Polygalacturonase arrestin gene CaPGIP1 and disease resistance technology thereof

A polygalactose and inhibitory protein technology, which is applied in the field of molecular biology to achieve the effects of improving disease resistance and inhibiting the infection of pathogens

Inactive Publication Date: 2011-11-23
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the prior art has not disclosed the research information on the inhibitory effect of the capsicum polygalacturonase inhibitory protein PGIP gene on the activity of polygalacturonase PG secreted by plant pathogenic bacteria and the improvement of disease resistance of transgenic plants.

Method used

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  • Polygalacturonase arrestin gene CaPGIP1 and disease resistance technology thereof
  • Polygalacturonase arrestin gene CaPGIP1 and disease resistance technology thereof
  • Polygalacturonase arrestin gene CaPGIP1 and disease resistance technology thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1: Gene Cloning

[0066] Primers were designed according to the reported PGIP gene sequence, PGIP1-1 (its sequence is shown in Seq ID No: 3) and PGIP1-2 (its sequence is shown in Seq ID No: 4), with pepper genomic DNA as template, PCR Amplify the CaPGIP1 gene fragment, according to the principle of Tail-PCR, design specific primers J5-1 (its sequence is shown in Seq ID No: 5), J5-2 (its sequence is shown in Seq ID No: 6) according to the target gene , J5-3 (its sequence is shown in Seq ID No: 7) and universal primer AD4 (its sequence is shown in Seq ID No: 8) to obtain the 5' flanking sequence of the gene, and primer J3-1 (its sequence is shown in Seq ID No: 8) is designed ID No: 9), J 3-2 (its sequence is shown in Seq ID No: 10), J 3-3 (its sequence is shown in Seq ID No: 11) and universal primer AD2 (its sequence is shown in Seq ID No: 12) to obtain the 3' flanking sequence of the gene, and then design full-length primers PGIP1SP (its sequence is shown in Seq...

Embodiment 2

[0101] Example 2: Analysis of CaPGIP1 Gene Expression Differences

[0102] 1. Sample preparation

[0103] Take different tissues and organs (roots, stems, leaves, flowers, fruits) of peppers and store them at -80°C for later use; treat peppers at the 4-6 leaf stage with stress factors (salicylic acid, methyl jasmonate, abscisic acid, trauma, freezing) Leaves were sampled after 0, 1, 2, 6, 12, 18, 24, 36, and 48 hours, and stored at -80°C for later use.

[0104] 2. Capsicum RNA extraction

[0105] Total RNA was extracted according to the RNA extraction kit (purchased from Tiangen Biochemical Technology Co., Ltd.)

[0106] (1) Homogenization treatment:

[0107] Grind 50-100 mg of capsicum plant leaves into powder with liquid nitrogen, add 450 μl RL (check whether β-mercaptoethanol has been added before use), and vortex vigorously to mix.

[0108] Note 1: Water bath at 56°C for 1-3 minutes is helpful for plant tissue lysis.

[0109] Note 2: Due to the rich plant diversity, a...

Embodiment 3

[0151] Example 3: Gene prokaryotic expression and protein purification

[0152] 1. Prokaryotic expression of CaPGIP1 gene

[0153] The expression vector pET28a(+) plasmid was extracted for use, and the CaPGIP1 gene amplified by PCR was purified and recovered by agarose gel electrophoresis before use.

[0154] Carry out double digestion of the gene and vector plasmid, the enzyme digestion system is: CaPGIP1 gene / pET28a plasmid 10-20g, 10×Buffer 10μL, BamHI 4μL, EcoRI 4μL, add ddH2O to 40ul

[0155] After mixing, they were digested in a water bath at 37°C for more than 4 hours, and then purified and recovered by agarose gel electrophoresis, ready for use.

[0156] Ligation transformation of gene and vector: 1-3 μL of pET-28a(+) after digestion, 5-7 μL of CaPGIP1 gene after digestion, 1ul of Solution I, 10ul in total.

[0157] After mixing, they were reacted in a water bath at 16°C for 4 hours and then connected.

[0158] Connection steps:

[0159] Take 10 μL of the ligation ...

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Abstract

The invention belongs to the field of plant biotechnology, in particular provides a polygalacturonase arrestin gene CaPGIP1 cloned from hot pepper, the gene can specially inhibit the activity of polygalacturonase (PGs) secreted by pathogenic bacteria. The invention provides the function verification techniques based on the coding of the gene, such as protein making, enzyme activity testing, gene silencing, transgenic excessive expression and the like and the application of the techniques. Based on the gene and protein operation technology, the CaPGIP1 gene is proved to effectively participate in the defense reaction of hot pepper under the induction of a stress facto, and can inhibit the activity of polygalacturonase (PGs) secreted by various pathogenic bacteria. The gene silence and transgenic technology are further used for proving that the silence or less expression of the gene in the hot pepper reduces the disease resistance of a host, and the excessive expression of the gene in the transgenic tobacco can enhance the resistance; therefore, the gene codes an important disease-resistance related protein. The invention provides an important technological reserve for transforming the polygalacturonase arrestin (PGIP) gene and cultivating a new disease-resistant variety.

Description

technical field [0001] The invention belongs to the field of molecular biology. Specifically, the invention relates to the gene cloning, function verification and application technology of polygalacturonase inhibitory protein gene CaPGIP1. Background technique [0002] During the interaction between plant pathogens and hosts, they can secrete a variety of cell wall degrading enzymes, mainly including polygalacturonase (PG), pectate lyase (Pel), pectin methylesterase (Pme) and β-galactosidase (LacZ), in which PG is an important cell wall degrading enzyme, these enzymes mainly destroy the cell wall by degrading pectin, an important component of the cell wall, and promote the infection of pathogenic bacteria, causing the host to produce disease symptoms or Accelerate the development of the disease process, and at the same time provide sugar source nutrition for the growth and reproduction of pathogenic bacteria. Many studies have proved the important role of PG in the infection...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415A01H5/00
Inventor 张修国
Owner SHANDONG AGRICULTURAL UNIVERSITY
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