Gene of Lygus lucorum polygalacturonase and application thereof

Inactive Publication Date: 2012-06-13
DALIAN UNIV OF TECH
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Problems solved by technology

Both nymphs and adults of mirid bugs can harm cotton plants, sucking flowers, fruits, and seed juices with p

Method used

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  • Gene of Lygus lucorum polygalacturonase and application thereof
  • Gene of Lygus lucorum polygalacturonase and application thereof
  • Gene of Lygus lucorum polygalacturonase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0226] Example 2 Expression of polygalacturonase from Lygus chlorophylla

[0227] 1. Escherichia coli expression system to obtain soluble expression products

[0228] 1. The three PG genes of PG1, PG2-1 and PG2-2 were respectively cloned into the NcoI and XhoI sites of the vector pET-28a(+) (Novagen Company), and 6 His tags (CACCACCACCACCACCAC) were added to the C-terminus of the protein , construct expression vectors pET-28a-PG1, pET-28a-PG2-1 and pET-28a-PG2-2 respectively.

[0229]

[0230] The detailed steps are:

[0231] 1. Design NcoI and XhoI restriction sites on the primers of PG1, PG2-1 and PG2-2. After the PCR reaction, use NcoI and XhoI to digest; use NcoI and XhoI to pET-28a(+) Enzyme digestion; using Solution I in the DNA Ligation Kit (Dalian Bao Biological Company, Code No. D6020A) kit, respectively ligate the digested PCR product to the digested pET-28a(+).

[0232] 2. Transform E.coli DH5α, screen positive recombinants, identify by enzyme digestion and de...

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Abstract

The invention discloses a gene sequence of Lygus lucorum polygalacturonase (PG) and a method for preparing the Lygus lucorum polygalacturonase. The method comprises the following steps of: extracting total RNA (ribonucleic acid) of Lygus lucorum; designing a primer according to the conserved sequences of the Lygus lucorum polygalacturonase; amplifying by utilizing an RT-PCR (reverse transcriptase-polymerase chain reaction) method to obtain homologous gene sequences of three PGs; obtaining 5' and 3' unknown sequences by utilizing an RACE experiment; and finally, respectively carrying out the recombinant expression, purification and property analysis of the Lygus lucorum polygalacturonase by utilizing an Escherichia coli expression system and a Pichia pastoris expression system. By using the method, the obtaining problem of the gene sequence of the polygalacturonase of the Lygus lucorum is solved firstly, and the recombinant expression problem of the polygalacturonase derived from insects is also solved for the first time. The gene disclosed by the invention has the application prospect in the aspects of food (fruit juice squeezing), oil material extraction, traditional Chinese medicinal material treatment, paper-making industry, oligo-pectin health care products and the like. In addition, an inhibitor aiming at the Lygus lucorum polygalacturonase can be used as a policy for preventing and controlling a piercing-sucking type pest, thereby achieving the prevention and control on the target pest.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to the gene of polygalacturonase of Lygus lucidum, the construction of an expression vector containing the gene, and the protein expression of the gene in host cells. Background technique [0002] Polygalacturonase (polygalacturonases, PG) belongs to a kind of pectinase (pectolytic enzyme or pectinase), which has similar properties to pectinase in application. [0003] Pectinase refers to the general term for various enzymes that can decompose pectin substances. It is roughly divided into pectin hydrolase, pectin lyase, pectin esterase and protopectinase, among which pectin hydrolase can be divided into It is polygalacturonase (polygalacturonases, PG), polygalacturonate methyl ester hydrolase (polymethylgalacturonases, PMG), arabinase, galactanase and the like. [0004] With the deepening of the research on pectin and pectinase, its application fields have b...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/26C12N15/63C12N15/70C12N15/81C12N1/21C12N1/19A23L2/84C12R1/19C12R1/84
Inventor 杨青姜秀萍贾秋磊杨君刘田
Owner DALIAN UNIV OF TECH
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