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
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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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