Two types of plant proteins as well as coding genes and application thereof
A technology for encoding genes and proteins, applied to two kinds of plant proteins and their encoding genes and applications, can solve the problems of cloning and functional studies of sucrose synthase encoding genes that have not been reported
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Embodiment 1
[0050] Embodiment 1, gene cloning of Cizhu MYB transcription factor and sucrose synthase
[0051] 1. Take the tender stem of Neosinocalamus affinis, extract the total RNA with Trizol reagent, and obtain cDNA by reverse transcription.
[0052] 2. Using the cDNA obtained in step 1 as a template, use specific primers MYB-F1 and MYB-R1 for PCR amplification, recover and purify the obtained PCR products, and connect them into the vector pMD18-T (purchased from Bao Biotechnology (Dalian) Co., Ltd.), obtained the intermediate vector pMD18-T-MYB, which was confirmed by sequencing. The intermediate vector pMD18-T-MYB was inserted at the TA cloning site of the vector pMD18-T with a length of 1299bp shown in Sequence 2 of the Sequence Listing A DNA fragment, the DNA fragment is named gene NaMYB1; the gene encodes a protein NaMYB1 consisting of 432 amino acid residues shown in Sequence 1 of the Sequence Listing.
[0053] 3. Using the cDNA obtained in step 1 as a template, use specific pr...
Embodiment 2
[0059] Embodiment 2, construction of recombinant vector
[0060] 1. Construction of eukaryotic bivalent recombinant plasmids
[0061] 1. Fusion of NaMYB1 and NaSuSY genes by overlapping PCR method
[0062] 1) Using the intermediate vector pMD18-T-MYB obtained in step 2 of Example 1 as a template, and MYB1 and MYB2 as primers, PCR amplification was performed to obtain DNA fragment A with the following sequence: SmaI restriction enzyme digestion sequence, sequence listing sequence 2 The full-length sequence at positions 1-1296 (excluding the stop codon tag) and the sequence at positions 1-25 of sequence 4 in the sequence listing;
[0063] 2) Using the intermediate vector pMD18-T-SuSY obtained in Step 3 of Example 1 as a template, and SuSY1 and SuSY2 as primers, PCR amplification was performed to obtain a DNA fragment B with the following sequence: SalI digestion recognition sequence, sequence listing sequence The full-length sequence of positions 1-2451 of 4, the sequence of p...
Embodiment 3
[0076] Embodiment 3, the acquisition of recombinant Agrobacterium
[0077] Take the recombinant vector pCAMBIA2301-MYB-SuSY obtained in Example 2, transform Agrobacterium tumefaciens EHA105 (purchased from Shanghai Beinuo Biotechnology Co., Ltd.) by freeze-thaw method, and obtain Agrobacterium tumefaciens EHA105 containing the recombinant vector pCAMBIA2301-MYB-SuSY , named recombinant Agrobacterium EHA105 / pCAMBIA2301-MYB-SuSY.
[0078] The recombinant vector pCAMBIA2301-MYB obtained in Example 2 was used to transform Agrobacterium tumefaciens EHA105 by the freeze-thaw method to obtain Agrobacterium tumefaciens EHA105 containing the recombinant vector pCAMBIA2301-MYB, which was named recombinant Agrobacterium EHA105 / pCAMBIA2301-MYB.
[0079] The recombinant vector pCAMBIA2301-SuSY obtained in Example 2 was used to transform Agrobacterium tumefaciens EHA105 by the freeze-thaw method to obtain Agrobacterium tumefaciens EHA105 containing the recombinant vector pCAMBIA2301-SuSY, w...
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