysis solution, and culture them in the dark at 28°C and 80rpm for 4-5h. Add 10 ml of W5 solution to the enzymolysis so...
Embodiment 3
[0056] [Example 3] Construction of OsNDU gene overexpression vector and genetic transformation mediated by Agrobacterium
[0057] 1. Construction of OsNDU overexpression vector
[0058] The inventors intercepted a section at both ends of the ORF of OsNDU to design primers, the sequence of which is as follows:
[0059] OEV-F: ATGCTGCCGCCGGCCGCC (5'-3'), as shown in SEQ ID NO.6;
[0060] OEV-R: GCAAGCATCTAGATCCCT (5'-3'), as shown in SEQ ID NO.7;
[0061] The vector used is pCXUN (provided by Professor Wang Guoliang of Ohio State University in the United States). The pCXUN vector is cut with XcmI, and the foreign fragment can be directly connected after adding A. According to the sequence of SEQ ID No.2, the ORF was directly amplified by PCR, and connected to the vector after adding A. After the sequence verification is correct, the obtained vector is the OsNDU gene overexpression vector, which is electrotransformed into Agrobacterium EHA105. Pick a single clone for expansio...
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