Ananas comosus sucrose phosphate synthase (Ac-SPS1) gene, coding protein and gene cloning method thereof

A technology of phosphate synthase and gene coding, which is applied in the direction of biochemical equipment and methods, enzymes, DNA preparation, etc., to achieve the effect of improving quality

Inactive Publication Date: 2010-12-01
SOUTH SUBTROPICAL CROPS RES INST CHINESE ACAD OF TROPICAL AGRI SCI
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0005] However, there is no related report about pineapple fruit sucrose phosphate synthase (SPS) gene. Therefore, the cloning of pineapple sucrose phosphate synthase gene and the study of their biological functions will help to clarify the molecules of pineapple fruit sugar metabolism. mechanism, laying the foundation for research on fruit quality regulation

Method used

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example 1

[0025] Molecular Cloning of Pineapple Sucrose Phosphate Synthase Gene Ac-SPS1

[0026] 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. According to the EST information in the NCBI database, primers were designed for the coding region of the pineapple Ac-SPS1 gene: Forward primer: 5'-GGA(G / A)CTTGG(T / A / G / C)CG(T / G)GATTCTGATA-3' , reverse primer: 5'-CAGGA AC(A / T / C)TC(A / T)GA(C / T)TG(C / T)TTGTG-3'. Takara's Ex-Taq enzyme was used for PCR amplification, and the PCR product was recovered and ligated with pGEM-T easy Vector (Promega). The ligated product was transformed into E.coli DH 5α competent cells, and positive clones were screened by blue and white spots. The screened positive clones were further verified by PCR and then sequenced to obtain the Ac-SPS1 gene.

example 2

[0028] Sequence information and characteristic analysis of Ac-SPS1

[0029] The Ac-SPS1 gene of the present invention has a length of 1133bp, and its gene fragment encodes 376 amino acids and contains a typical glycosyltransferase domain. By comparison of homologous sequences, it was found that the amino acid sequence of the cloned pineapple Ac-SPS1 gene contained two conserved serine domains of the SPS protein family, one for the 14-3-3 protein-specific binding site, and the other for osmotic stress They can be phosphorylated or dephosphorylated by corresponding protein kinases or phosphatases to change the enzyme activity, and can also specifically bind to the 14-3-3 protein to change the enzyme activity. Sequence comparative analysis also showed that Ac-SPS1 had high homology with rice, sugarcane, sand pear, peach, potato, melon, tomato, tobacco, kaoshun bamboo and ryegrass.

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Abstract

The invention provides an ananas comosus sucrose phosphate synthase gene shown as SEQ ID NO:1 and coding protein thereof shown as SEQ ID NO:2, belonging to the technical field of biology. The invention also relates to a cloning method of the gene, and the method comprises the following steps of: extracting total RNA in pineapple fruits; detecting the total RNA in the ananas comosus fruits with agarose gel electrophoresis; carrying out reverse transcription to synthesize a first chain cDNA by using the total RNA as a template and Oligo dT as a primer; carrying out PCR (Polymerase Chain Reaction) amplification, and connecting a PCR product with a pGEM-T easy vector; converting; screening positive clones; and further verifying the screened positive clones by a PCR and then sequencing to obtain an Ac-SPS1 gene. The gene can provide a theoretical basis for researching the mechanism of sugar accumulation of the pineapple fruits from the molecular level.

Description

technical field [0001] The invention relates to a gene and a cloning method thereof, as well as a gene-encoded protein, belonging to the field of biotechnology, in particular to a pineapple sucrose phosphate synthase gene Ac-SPS1, its encoded protein and a gene cloning method. Background technique [0002] Pineapple (Ananas comosus) is a perennial evergreen herb of Bromeliaceae. Its fruit is rich in various sugars, organic acids, mineral elements and vitamins necessary for the human body. It has high nutritional value and is deeply loved by people. Today, pineapple has become an important fruit worldwide. According to FAO statistics, the world's pineapple output in 2006 was 18.26 million tons, making it the third largest tropical fruit in the world after bananas and mangoes. my country has introduced pineapples since the beginning of the 17th century, mainly distributed in Guangdong, Guangxi, Hainan, Yunnan, Fujian and other provinces. In recent years, my country's pineappl...

Claims

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Application Information

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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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