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Function and application of xylose isomerase gene of non-glutinous rice

A technology of xylose isomerase and japonica rice, which can be applied in application, genetic engineering, plant genetic improvement, etc., can solve the problem of low activity

Inactive Publication Date: 2012-08-22
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far only a few xylose isomerase genes, most of which are thermophilic bacteria, have been actively expressed in the traditional ethanol production strain Saccharomyces cerevisiae. The rate-limiting step in the glucose metabolic pathway

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Obtaining japonica rice cDNA

[0024] (1) Plant total RNA extraction-Trizol method

[0025] 1. After grinding the tissue into powder in liquid nitrogen, take 50-100 mg of tissue and add 1 ml Trizol solution for grinding. Note that the total volume of the sample should not exceed 10% of the volume of Trizol used.

[0026] 2. Keep the grinding solution at room temperature for 5 minutes, then add 0.2ml of chloroform, cover the centrifuge tube tightly, and shake the centrifuge tube vigorously by hand for 15 seconds.

[0027] 3. Take the upper aqueous phase into a new centrifuge tube, add 0.5ml of isopropanol, place at room temperature for 10 minutes, and centrifuge at 12000g for 10 minutes.

[0028] 4. Discard the supernatant, add 1ml of 75% ethanol, vortex and mix well, and centrifuge at 7500g for 5min at 4°C.

[0029] 5. Carefully discard the supernatant, then dry at room temperature or in vacuum for 5-10 minutes, be careful not to dry too much, otherwise the ...

Embodiment 2

[0054] Example 2 The sequence of the xylose isomerase gene

[0055] A fragment of about 1.5kb was cloned from the japonica rice cDNA obtained by reverse transcription with conservative primers. Sequencing analysis found that it contained a 1440bp ORF sequence, which was confirmed to be the xylose isomerase gene of japonica rice (GenBank accession number 4344231).

[0056] DNA sequencing was completed by Shanghai Sangon Biotechnology Co., Ltd. The nucleotide and amino acid analysis software was mainly DNAMAN and the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / , National Center for Biotechnology Information, NCBI) The Blast program.

Embodiment 3

[0057] Example 3 Construction of Xylose Isomerase Gene Expression Vector Plasmid

[0058] Primers were designed using the 5' and 3' end sequences of the gene encoding japonica xylose isomerase, including the Mfe I and Spe I sites. The PCR product was digested with Mfe I and Spe I. The final product was cloned into a vector generated by pYES2. In this vector, the GAU promoter on pYES2 was replaced with the TPI1 promoter to ensure constitutive expression of xylose isomerase, thereby eliminating the need for galactose in the medium. The TPI1 promoter was cloned from the Saccharomyces cerevisiae genome. This promoter was enzymatically cleaved into the Nhe I-EcoRI fragment. The TPI1 promoter and the PCR product of the gene encoding xylose isomerase were all connected to pYES2 cut with Spe I and Xba I to finally obtain the recombinant plasmid pYES-RXI containing the xylose isomerase gene.

[0059] Preparation of Escherichia coli Competent Cells and Transformation of Plasmids

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Abstract

The invention discovers that a xylose isomerase gene of non-glutinous rice has the function of converting xylose into xylulose. According to the invention, a recombinant plasmid containing the gene is constructed and is introduced into saccharomyces cerevisiae to obtain new engineered saccharomyces cerevisiae. Experiments prove that the saccharomyces cerevisiae which does not have the function ofconverting xylose into xylulose obtains the conversion capacity after the xylose isomerase gene is expressed in a host cell.

Description

Technical field: [0001] The invention relates to the function of converting xylose into xylulose by the japonica rice xylose isomerase gene, and also relates to a recombinant plasmid containing the gene and an engineering strain, and using the engineering bacteria to prepare ethanol and xylulose by fermenting a culture medium containing xylose Application of other fermentation products. Background technique: [0002] The industrial production of ethanol from lignocellulose is an economical and environmentally friendly way. Because lignocellulosic raw materials from plants are renewable resources that can be obtained in large quantities. But many yeasts that can ferment ethanol (such as Saccharomyces cerevisiae) cannot use xylose as a carbon source. Therefore, there is a need to provide yeast capable of ethanol fermentation using xylose as a carbon source. [0003] If xylose in plant lignocellulose can be isomerized to generate xylulose, xylose can be used as a carbon sour...

Claims

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Application Information

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IPC IPC(8): C12N15/61C12N15/81C12N1/19C12P7/06C12P19/02C12R1/865
CPCY02E50/10
Inventor 顿宝庆王智李佳英路明
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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