Gene of lygus lucidum polygalacturonase (PG) and application of gene

A technology of polygalactose and chlorophyll, which is applied in the fields of separation and purification and enzyme activity analysis, and can solve problems such as economic loss and harm to cotton plants.

Inactive Publication Date: 2013-01-16
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 piercing-sucking mouthparts, forming "crazy heads" and "leafcrats" on cotton, causing serious economic losses

Method used

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  • Gene of lygus lucidum polygalacturonase (PG) and application of gene
  • Gene of lygus lucidum polygalacturonase (PG) and application of gene
  • Gene of lygus lucidum polygalacturonase (PG) and application of gene

Examples

Experimental program
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Effect test

Embodiment 2

[0234] Example 2 Pichia pastoris expression system to obtain soluble expression of polygalacturonase from Lygus green bug (represented by PG1)

[0235] 1. Construction of expression vector pPIC9-PG1

[0236] 1. Addition of enzyme cleavage sites and histidine tags

[0237] On the primers of PG1, the enzyme cutting sites of EcoR Ⅰ and Not Ⅰ and the nucleotide sequences of 6 His tags were designed. Using PCR reaction, EcoR Ⅰ and Not Ⅰ restriction sites were added to both ends of the protein, and 6 His tags (CACCACCACCACCACCAC) were added to the C-terminus of the protein.

[0238] The primers used in the experiment were synthesized in Dalian Bao Biological Co., Ltd., and the sequences are as follows:

[0239]

[0240] Reaction system 1: PCR amplification using PIC9-PG1-F and PIC9-PG1-R primers

[0241]

[0242]

[0243] Reaction conditions: 94°C for 5 min; 94°C for 30 sec, 55°C for 30 sec, 72°C for 1 min, 25 cycles; 72°C for 7 min.

[0244] Reaction product: Take 5ul ...

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Abstract

The invention discloses a gene sequence of lygus lucidum polygalacturonase (PG) and a method for preparing the PG. According to the method, firstly, the total RNA (ribose nucleic acid) of lygus lucidum is extracted, a primer is designed according to the enzyme conserved sequence, an RT-PCR (reverse transcription-polymerase chain reaction) method is utilized for amplification to obtain three PG homologous gene sequences, then, a RACE (rapid-amplification of cDNA ends) method is utilized for obtaining 5' and 3' unknown sequences; and finally, the PG1 is used as the representative, a pichia pastoris expression system is used for carrying out the recombination expression, separation and purification and enzyme activity analysis of the enzyme. Firstly, the gene sequence obtaining problem of the lygus lucidum PG is solved, and the recombination expression problem of the PG of the inspect source is also solved for the first time. The obtained lygus lucidum PG has Pectin decomposition activity. The enzyme has application prospects in the application of the plant pectin aspect in the aspects including food (juice) squeezing, oil material extraction, traditional Chinese medicine treatment, papermaking industry, oligomerization pectin health-care products and the like.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to the polygalacturonase gene of Lygus lucidum, the construction of an expression vector containing the gene, the protein expression of the gene in host cells, separation and purification and enzyme activity analysis. 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, etc. [0004] With the deepening of the research on pectin a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N9/26C12N15/63C12N1/15C12N1/19C12N1/21
Inventor 杨青姜秀萍贾秋磊杨君刘田
Owner DALIAN UNIV OF TECH
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