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Oryza sativa thioredoxin gene OsNDU, protein, vector, host cell, molecular marking method and application of molecular marking method

A thioredoxase, host cell technology, applied in the field of plant genetic engineering

Active Publication Date: 2020-03-06
WUHAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

OsNDU is an interacting protein of BPH6, and its function in rice resistance to BPH has not been studied

Method used

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  • Oryza sativa thioredoxin gene OsNDU, protein, vector, host cell, molecular marking method and application of molecular marking method
  • Oryza sativa thioredoxin gene OsNDU, protein, vector, host cell, molecular marking method and application of molecular marking method
  • Oryza sativa thioredoxin gene OsNDU, protein, vector, host cell, molecular marking method and application of molecular marking method

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention provides an oryza sativa thioredoxin gene OsNDU, a protein, a vector, a host cell, a molecular marking method and an application of the molecular marking method. The OsNDU has a nucleotide sequence shown in SEQ ID No.1, a cDNA sequence of the OsNDU is shown in SEQ ID No.2, and a protein sequence of the OsNDU is shown in SEQ ID No.3. A scheme provided by the invention is a good example that thioredoxin participates in nilaparvata lugens resistance of oryza sativa, and the scheme has a certain reference value for understanding a function of thioredoxin and regulating and controlling resistance. A research of the OsNDU gene provides a good theoretical basis for a downstream molecular mechanism of nilaparvata lugens resistant genes of oryza sativa, and has reference significancefor researching a gene molecular function and breeding.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, in particular to a rice thioredoxinase gene OsNDU, protein, carrier, host cell, molecular marker method and application. Background technique [0002] Rice (Oryza sativa) is an important food crop, and its planting area ranks first in the world. Brown planthopper (BPH; Nilaparvata lugens ) is a migratory pest in rice-growing areas in Asia, and it is also a major pest in rice-growing areas in the Yangtze River Basin of China and the vast rice-growing areas of Southwest and South China. There are three main ways for N. lugens to damage rice: one is to directly damage rice by sucking rice phloem juice through a stylet (Wang et al., 2008; Cheng et al., 2012). The second is egg damage. The brown planthopper inserts the ovipositor into the vascular tissue of rice, destroying the transportation system of rice. The third is to induce rice diseases, such as spreading straw dwarf and leaf dwarf ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/82C12N5/10A01H5/00A01H6/46C12Q1/6895
CPCC12N9/0051C12N15/8286C12Q1/6895C12Q2600/13C12Y108/01008
Inventor 何光存徐春雪郭建平杜波陈荣智祝莉莉
Owner WUHAN UNIV
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