Pineapple neutral invertase gene Ac-NI1, coded protein and gene cloning method thereof

An invertase and protein technology, applied in the field of gene technology, can solve the problems of undiscovered research reports on pineapples, etc.

Inactive Publication Date: 2010-12-01
SOUTH SUBTROPICAL CROPS RES INST CHINESE ACAD OF TROPICAL AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there have been many relevant reports on invertase genes in peaches, grapes, Japanese pears, carrots, sugarcane and other crops, but no relevant research reports have been found on pineapple

Method used

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Embodiment Construction

[0020] The present invention will be described in further detail below in conjunction with specific examples, and these examples are only used to illustrate the present invention, and do not limit the scope of the present invention.

[0021] Molecular Cloning of Neutral Invertase Gene Ac-NI1 in Pineapple Fruit

[0022] 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, the primers for the coding region of the pineapple Ac-NI1 gene were designed: forward primer: 5'-CAG(T / C)T(T / G / A)CAGAG(C / T)TGGGAGA-3', reverse Direction primer: 5'-GTGTC(A / G)TA(A / C)TA(T / C)TCAGGCCA-3'. Takara's Ex-Taq enzyme was used for PCR amplification, and the PCR product was recovered and ligated with the pGEM-Teasy (Promega) vector. The ligated product was transformed into E.coli DH 5α competen...

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Abstract

The invention relates to a pineapple neutral invertase gene Ac-NI1 with a nucleotide sequence shown as SEQ ID NO:1, pineapple neutral invertase gene Ac-NI1 coded protein with a nucleotide sequence shown as SEQ ID NO:2 and a pineapple neutral invertase gene cloning method. The method comprises the following steps of: extracting total RNA in a pineapple fruit by adopting an improved SDS (Sodium Dodecyl Sulfate) method and detecting and RNA content and quality by adopting agarose gel electrophoresis; carrying out reverse transcription by adopting the obtained total RNA as a template to obtain cDNA; by adopting a first chain reverse transcription product 10 mul as a template of PCR, carrying out PCR (Polymerase Chain Reaction) amplification, converting and screening a positive clone; after carrying out further PCR verification, sequencing to obtain an Ac-NI1 gene; and after carrying out further PCR verification on the screened positive clone, sequencing to obtain an Ac-NI1 gene. The great expression of the pineapple fruit neutral invertase gene is beneficial to accumulating fructose and glucose and changing the composition and proportion of sugar in the fruit, thereby changing the quality of the fruit.

Description

technical field [0001] The invention relates to a gene technology, which belongs to the field of biotechnology, in particular to a pineapple neutral invertase gene Ac-NI1, its encoded protein and its gene cloning method. Background technique [0002] The molecular weight of invertase is between 50kD and 80kD, and it is a monomer or dimer. It can be divided into acid invertase (AI) and neutral or alkaline invertase (NI). It catalyzes the following reaction: Suc+H 2 O←→Fru+Glu. NI is mostly considered as a cytoplasmic enzyme, its optimum pH value is around 7.0, and can also be divided into soluble and insoluble, the former is distributed in the cytoplasm, and the latter is present on the cell wall (Lowell and Tomlinson, 1989 ; Klann et al., 1996; Xu et al., 1995). There is highly active Ivr in the vacuole of L. esculentum tomato fruit, so a large amount of fructose and glucose are accumulated in the vacuole, and the content of sucrose is very little. Vacuole Ivr activity is...

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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