Solanum torvum StoWRKY6 gene and expression vector and applications thereof

A technology of expression vector and wild eggplant, applied in application, genetic engineering, plant gene improvement, etc., can solve problems such as low disease resistance and yield loss

Inactive Publication Date: 2014-02-19
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Eggplant (Solanum melongena) is a worldwide vegetable crop of the Solanaceae family. Most eggplant cultivars have low disease resistance and are often attacked by pathogens in nature, resulting in yield loss

Method used

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  • Solanum torvum StoWRKY6 gene and expression vector and applications thereof
  • Solanum torvum StoWRKY6 gene and expression vector and applications thereof
  • Solanum torvum StoWRKY6 gene and expression vector and applications thereof

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Embodiment 1

[0035] The cloning of embodiment 1StoWRKY6 gene

[0036] Total RNA was extracted from wild eggplant Solanum torvum Swartz by Trizol kit. The first strand of cDNA was synthesized using the reverse transcriptase system of Bao Bioengineering Company.

[0037] Design intermediate conserved sequence primer P1 (forward primer W1: 5'-ATATGGGTAACTCATCGAMTSATC-3' (SEQ ID NO.3) (M stands for A or C; S stands for C or G) and reverse primer W2: 5'-TGTTTTTGTCCATCTTGTCC- 3' (SEQ ID NO.4)), the middle conserved region of StoWRKY6 cDNA was obtained by PCR amplification of the first strand of synthetic cDNA. After electrophoresis on 1.0% agarose, the PCR product was recovered with the gel recovery kit of Bao Bio Company, connected to pMD18-T Vector, transformed into Escherichia coli DH5α, single clone was detected by PCR, and the positive clone was sent to BGI for sequencing. The middle fragment of about 382bp.

[0038] According to the blastn alignment results of the wild eggplant StoWRKY6...

Embodiment 2

[0039] Example 2 StoWRKY6 Gene Tissue Expression Analysis

[0040] Total RNA was extracted from wild eggplant roots, stems, leaves, senescent leaves, mature leaves, and young leaves at the 6-leaf stage, and the cDNA obtained after reverse transcription was used as a template. ID NO.7) and reverse primer 5'-CAAAATTCCAAAGACCCTCC-3' (SEQ ID NO.8)), semi-quantitative RT-PCR was performed to detect the expression of StoWRKY6 gene in different tissues of wild eggplant. PCR amplification conditions Pre-denaturation at 94°C for 4min, followed by 30 cycles at 94°C for 40s; 58°C for 40s; 72°C for 30s, and finally 72°C for 10min. PCR products were spotted and detected by 1% agarose gel electrophoresis. The internal reference used the constitutively expressed EF1α gene, and the primers were EF1α1:5′-GTGGTGGAGTCAATAATGAGGAC-3′ (SEQ ID NO.9) and EF1α2:5′-TCGACAACAGAAACATCAGCAGT-3′ (SEQ ID NO.10). The PCR conditions were: 94°C pre-denaturation for 4min, then 94°C for 40s; 58°C for 30s; 72°...

Embodiment 3

[0041] Semi-quantitative analysis of StoWRKY6 gene expression under embodiment 3SA, JA and Verticillium dahliae induction

[0042] To study the expression of StoWRKY6 under the stress of salicylic acid (SA) and jasmonic acid (JA). The healthy and disease-free 6-leaf stage seedlings were taken for chemical treatment as follows: the final concentrations of salicylic acid and jasmonic acid were 300 mM and 200 μM, respectively, and the control treatment was Tween20 / water to make it similar to the corresponding chemical treatment conditions. The leaves of all plants were sampled at 0h, 2h, 4h, 12h, and 48h after treatment, and all were frozen and stored in a -70 refrigerator. RNA was extracted for semi-quantitative reverse transcription PCR (RT-PCR) analysis, and the constitutively expressed EF1α gene was used as an internal reference. Semi-quantitative RT-PCR using primer QN was used to detect the expression of StoWRKY6 gene in wild eggplant plants under SA and JA treatment, resp...

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Abstract

The invention belongs to the field of biology, and discloses a solanum torvum StoWRKY6 gene and an expression vector and applications thereof. The nucleotide sequence of the solanum torvum StoWRKY6 gene is shown in SEQ ID NO.1. An agrobacterium-mediated method is adopted to transfer StoWRKY6 gene overexpression and a RNAi vector into potatoes to respectively obtain 9 strains of overexpression and 14 strains of RNAi transgenic plants. The morbidity and disease index of overexpressed transgenic potatoes are obviously lower than those of a wild type control group, but the morbidity and disease index of RNAi transgenic potatoes are higher than those of the wild type control group, and almost all of the RNAi transgenic potatoes are attacked. The result shows that the StoWRKY6 gene plays an important role on defensive reaction of plants for verticillium wilt. The StoWRKY6 gene can be used for expressing in the plants to enhance the disease resistance of plants for verticillium wilt, thereby having an important breeding value.

Description

technical field [0001] The invention belongs to the field of biology, and relates to wild eggplant StoWRKY6 gene and its expression vector and application. Background technique [0002] Verticillium wilt (Verticillium Wilt) is one of the important diseases of eggplant caused by Verticillium dahliae Kleb infection. It makes the fruit of the plant small, hard, and has brown vascular bundle lesions inside. Tube bundles turn brown or tan. Plants can get sick at any growth stage, and the disease usually starts to show symptoms after fruit setting, and spreads from bottom to top or from one side to the whole plant, which is a very harmful plant vascular disease. In the early stage of the disease, the leaf margins or between the veins become chlorotic and yellow, and gradually develop to the half leaf or the whole leaf becomes yellow, or yellow mottled, or palm-shaped macula. With the development of the disease, the diseased leaves on the diseased plants gradually turn from yello...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/84C12N1/21A01H5/00
Inventor 杨清决登伟陈敏
Owner NANJING AGRICULTURAL UNIVERSITY
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