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Application of persimmon WRKY transcription factor gene in improving persimmon anthracnose resistance

A transcription factor, anthracnose technology, applied in the field of genetic engineering, can solve problems such as lack of research on the resistance mechanism of persimmon anthracnose

Pending Publication Date: 2021-03-12
SHANDONG YANTAI AGRI SCI & TECH INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The pathogenesis of persimmon anthracnose and the isolation and identification of anthracnose pathogens have been studied to

Method used

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  • Application of persimmon WRKY transcription factor gene in improving persimmon anthracnose resistance
  • Application of persimmon WRKY transcription factor gene in improving persimmon anthracnose resistance
  • Application of persimmon WRKY transcription factor gene in improving persimmon anthracnose resistance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Extraction of total RNA

[0028] Persimmon total RNA extracts the total RNA extract kit (Tiangen DP441) with RNA PREP PURE polysaccharide polyphenol plants, and the grinding operation is carried out in the ultra-clean workbench. Specific steps are as follows:

[0029] 1. Add beta-mercaptoethanol to a final concentration of 5% in advance: 25 μl of beta-mercaptoethanol (cleavage liquid is currently prepared) in a 475 μl lysate. The mortar is so dipped in liquid nitrogen to allow liquid nitrogen to sufficient cooling;

[0030] 2, 100 mg of left and right plants tissue blades with liquid nitrogen quickly ground into powder in pre-cooling, immediately premanely pre-cooling and adding 500 μL of lysate of 1.5 ml of centrifuge tube, immediately vortex oscillation makes it sufficient Crack;

[0031] 3, the centrifuge 12,000 × g centrifuge for 10 min, transfer the supernatant to the filter column CS, 12,000 × g from 2min, and absorb the supernatant in the collection tube t...

Embodiment 2

[0038] Example 2: Reverse composite synthesis cDNA

[0039] RNA extracted with infecting persimmon branch material is template, using kits primescript TM Rt Reagentkit with GDNA ERASER (Takara Baby Beijing), the following operations are carried out on ice, and the specific operation is as follows:

[0040]1, the genome DNA removal, the removal of the GDNA reaction system is shown in Table 1-1.

[0041] Table 1-1 Remove GDNA Reaction System

[0042] Table 1-1 Reaction Condition of Genomic DNA Elimination

[0043]

[0044] 2, the synthesis of single-stranded cDNA, the reverse transcription reaction system is shown in Table 1-2.

[0045] Table 1-2 Reverse Reaction System

[0046] Table 1-2 Reverse Transcription Reaction Condition

[0047]

[0048] 20 μl of the reaction system, 37 ° C, 15 min; 85 ° C, 5SEC; cooled to 4 ° C. After the reaction, the cDNA concentration was detected, using DDH 2 O Perform a concentration dilution, preserved - 20 ° C spare.

Embodiment 3

[0049] Example 3: DKCAD1 participates in persimmon anthrax

[0050] (1) DKCAD1 full length acquisition and promoter clone

[0051] Lignin has an important role in plants to resist the external pathogens, of which CAD is a key enzyme of lignin metabolism. Based on known DKCAD1 sequence information, the primer, PCR amplification, clonal sequencing was sequenced, and DKCAD1 was obtained, and the total length of CDNA was 975 bp, and the promoter sequence length was 828 bp.

[0052] (2) DKCAD1 up-regulate the expression of anthrax in 'anti-illneus "

[0053] QRT-PCR analysis showed that DKCAD1 DKCAD1 was obviously up-regulated in the extreme anti-species 'anti-disease peak, and the expression amount was induced by 9.5 times after 1D, and the expression amount decreased slightly, but far above' rich pacific Persimmon '; DKCAD1 is basically unchanged in the high-sensitive variety' rich pepper ', and the induction of anthrax is not obvious, such as figure 1 A shown. Klaason method is det...

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Abstract

The invention discloses application of a persimmon WRKY transcription factor gene in improving anthracnose resistance of persimmon, relates to the technical field of gene engineering, and adopts the technical scheme that the WRKY transcription factor gene is any one of DkWRKY3, DkWRKY5, DkWRKY7, DkWRKY8 and DkWRKY10, and nucleotide sequences of the genes are respectively shown as SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 According to the invention, through an infection experiment, anthracnose resistance of common persimmon varieties is evaluated; five transcription factors DkWRKY3, DkWRKY5, DkWRKY7, DkWRKY8 and DkWRKY10 interacting with DkCAD1 have obvious expression changes after the high-resistance variety disease-resistant pointed persimmon is inoculated with anthracnose pathogenic bacteria, and participate in persimmon anthracnose resistance. The research result provides a scientific basis for persimmon anthracnose resistance molecular research.

Description

Technical field [0001] The present invention relates to the field of genetic engineering technology, and more particularly, it involves an application of a persimmon WRKY transcription factor gene in improving persimmon influence resistance. Background technique [0002] Persimmon (Diospyros Kaki) belongs to the Gemini Formation, and the tall deciduous trees, the whole catasmon is more than 250, and more than 3,000 years of cultivation history, tropical and subtropical are mainly distributed areas. As the most cultivated in the primary production area, in China, Japan and South Korea. Persimners come from China, which is very rich in traditional fruits and germplasm resources, which is a widely cultivated and exploitable persimmon, Zhejiang persimmon, oil, and so on. According to the data released by the United Nations World Food and Agriculture 2018, the harvest area of ​​my country's persimmon accounted for 90.16% of the world's total harvest area, and persimmon production acco...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/29A01H5/12A01H6/82A01H6/00
CPCC12N15/8281C07K14/415
Inventor 杜晓云关长飞王彦波杨勇王孟珂于晓丽张硕赵玲玲牟红梅慈志娟
Owner SHANDONG YANTAI AGRI SCI & TECH INST
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