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
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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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