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Sucrose synthetase gene Ac-Sy1 in pineapple fruits, encoded protein and gene cloning method thereof

A technology of sucrose synthase and ac-sy1, which is applied in the field of gene research for fruit quality improvement, and can solve problems such as blanks

Inactive Publication Date: 2010-12-01
SOUTH SUBTROPICAL CROPS RES INST CHINESE ACAD OF TROPICAL AGRI SCI
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Problems solved by technology

[0005] At present, there are many studies on the gene cloning and expression of sucrose synthase in citrus, tomato, coffee, corn and cotton, but it is still blank in the field of pineapple research

Method used

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Embodiment Construction

[0018] The present invention will be described in further detail below through examples, and these examples are only used to illustrate the present invention, and do not limit the scope of the present invention.

[0019] Molecular Cloning of Pineapple Sucrose Synthase Gene Ac-Sy1

[0020] The Bali pineapple fruit was selected as the experimental material, and the total RNA in the pulp was extracted. The obtained total RNA was used as a template, and cDNA was synthesized by reverse transcription. The primers for the coding region of the pineapple Ac-Sy1 gene were designed according to the EST information in the NCBI database: forward primer: 5'-GACCGGTCTAAGCCCATCATCT-3', reverse primer: 5'-ACAAGTCATAGCCTCCACCACAGT-3'. Takara's Ex-Taq enzyme was used for PCR amplification, and the PCR product was recovered and ligated with pGEM-T easyVector (Promega). The ligated product was transformed into E.coli DH 5α competent cells, and positive clones were screened by blue and white spots...

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Abstract

The invention relates to a gene sequence of a sucrose synthetase gene Ac-Sy1 in pineapple fruits and a protein sequence encoded by the same, belonging to the technical of biology. The invention further relates to a cloning method of the gene, which comprises the following steps of: extracting the total RNA in the pineapple fruits by using a modified SDS method; detecting the total RNA in the pineapple fruits by using agarose gel electrophoresis; carrying out reverse transcription to synthesize a first chain cDNA by using the total RNA as a template and using Oligo dT as a primer for; using the cDNA as a template and carrying out PCR amplification by using the Ex-Taq of Takara; connecting a PCR product with a pGEM-T easy Vector, converting, screening the positive for cloning, further validating by PCR and sequencing to obtain the Ac-Sy1 gene. The gene can provide the theoretical evidence for scientifically adjusting and controlling the quality of the pineapple fruits.

Description

technical field [0001] The present invention relates to a gene and its cloning method, and at the same time relates to the coded protein of the gene, in particular to a pineapple sucrose synthase gene Ac-Sy1, the coded protein and a gene cloning method, which belong to the field of biotechnology, and in particular to the gene research for fruit quality improvement field. Background technique [0002] Sucrose synthase (SS) is a soluble enzyme that exists in the cytoplasm. It is a tetramer composed of subunits with a molecular weight of 83-100kD. The optimum pH value for the sucrose synthesis direction is 8.0-9.5, and the optimum pH value for the sucrose cracking direction The value is 5.5-6.5, which catalyzes the following reversible reaction: Suc+UDP←→Fru+UDPG, whether it plays a role in synthesis or decomposition in sucrose metabolism is related to whether the enzyme protein is phosphorylated (Amor et al., 1995; Tanase and Yamaki, 2000 ; Tanase et al., 2002). Since sucros...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N9/00C12N15/10
Inventor 杜丽清张秀梅孙光明谢江辉姚艳丽
Owner SOUTH SUBTROPICAL CROPS RES INST CHINESE ACAD OF TROPICAL AGRI SCI
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