Protein coded sequence of cucumber TTG1-like gene
A ttg1-like, coding sequence technology, applied in the field of protein coding sequence of cucumber TTG1-like gene
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Embodiment 1
[0050] Embodiment 1, the cloning of cucumber TTG1-like gene
[0051] 1. Organization separation
[0052] Common cucumber variety Shenlv 64 (commercially available product), which belongs to the European greenhouse type of cucumber, is all female, has fruit thorns, and has no fruit tumors. Sow the cucumber seeds in the plug trays of the greenhouse, and when the plants grow to two leaves and one heart, prepare to extract DNA or RNA.
[0053] 2. Isolation of RNA
[0054] Take part of the tissue, grind it with liquid nitrogen, put it into a 1.5ml centrifuge tube, add Trizol, shake fully, and use the RNA extraction kit (Shanghai Sangong UNIQ-10 Column Total RNA Extraction Kit, product number: Cat.No .SK1361) to extract total RNA, the extraction steps and reagents are detailed in the kit instructions. The quality of total RNA was detected by formaldehyde denaturing gel electrophoresis, and then the RNA content was determined on a spectrophotometer.
[0055] 3. Full-length clonin...
Embodiment 2
[0065] Example 2, Sequence Information and Homology Analysis of Cucumber TTG1-like Gene
[0066] The cucumber TTG1-like gene obtained in the present invention is a new gene, the full length of the cDNA is 1414bp, the sequence is shown in SEQ ID NO.3, and the open reading frame is located at 160-1158 nucleotides, a total of 999 nucleotides. According to the full-length cDNA sequence, the amino acid coding sequence of cucumber TTG1-like gene was deduced, with a total of 333 amino acids, a molecular weight of 37192.27 Daltons, and an isoelectric point of 4.95. See SEQ ID NO.4 for the detailed sequence.
[0067] The protein sequence encoded by the cucumber TTG1-like gene was compared with protein homology at NCBI using the BLAST program, and it was found that it had 78% homology with the Arabidopsis gene AtTTG1 gene at the protein level (attached table 2), and Cotton GhTTG1 gene is also 78% homologous (Supplementary Table 3), both of these genes have been proven to have the funct...
Embodiment 3
[0068] Example 3. Eukaryotic cell expression of cucumber TTG1-like gene protein in Arabidopsis ttg1 mutant and functional verification of transgenic plants
[0069] One, the construction of the plant expression vector containing cucumber TTG1-like gene
[0070] According to the full-length sequence of the cucumber TTG1-like gene (SEQ ID NO.3), design and amplify the complete coding frame primers, and combine with the multiple cloning site of the expression vector, add HindIII and BamH I to the upstream and downstream primers respectively Two restriction endonuclease sites (upstream primer eCsTTG1-F sequence is 5'-CTTC AAGCTT GGAATC GAACACGCAGC-3'; downstream primer eCsTTG1-R sequence is 5'-CTC GGATCC CAAACTT AAAGCTGCATT-3') to facilitate the construction of expression vectors. Using the cDNA obtained in Example 1 as a template, after PCR amplification, separate by 1% agarose gel electrophoresis, use a DNA recovery kit (Shanghai Sangong UNIQ-10 Column DNA Gel Recovery K...
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