Cloning of a kind of Sophora japonica samet6 gene and its application
A kind of technology of bitter beans and amino acids, applied in the fields of application, genetic engineering, plant genetic improvement, etc.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0027] Example 1: Acquisition and sequence analysis of the full-length cDNA of SaMET6 gene
[0028] 1. Stress treatment of bitter beans:
[0029] Select 10 g of Sophora japonica seeds with full grains, add 5 ml of concentrated sulfuric acid with a concentration of 98%, and soak for 20 min. The seeds were washed and sown in soil (peat:vermiculite=1:1), and the cultivation conditions were: 16h light, temperature 26°C, humidity 65%, light intensity 30000 lux. After 4 weeks of germination, the seedlings were transferred to hoagland nutrient solution containing 200 mM NaCl for 0h, 1h, 2h, 4h, 8h, 12h, 24h, and 48h, respectively. The roots of Sophora japonica under each treatment were taken and stored at -80°C for library construction.
[0030] 2. Construct cDNA library:
[0031] Total RNA was extracted from the Sophora japonica samples (young roots) after NaCl treatment for each period, and then the single-stranded cDNA containing the complete cap structure was enriched using th...
Embodiment 2
[0047] Example 2: Sequence analysis of the SaMET6 gene
[0048] After sequencing analysis, the full-length cDNA sequence of SaMET6 gene is 1041bp, including an open reading frame of 507bp. According to DNAMAN analysis, the protein is composed of 170 amino acids. According to Hphob. / Kyte&Doolittle of Expasy protscale, the possible surface of globulin was analyzed. Region is N-terminal. PSORT Prediction predicts that proteins are most likely to localize to the cytoplasm and mitochondrial matrix, a major feature of the cysteine protease family. Analysis showed that the full-length cDNA sequence of SaMET6 gene was obtained.
[0049] Using NCBI (http: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi) to analyze the conserved region of the coding gene, the protein encoded by this gene belongs to 5-methyltetrahydropterinylglycerol Triester homocysteine methyltransferase family proteins. BLASTX was performed on the amino acid sequence encoded by the SaMET6 gene by NCBI, homology a...
Embodiment 3
[0050] Example 3: Transformation and detection of recombinant yeast
[0051] Saccharomyces cerevisiae strain INVSc1 is ureaine auxotroph (Ura - ) type strain in yeast minimal medium lacking uridine (SC-Ura - ), the yeast strain could hardly grow and reproduce. The yeast expression vector (pYES2-DEST52) contains URA3 gene, and the expression of this gene can make yeast transformants in SC-Ura - grow normally on the medium. Therefore, SC-Ura - The selection medium allows for the selection of positive and non-positive yeast transformants. The vector plasmids pYES-SaMET6 and pYES-DEST52 were respectively transformed into yeast competent strain INVSc1 by the lithium acetate method, and the transformed yeast was spread on SC-Ura - On the solid selection medium, the untransformed yeast was used as a control and cultivated. After 2 days, the yeast transformed with the two plasmids could grow and colonies grew, except that the empty yeast did not grow at all, indicating that the y...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More