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Recombinant vector and recombinant bacterium containing tomato LeEXP2 gene, and expression of LeEXP2 gene in recombinant bacterium

A technology of recombining vectors and genes, applied in the field of bioengineering, can solve the problems of cumbersome and difficult steps of Expansin protein, and the difficulty of plant expansin, and achieve the effects of solving energy crisis, improving efficiency, and avoiding pollution

Inactive Publication Date: 2013-02-13
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 and recombinant bacterium containing tomato LeEXP2 gene, and expression of LeEXP2 gene in recombinant bacterium
  • Recombinant vector and recombinant bacterium containing tomato LeEXP2 gene, and expression of LeEXP2 gene in recombinant bacterium
  • Recombinant vector and recombinant bacterium containing tomato LeEXP2 gene, and expression of LeEXP2 gene in recombinant bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Cloning of tomato LeEXP2 gene

[0043] Extract the total RNA of tomato with Trizol reagent according to the kit instructions: take 2ug of total RNA, reverse-transcribe it into cDNA (Promega company) with a reverse transcription kit, use this cDNA as a template, and use SEQ ID NO. 1 and the nucleotide sequence shown in SEQ ID NO.2 are upstream primers and downstream primers to carry out PCR to amplify the 744bp LeEXP2 gene shown in SEQ ID NO.3 in the sequence listing (see figure 1 ). The PCR product was recovered and connected to the TA vector 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 into Pichia pastoris expression vector

[0045] Using plasmid pGEM-LeEXP2 as a template, using the nucleotide sequence shown in SEQ ID NO.4 in the sequence table as the upstream primer, and using the nucleotide sequence shown in SEQ ID NO.5 in the sequence table as the downstream primer to carry out PCR amplification, Obtain the 703bp fragment shown in SEQ ID NO.6 in the sequence listing, connect the 703bp fragment with the TA cloning vector pGEM-T and transform the transformant obtained by Escherichia coli TOP10F', it is identified that the plasmid in the transformant is composed of LeEXP2 gene and pGEM The new plasmid obtained from the transformant was named pTLeEXP2, and the plasmid pTLeEXP2 was extracted from the above transformant, and the pPICZalpha A plasmid was extracted from the E. coli containing the yeast expression vector pPICZalpha A plasmid. Cut plasmids pTLeEXP2 and pPICZalpha A; agarose gel electrophoresis digestion ...

Embodiment 3

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

[0047] Take 10-15μg pPICZalpha A-LeEXP2 plasmid, cut into the linear 4219bp fragment shown in SEQ ID NO.9 with PmeI enzyme, and detect by electrophoresis as follows Figure 7 The 4219bp fragment was produced as shown. After extraction with phenol / chloroform, the linear DNA fragment shown in SEQ ID NO. 9 was precipitated with ethanol, and after drying, it was dissolved in sterile water to prepare a linear DNA solution with a DNA concentration of 0.5-1 μg / μL. ;

[0048] Competent cells of Pichia pastoris host strain X-33 were prepared by referring to the method for preparing yeast competent cells by electroporation transformation in the Experiment Guide for Molecular Biology (Fourth Edition) (P512-513). After mixing the competent cells of Pichia pastoris host strain X33 with 10 μL of 0.5-1 μg / μL linear DNA solution, they were transferred to a pre-cooled electrode cup at 4 °C, ice bathed for 5 min, vo...

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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, in particular to a recombinant vector containing tomato LeEXP2 gene, a recombinant Pichia pastoris containing tomato LeEXP2 gene and the induced expression of tomato LeEXP2 gene in recombinant Pichia pastoris . Background technique [0002] Cellulose is the main component of plant cell walls and is the largest renewable carbon source on earth. Using it for biotransformation to produce fuel ethanol and other chemicals is of great significance to 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 bond inside the cellulose molecule, and enzymatic hydrolysis consumes a lot of cellulase and time has become the bottleneck of biomass conversion. [1] . At present, the use of chemical reagents or high temperature and high pressure to ch...

Claims

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Application Information

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