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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

Inactive Publication Date: 2008-08-27
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Found in the prior art document retrieval: magazine " Plant Cell (plant cell) ", published article " The TRANSPARENT TESTA GLABRA1 Locus, Which Regulates Trichome Differentiation and Anthocyanin Biosynthesis in Arabidopsis, Encodes a WD40 Repeat Protein (Arabidopsis TTG1 locus encodes a WD40 repeat protein, which regulates epidermal hair differentiation and anthocyanin synthesis)” although the document explained the important role of TTG1 gene in Arabidopsis thaliana, but TTG1 gene in cucumber fruit Whether it exists in this multicellular epidermis plant, and the research on the cucumber TTG1 gene has never been reported, because the TTG1 gene not only controls the formation of epidermal hairs, but also controls the synthesis of anthocyanins, so this gene can use genetic engineering technology Improving the traits of cucumber fruit thorns and improving the flower color of plants

Method used

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  • Protein coded sequence of cucumber TTG1-like gene
  • Protein coded sequence of cucumber TTG1-like gene
  • Protein coded sequence of cucumber TTG1-like gene

Examples

Experimental program
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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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Abstract

The invention relates to a protein coding sequence of a cucumber TTG1-like gene in the technical field of genetic engineering, which comprises: encoding a nucleotide sequence of polypeptide which has the activity of cucumber TTG1-like protein, wherein the nucleotide sequence has the homology 70% of a 160-1158th nucleotide sequence from nucleotide in SED ID NO.3, or the nucleotide sequence can be hybridized with the 160-1158th nucleotide sequence from the nucleotide in the SED ID NO.3 under the condition that the temperature is 45-55DEG C. A gene which is homologous with TTG1 which determines the initiation of epidermal hair by arabidopsis thaliana is cloned in cucumbers, which is not only beneficial for understanding a molecular mechanism which is built by plant epidermal hair form, but also helpful for recognizing the molecular mechanism which is formed by cucumber fruit thorn which is an important quality gene, theoretical foundation is provided for breeding the cucumber quality, and the variety breeding process is accelerated. The protein coding sequence of the invention also plays an important role of improving plant flower color and has great application value.

Description

technical field [0001] The invention relates to a protein coding sequence in the technical field of genetic engineering, in particular to a protein coding sequence of a cucumber TTG1-like gene. Background technique [0002] Trichomes are surface appendages of many land plants, which develop from epidermal cells. It is divided into unicellular or multicellular, and some have glands in the epidermis, which can secrete some lipids or secondary metabolites, and some have no glands. The shape of plant epidermis varies greatly, such as star, pinnate, shield and head, etc. Different types of epidermis can be produced on the same plant. Plant epidermal trichomes have a series of functions: non-glandular epidermal trichomes can dissipate heat, increase tolerance to chilling damage, facilitate seed dispersal, absorb water, protect from ultraviolet rays and damage plant tissues from some herbivorous insects; glandular trichomes Cuticle hairs help plants resist herbivorous insects and...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82C12N5/10C12N1/19C07K14/415C12Q1/68
Inventor 关媛蔡润何欢乐陶倩怡赵俊龙
Owner SHANGHAI JIAO TONG UNIV
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