Verticillium dahliae phospholipase target gene fragment and interference vector and application thereof

A technology of Verticillium dahliae and phospholipase, which is applied in the fields of application, genetic engineering, plant gene improvement, etc., to achieve a significant effect of improving disease resistance and disease resistance

Active Publication Date: 2016-01-20
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the functional analysis of the phospholipase of Verticillium dahliae has not been reported, so the utilization has been successfully sequenced and published in the BroadInstitute (http: / / www.broadinstitute.org / annotation / genome / verticillium_dahliae / GenomeDescriptions.html#VD_VdLs. 17) Genome data information of Verticillium dahliae race VdLs.17 (KlostermanSJ, AtallahZK, ValladGE, SubbaraoKV: Diversity, pathogenicity, and management of Verticillium species. Annual review of phytopathology 2009, 47:39–62.), found that they are located on different chromosomes ( The first chromosome, the third chromosome), and the two phospholipase genes with different sequence information, named phospholipase 1 (PL1, located on the first chromosome), and phospholipase 3 (PL3, located on the third chromosome) ), from these phospholipase genes, the target genes related to the resistance to Verticillium dahliae will be of great significance for improving the disease resistance of plants to Verticillium dahliae

Method used

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  • Verticillium dahliae phospholipase target gene fragment and interference vector and application thereof
  • Verticillium dahliae phospholipase target gene fragment and interference vector and application thereof
  • Verticillium dahliae phospholipase target gene fragment and interference vector and application thereof

Examples

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Effect test

Embodiment 1VI

[0040] Example 1 Construction of VIGS-PL1 and VIGS-PL3 Transient Interference Vectors, Agrobacterium Injection of Nicotiana benthamiana and Inoculation of Verticillium dahliae

[0041] 1. Experimental method

[0042] Using the total RNA of Verticillium dahliae as a template, 3 pairs of phospholipase 1 (PL1, VDAG_00942) gene and 4 pairs of phospholipase 3 (PL3, VDAG_02397) gene primers were designed (see Table 1 for the primer sequences) to amplify the phospholipids respectively. The target segment information of the enzyme gene, the length is about 400bp. The 7 fragments obtained by the amplification were connected to the multiple cloning site region of the vector TRV2 ( figure 1 ), obtain recombinant vectors TRV2-VdPL1-1, TRV2-VdPL1-2, TRV2-VdPL1-3, TRV2-VdPL3-1, TRV2-VdPL3-2, TRV2-VdPL3-3, TRV2-VdPL3-4, after the sequence is correct Electric shock transformation of Agrobacterium GV3101, mixed with Agrobacterium GV3101 strain TRV1 (carrying another part of TRV virus gene i...

Embodiment 2

[0051] Example 2 Stable Genetic Interference Vector Construction of Phospholipase Target Gene Sequence and Transformation of Nicotiana benthamiana

[0052] 1. Experimental method

[0053] 1.1 Cloning of target fragments and construction of interference vectors

[0054] In order to obtain stably inherited transgenic plants containing target gene dsRNA, the present invention utilizes Gateway technology to combine seven gene fragments VdPL1-1 (nucleotide sequence shown in SEDNo.1), VdPL1-2 ( Nucleotide sequence is shown in SEDNo.2), VdPL1-3 (nucleotide sequence is shown in SEDNo.3), VdPL3-1 (nucleotide sequence is shown in SEQIDNo.4), VdPL3-2 (nucleoside The acid sequence is shown in SEQIDNo.5), VdPL3-3 (nucleotide sequence is shown in SEQIDNo.6), VdPL3-4 (nucleotide sequence is shown in SEQIDNo.7) is the target gene, and the length is about 300bp. Design primers VdPL1-1-F / R, VdPL1-2-F / R, VdPL1-3-F / R, VdPL3-1-F / R, VdPL3-2-F / R containing BP sites at both ends of the seven segmen...

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Abstract

The invention discloses a verticillium dahliae phospholipase target gene fragment and an interference vector and application thereof, and belongs to the fields of clone and application of verticillium dahliae disease course key genes. A gene sequence of a verticillium dahliae phospholipase target gene is introduced into transient expression ds RNA of nicotiana benthamiana through a virus-induced gene silencing technology, and the target gene which can extremely significantly improve the plant disease resistance is screened; an RNAi technology is further utilized to establish the stably inherited interference vector and transform the nicotiana benthamiana, and transforming results show that transgenic tobacco plants extremely significantly improve the disease resistance of tobaccos to verticillium dahliae, show the highly resistance and even reach the immune level. The verticillium dahliae phospholipase target gene fragment and the RNA interference vector can be applied to improving the disease resistance of the plants to the verticillium dahliae and cultivating new transgenic plant species with the verticillium dahliae resistance.

Description

technical field [0001] The present invention relates to a target gene fragment of Verticillium dahliae phospholipase and the RNA transcribed by the target gene fragment, and also relates to an RNA interference carrier containing the target gene fragment of phospholipase, and the present invention further relates to the phospholipid of Verticillium dahliae The application of the enzyme target gene and the RNA transcribed by the target gene fragment or the interference carrier in improving the disease resistance of plants to Verticillium dahliae belongs to the field of cloning and application of key genes in the course of Verticillium dahliae. Background technique [0002] Verticillium dahliae (Verticilliumdahliae) is an extremely harmful soil-borne vascular fungal disease with a wide range of hosts, which can infect cotton, sunflower, eggplant, pepper, tomato, tobacco, potato, melon, watermelon, cucumber, peanut More than 200 kinds of economic crops such as , kidney bean, mun...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N15/82C12N15/66A01H5/00
Inventor 程红梅齐希梁苏晓峰郭惠明
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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