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Rice blast resistance related gene OsPLUS3 and application thereof in gene engineering

A technology of genetic engineering and rice blast, applied in the field of genetic engineering, can solve the problem that the disease resistance function has not been reported, and achieve the effect of improving disease resistance

Pending Publication Date: 2022-03-15
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The Plus-3 domain plays a very important role in the growth and development of rice, but its function in disease resistance has not been reported

Method used

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  • Rice blast resistance related gene OsPLUS3 and application thereof in gene engineering
  • Rice blast resistance related gene OsPLUS3 and application thereof in gene engineering

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Experimental program
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Effect test

Embodiment 1

[0026] 1) Extraction of total RNA and DNA

[0027] The rice indica variety 9311 was selected, and the leaves were taken after the rice seedlings grew to the 3-4 leaf stage. Some were immediately frozen with liquid nitrogen and stored in a -80°C freezer. Take the leaves and grind them with a mortar, transfer them into a 1.5mL EP tube (TRIzol Reagents, purchased from TIANGEN, CHINA) filled with Trizol lysate, shake fully, extract the total RNA, and identify the quality of the total RNA by electrophoresis. Part of the leaves were ground with a grinder, and the DNA in the leaves was extracted with the CTAB method. The quality of the extracted DNA and RNA was tested by electrophoresis.

[0028] 2) Cloning of rice gene OsPLUS3

[0029] 9311 is an indica rice variety that has been sequenced. Search the 9311 genome sequence online to obtain the full-length sequence of the 5121bp rice gene OsPLUS3, and design primers P3 and P4 at both ends:

[0030] P3:5-ATGGACGGGGGGACGGGGCC-3 (SEQ...

Embodiment 2

[0039] Using multi-fragment recombinase (purchased from Nanjing Novozyme Co., Ltd.), the promoter and cDNA of the amplified OsPLUS3 gene were connected, and then the restriction sites Hind III and Kpn1 were introduced, and homologous recombinase (purchased from Nanjing Novozyme) the gene was connected to the pCAMBIA1300S vector. Homologous arm amplification primers P5 and P6 primer sequences are:

[0040] P5:5-ACGGCCAGTGCCAAGCTTATGGACGGGGGGACGGGGCC-3 (SEQ ID NO. 7)

[0041] P6:3-TCTAGAGGATCCCCGGGTACCTCATTTAGGGCTGTACAAAA-5 (SEQ ID NO.8) was sequenced and identified to ensure that the reading frame of the coding region in the vector was correct, and then it was transformed into Agrobacterium, and further transformed into Su Yunuo, a rice blast-susceptible strain.

[0042] (The vector has been constructed, but the transgenic plants have not yet been obtained, so there is no phenotypic identification result of the transgenic material for the time being)

Embodiment 3

[0044] The designed primers P7 and P8 were used for real-time quantitative RT-PCR to analyze the expression of rice seedlings at the 3-4 leaf stage after inoculation. The results showed that the expression of OsPLUS3 was significantly enhanced 24 hours after inoculation with Magnaporthe grisea spores, indicating that the expression of OsPLUS3 gene related to Magnaporthe grisea ( figure 1 ). The sequences of P7 and P8 primers are:

[0045] P7:5-ACGATCACGTAGATGGCTCAGTC-3 (SEQ ID NO.9)

[0046] P8:3-CCGGCTTACTTGGTATCTGCTG-5 (SEQ ID NO. 10)

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Abstract

The invention belongs to the field of gene engineering, and relates to a rice blast resistance related gene OsPLUS3 and application thereof in gene engineering. The rice gene OsPLUS3 is a transcription factor with a plus-3 structural domain, the cDNA (complementary deoxyribonucleic acid) sequence of the gene is as shown in SEQ ID NO.1, and the amino acid sequence of the gene is as shown in SEQ ID NO.2. The invention discloses a rice gene OsPLUS3 which is reported for the first time in rice, and the gene is a rice gene which is screened through 240 parts of natural population whole genome association analysis and is significantly related to seedling blast resistance and has haplotype significant difference. MRNA expression analysis shows that the gene is obviously induced by inoculation of magnaporthe oryzae, and the seedling blast resistance of a rice plant with the T-NDA inserted mutant is lower than that of a wild Dongjin material. Therefore, the gene can be used as a target gene to be introduced into a susceptible material to improve the plant disease resistance so as to improve the plant variety.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a rice blast resistance-related gene OsPLUS3 and its application in genetic engineering. Background technique [0002] Plants contain many transcription factor families, whose main function is to recognize and bind to the nucleotide sequence in the promoter of the target gene, activate or repress the expression of the target gene, regulate the process of plant growth and stress defense response. Plus-3 is a conserved domain in the plant RNA polymerase-associated factor Paf1 (polymerase-associated factor 1 complex), which can bind DNA and RNA, regulate target genes, and function as a transcription factor. The Plus-3 domain plays a very important role in the growth and development of rice, but its function in disease resistance has not been reported. [0003] The OsPLUS3 cloned in the present invention is a rice blast resistance-related gene obtained by GWAS. It is a transcription ...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/54C07K14/415C12N9/12C12N15/82A01H5/00A01H6/46
CPCC07K14/415C12N15/8279C12N15/8205C12N2800/22
Inventor 鲍永美马璐余耀张红生黄骥王建飞唐海娟董世楠
Owner NANJING AGRICULTURAL UNIVERSITY
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