Method for improving beauveria bassiana chitinase gene disease resistance and culturing disease resistance plants adopting method

A technology of Beauveria bassiana and chitinase, which is applied in the field of molecular biology and plant genetic engineering, achieves huge economic and social benefits, remarkable effects, and the effect of improving resistance

Inactive Publication Date: 2012-07-04
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In view of the above-mentioned deficiencies in the prior art, the object of the invention is to solve the problem that the Beauveria bassiana chitinase gene improves plant disease resistance, and provides a method for improving the disease resistance of the Beauveria bassiana chitinase gene

Method used

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  • Method for improving beauveria bassiana chitinase gene disease resistance and culturing disease resistance plants adopting method
  • Method for improving beauveria bassiana chitinase gene disease resistance and culturing disease resistance plants adopting method
  • Method for improving beauveria bassiana chitinase gene disease resistance and culturing disease resistance plants adopting method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Extraction of DNA

[0056] 1.1 DNA extraction buffer

[0057] (1) Fungal DNA extraction buffer

[0058] Tris-HCl (pH 7.5) 0.2 mol / L, NaCl 0.5 mol / L, EDTA 0.01 mol / L, SDS 1% (w / v).

[0059] (2) Plant DNA extraction buffer

[0060] CTAB extract: 100 mmol / L Tris-HCl (pH8.0), 20 mmol / L EDTA (pH8.0), 1.5 mol / L NaCl, 2% CTAB (w / v), 4% PVP40 (w / v ) and 2% mercaptoethanol (v / v), PVP and mercaptoethanol were added just before use.

[0061] (3) Alkaline lysis plasmid extraction buffer

[0062] STE: 0.1 mol / L NaCl, 10 mmol / L Tris-HCl (pH 8.0), 1 mmol / L EDTA (pH 8.0).

[0063] Solution I: 50 mmol / L glucose, 25 mmol / L Tris-HCl (pH 8.0), 10 mmol / L EDTA (pH 8.0).

[0064] Solution II: 0.2 mol / L NaOH, 1% (w / v) SDS.

[0065] Solution III: 50 mL 5 mol / L potassium acetate, 11.5 mL glacial acetic acid, 28.5 mL water.

[0066] 1.2 Extraction method of Beauveria bassiana genome DNA

[0067] Collect the Beauveria bassiana bacteria liquid in a 1.5 ml centrifuge ...

Embodiment 2

[0074] Example 2: Extraction of RNA

[0075] 2.1 RNA extraction buffer

[0076] CTAB extraction buffer: 2% CTAB (w / v), 2% polyvinylpyrrolidone PVP40 (w / v), 100mmol / L Tris-HCl (pH8.0, prepared in DEPC-treated water), 25mmol / L EDTA, 0.5 g / L Spermidine, 2.0mol / L NaCl, 2% mercaptoethanol (v / v, add before use).

[0077] SSTE solution: 1mol / L NaCl, 0.5% SDS (w / v), 10mmol / L Tris-HCl (pH8.0), 1.0mmol / L EDTA.

[0078] 2.2 Plant RNA extraction method

[0079] Total RNA was extracted from cotton or tomato tissues by CTAB method. Take about 3g of fresh cotton or tomato tissue, quickly grind it into powder in liquid nitrogen, put it into a 50ml centrifuge tube treated with DEPC water, then add 15ml of RNA extraction solution preheated at 65°C, mix it upside down, and then bathe in water at 65°C for 3 minutes. Centrifuge at 8,000 rpm and 4°C for 10 min, transfer the supernatant to a new 50ml centrifuge tube treated with DEPC water, and extract twice with an equal volume of chlorof...

Embodiment 3

[0082] Example 3 Obtaining the chitin-binding domain (SwChBD) of the silkworm chitinase gene

[0083] RNA was extracted from the molting stage of the 4th to 5th instar silkworm (Bombyx mori), and cDNA was synthesized by reverse transcription (product of TaKaRa Company) according to the instructions of the reverse transcription kit. According to the cDNA sequence of the silkworm chitinase gene (Genebank accession number: AF273695), primer sequence 3 and sequence 4 were designed to amplify the chitin-binding domain of the silkworm chitinase gene and introduce Speech I restriction site, C-terminal introduction EcoR I restriction site.

[0084] Components of the amplification reaction: 10×DNA polymerase Buffer (product of Dingguo Company) 2.5 μL, 10 mmol / L dNTP 0.5 μL, 5 μmol / L upstream and downstream primers 1 μL each, DNA polymerase 0.7U, cDNA 1 μL, add water to 25 μL . Cycle conditions: 95°C, 5min; 94°C, 1min; 55°C, 1min; 72°C, 1min, 32 cycles; 72°C, 20min, agarose gel e...

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Abstract

The invention provides a method for improving the beauveria bassiana chitinase gene disease resistance and culturing disease resistance plants adopting the method. The method is characterized in that a chitin binding structure domain of silkworm chitinase genes is fused with beauveria bassiana chitinase genes, and the disease resistance of the beauveria bassiana chitinase genes is improved. A 35S promoter is used for controlling fusion genes SwBbchit1, in addition, plant expression vectors are built and are respectively transferred into cotton and tomatoes, and the disease resistance of the transgenic cotton and tomatoes can be further improved. Compared with the disease resistance of the Bbchit1 transgenic cotton and tomatoes, the disease resistance of the SwBbchit1 cotton and tomatoes obtained by the method provided by the invention is obviously improved.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and plant genetic engineering. It mainly involves the use of molecular biology technology to modify genes to obtain new genes with better performance, and the use of plant genetic engineering technology to use the obtained new genes to prepare disease-resistant transgenic plants to further improve plant disease resistance. [0002] technical background [0003] Since humans began to cultivate crops, crop diseases caused by pathogenic bacteria have been one of the main factors limiting agricultural production. Diseases can cause more than 15% crop yield reduction in the world every year, and direct economic losses amount to 30-50 billion US dollars (Li Runzhi et al., 2001; Osusky et al., 2000). In addition, the disease also causes a decline in the quality of crop products, and the toxins produced by pathogenic bacteria seriously endanger human health. In the integrated crop disease ma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N15/10C12N15/70C12N15/84A01H5/00C12R1/645C12R1/19
Inventor 李先碧裴炎范艳华罗志兵李德谋侯磊罗明肖月华罗小英宋水清金丹张瑞芝梁爱敏
Owner SOUTHWEST UNIVERSITY
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