Coding sequence of taxus chinensis 2-C-Methyl-D-erythritol4-phosphatecytidyltransferase protein and application thereof
A kind of phosphoryl cystyl transferase protein, phosphoryl cystyl transferase technology, applied in the field of preparation of the protein and nucleic acid sequences
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Embodiment 1
[0050] Cloning of TcMCT Gene from Taxus chinensis
[0051] 1. tissue separation
[0052] The southern yew bulbs were sourced from Southwest University, Beibei, Chongqing. After the inflorescence and young leaves of the southern yew plant were collected, they were immediately frozen in liquid nitrogen for preservation.
[0053] . RNA isolation
[0054] See the instructions for using RNA Plant Buffer, the method is as follows:
[0055] 1). Take 0.1 g of southern yew leaves and grind them into fine powder with liquid nitrogen in a mortar (burned with alcohol for 10 minutes before the experiment and cooled for later use);
[0056] 2). Quickly transfer to a 1.5 mL centrifuge tube that has been added with 0.5 mL RNA Plant Buffer in advance, shake and mix well; place horizontally at room temperature for 5 min;
[0057] 3). After centrifuging at 12000 rpm for 5-10 min at 4 °C, transfer the supernatant into a new 1.5 mL centrifuge tube;
[0058] 4). Add 0.1 mL of 5 mol·L-1...
Embodiment 2
[0068] Sequence bioinformatics analysis and functional verification of the TcMCT gene of Taxus chinensis
[0069] The length of the full-length cDNA of the southern yew TcMCT of the present invention is 1280 bp, and the detailed sequence is shown in SEQ ID NO. 3, wherein the open reading frame is located at nucleotides 59-1001. The amino acid sequence of Taxus chinensis TcMCT was deduced according to the full-length cDNA, with a total of 312 amino acid residues, a molecular weight of 34.33kDa, and a pI of 8.96. See SEQ ID NO. 4 for the detailed sequence.
[0070] TcMCT The subcellular location prediction of the gene shows that the gene is located in the plastid, which is consistent with the fact that the gene on the plant MEP pathway is located in the plastid. Further prediction using ChloroP shows that its N-terminus contains a length of 83 amino acid residues The plastid transit peptide, the predicted cleavage site is Arg88. The synthesis of paclitaxel precursor is als...
Embodiment 3
[0074] Introducing the Sequence of TcMCT Gene from Taxus southernis to Plants (In the example, the sweet potato variety Yushu 99)
[0075] The intermediate vector containing the 2-C-methyl-D-erythritol-4-phosphate cystyl transferase gene was further cloned into the plant binary expression vector (such as pCAMBIA1380), under the premise of ensuring the correct reading frame The identified expression vector is then transferred into Agrobacterium (such as EHA105, LBA4404, A4, R1000, ATCC15834, etc.) to transform sweet potato embryogenic cells. The transformation method is based on the literature of Zhang Peng et al. in Plant Science (2011), etc. in method.
[0076] Use PCR and Southern blotting to detect the transgenic regenerated plants, transplant the positive plants in the field in May, and use fluorescent quantitative PCR to detect the transgenic sweet potato 2-C-methyl-D-erythritol-4-phosphocystyl in August-October The expression level of transferase gene and according to...
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