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Wild vine ubiquitin binding enzyme gene sequence

A technology of ubiquitin-conjugating enzyme and wild grape is applied in the field of full-length sequence of wild grape ubiquitin-conjugating enzyme gene, which can solve the problems of low yield and low quality of total RNA.

Inactive Publication Date: 2008-07-16
NORTHWEST A & F UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the high content of polyphenols, polysaccharides and other compounds in grape tissue, the total RNA extracted with the existing RNA extraction kits has low yield and low quality, and cannot be used to construct cDNA libraries.

Method used

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  • Wild vine ubiquitin binding enzyme gene sequence
  • Wild vine ubiquitin binding enzyme gene sequence

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Embodiment Construction

[0017] The operating steps of the wild grape ubiquitin-conjugating enzyme gene sequence obtained in the present invention are as follows: after inoculation, 1d, 2d, 3d, 4d, 5d, 6d, and 7d were sampled respectively, and the total RNA of leaves of wild grape "Baihe-35-1" was extracted ; Carry out the purification and detection of total RNA (see Figure 2); according to SMART TM The cDNA library Construction Kit method is used to construct and detect long-distance PCR-cDNA libraries. The main steps include: synthesis of the first strand of cDNA; synthesis of the second strand of cDNA (see Figure 3); digestion with proteinase K to remove excess DNA polymerase ; Sfi1 enzyme digestion to obtain double-stranded DNA molecules with specific enzyme cleavage sites; cDNA fragments digested by Sfi1 are connected to λTrip1Ex2 vector; packaging reaction; cDNA library identification; cDNA library amplification; amplified cDNA library titer, Identification of library capacity and recombination...

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Abstract

The invention discloses a wild grape ubiquitin conjugating enzyme gene sequence, which firstly obtains a key gene-ubiquitin conjugating enzyme gene of a protein ubiquitination path by adopting the technology of constructing a cDNA library for carry out the cloning of a wild grape powdery mildew resistance gene; the encoded amino acid sequence of the gene has an HOTLOOPS structural sequence in 1 to 30 amino acid residues, a conservative catalytic domain UBCc structural domain which is shared by all the ubiquitin conjugating proteins exists between the 32 and 174 amino acid residues; the total length of the protein amino acid sequence is 183 amino acids, molecular weight is 20.8KD, isoelectric point is 8.23, and the encoded protein of the gene pertains to an ubiquitin conjugating protease E2. The construction of the sequence provides a molecular basis for the further study of the wild grape ubiquitin conjugating enzyme gene expression mechanism.

Description

technical field [0001] The invention relates to the full-length sequence of wild grape ubiquitin-conjugating enzyme gene, belonging to the field of bioengineering. Background technique [0002] The improved SDS / phenol method was used to extract the total RNA of wild grape leaves induced by pathogenic bacteria; according to the SMART of Clontech company TM The cDNA library Construction Kit method was used to construct the library; according to Epicentre Maxplax TM Packaging Extract completed the packaging of the cDNA library; Biosystems 3700 DNA analyzer was used for sequencing, and the sequencing primer was: 5'-CTCGGGAAGCGCGCCATTGTGTTGGT-3'; the obtained sequences were compared with DNASTAR software to check for duplication; using NCBI's VecScreen ( The BLASTN2.2.5) program identifies and removes the carrier sequence in the sequencing results. The retrieved database is UniVec, and the website is as follows: http: / / www.ncbi.nlm.nih.gom / vecscreen / vecscre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52
Inventor 王跃进徐炎
Owner NORTHWEST A & F UNIV
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