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Recombinant vector containing tomato leexp2 gene, recombinant bacteria and expression of leexp2 gene in recombinant bacteria

A recombinant vector and gene technology, applied in genetic engineering, plant genetic improvement, recombinant DNA technology, etc., can solve the problems of complicated steps, difficult steps, and difficult plant expansion factors for Expansin protein, so as to solve the energy crisis, improve efficiency, avoid The effect of pollution

Inactive Publication Date: 2011-12-21
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the purification of a single Expansin protein from the total protein of plants is not only cumbersome and difficult, but also the amount of purification is relatively limited, and it is difficult to express plant expansin in large quantities in bacteria. [11]
This limits the application of Expansin
[0004] The Expansins family of tomato includes LeEXP1, LeEXP2, LeEXP3 and other proteins. LeEXP2 is reported to be expressed in swollen and mature tissues, and participates in the physiological roles of cell elongation and other aspects [12-13] , but it has not been expressed in other hosts in vitro in tomato, nor has it been used to improve the efficiency of cellulase in degrading cellulosic materials

Method used

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  • Recombinant vector containing tomato leexp2 gene, recombinant bacteria and expression of leexp2 gene in recombinant bacteria
  • Recombinant vector containing tomato leexp2 gene, recombinant bacteria and expression of leexp2 gene in recombinant bacteria
  • Recombinant vector containing tomato leexp2 gene, recombinant bacteria and expression of leexp2 gene in recombinant bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Cloning of tomato LeEXP2 gene

[0043] Use the Trizol reagent to extract the total RNA of tomato according to the kit instructions: get 2ug of total RNA, use the reverse transcription kit to reverse-transcribe it into cDNA (Promega Company), use the cDNA as a template, and use the SEQ ID NO. 1 and the nucleotide sequence shown in SEQ ID NO.2 carry out PCR for upstream primer and downstream primer, amplify the LeEXP2 gene of 744bp shown in SEQ ID NO.3 in the sequence listing (see figure 1 ). The PCR product was recovered and connected to the TA carrier pGEM-T (Promega Company) to obtain a new plasmid, which was confirmed by sequencing to contain the tomato LeEXP2 gene, and the plasmid was named pGEM-LeEXP2, that is, the plasmid pGEM-LeEXP2 containing the tomato LeEXP2 gene was obtained. build strategy see figure 2 .

Embodiment 2

[0044] Example 2 Construction of LeEXP2 gene to Pichia pastoris expression vector

[0045] Using the plasmid pGEM-LeEXP2 as a template, using the nucleotide sequence shown in SEQ ID NO.4 in the sequence listing as an upstream primer, and using the nucleotide sequence shown in SEQ ID NO.5 in the sequence listing as a downstream primer for PCR amplification, Obtain the 703bp fragment shown in SEQ ID NO.6 in the sequence listing, and connect the 703bp fragment with the TA cloning vector pGEM-T to transform the transformant obtained from Escherichia coli TOP10F'. It has been identified that the plasmid in the transformant is composed of LeEXP2 gene and pGEM -T connection, the new plasmid obtained from the transformant was named pTLeEXP2, and the plasmid pTLeEXP2 was extracted from the above-mentioned transformant, and the pPICZalpha A plasmid was extracted from Escherichia coli containing the yeast expression vector pPICZalpha A plasmid, and simultaneously enzyme Plasmids pTLeEXP2...

Embodiment 3

[0046] Example 3 Integration of LeEXP2 gene into Pichia pastoris chromosome:

[0047] Take 10-15 μg of pPICZalpha A-LeEXP2 plasmid, use PmeI enzyme to cut into the 4219bp fragment shown in the linear SEQ ID NO.9, electrophoresis detection is as follows Figure 7 A 4219bp fragment is generated as shown. After extraction with phenol / chloroform, ethanol precipitates the linear DNA fragment shown in the linear SEQ ID NO.9. After drying, it is dissolved in sterile water to make a linear DNA solution with a DNA concentration of 0.5-1μg / μL ;

[0048] Prepare the competent cells of Pichia pastoris host strain X-33 by referring to the electroporation transformation preparation yeast competent cell method in the refined Molecular Biology Experiment Guide (Fourth Edition) (P512-513), and 80 μL Bath The competent cells of Pichia host strain X33 were mixed with 10 μL of 0.5-1 μg / μL linear DNA solution, then transferred to a pre-cooled electrode cup at 4 °C, ice-bathed for 5 min, and the v...

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Abstract

The invention discloses a recombinant vector and recombinant bacterium containing tomato LeEXP2 gene, and expression of the LeEXP2 gene in the recombinant bacterium. In the invention, the LeEXP2 gene is firstly cloned from the tomato; the LeEXP2 gene is further constructed to a Pichia pastoris expression vector; a recombinant Pichia pastoris bacterium containing tomato LeEXP2 gene is obtained by electrotransformation; and the tomato LeEXP2 gene is induced to express in the recombinant Pichia pastoris bacterium. The recombinant bacterium disclosed by the invention can effectively express the target protein LeEXP2. The invention overcomes the defect that the limited amount of expansin purified from the plants in the prior art can not be massively applied to cellulose degradation. Many wastelignocelluloses exist in the natural world; and the LeEXP2 protein obtained in the invention can enhance the cellulose degradation efficiency of cellulase, and has an important meaning for cleanly and effectively utilizing waste celluloses and solving the current energy crisis.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a recombinant vector containing tomato LeEXP2 gene, recombinant Pichia pastoris containing tomato LeEXP2 gene and induced expression of tomato LeEXP2 gene in recombinant Pichia pastoris . Background technique [0002] Cellulose, the main component of plant cell walls, is the largest renewable carbon source on Earth. The use of biotransformation to produce fuel ethanol and other chemicals is of great significance for the current human beings to solve problems such as energy crisis, food shortage, and environmental pollution. The special crystal structure of cellulose makes it difficult for cellulase molecules to approach the glycosidic bonds inside the cellulose molecule, and enzymatic hydrolysis consumes a large amount of cellulase and time, which has become the bottleneck of biomass conversion [1] . At present, the use of chemical reagents or high temperature and hig...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/66C12N1/19C12N15/29C07K14/415C12R1/84
Inventor 马媛媛邹少兰张鲲洪解放井欣张敏华
Owner TIANJIN UNIV
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