Apple V-ATPase subunit gene MdVHA-B1S396A and stress-resistance application thereof

A mdvha-b1s396a, apple technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve the problem of long cycle of resistant varieties

Active Publication Date: 2015-04-22
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, apple is a perennial woody plant, and it takes a long time to select resistant varieties through conventional breeding.

Method used

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  • Apple V-ATPase subunit gene MdVHA-B1S396A and stress-resistance application thereof
  • Apple V-ATPase subunit gene MdVHA-B1S396A and stress-resistance application thereof
  • Apple V-ATPase subunit gene MdVHA-B1S396A and stress-resistance application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1: Cloning of apple V-ATPase subunit gene MdVHA-B1.

[0070] 1) Gala tissue culture leaf RNA extraction and reverse transcription

[0071] 1. Extraction of plant total RNA

[0072] Use the RNAplant plus Reagent kit to extract RNA on a small scale (the material is generally a tissue containing more starch and sugar, such as apple tissue, and the sample mass is <0.1g):

[0073] Take less than 0.1g of frozen ground apple tissue material, add 0.5ml kit extraction reagent (4°C), shake until thoroughly mixed;

[0074] 1) Leave at room temperature for 5 minutes. Note: Lay centrifuge tubes flat to maximize surface area;

[0075] 2) Centrifuge at 12,000 rpm for 1 minute at 4°C, and transfer the supernatant to a new RNase-free centrifuge tube;

[0076] 3) Add 0.1ml 5M NaCl and mix gently;

[0077] 4) Add 0.3ml chloroform, mix up and down;

[0078] 5) Centrifuge at 12,000 rpm for 10 minutes at 4°C, take the upper aqueous phase and transfer it to a new RNase-free cent...

Embodiment 2

[0108] Example 2: Apple MdVHA-B1 Gene Chromosomal Location and Genome Structure

[0109] Using the nucleotide sequence of the V-ATPase subunit gene AtVHA-B1 in Arabidopsis thaliana published in the Arabidopsis genome database, the homologous sequence (serial number MDP0000945182) was found in the apple genome database blast. Chromosomal location of the MdVHA-B1 gene uses the genome database of the Apple website (http: / / genomics.research.iasma.it / ); and genome structure prediction uses (http: / / gsds.cbi.pku.edu.cn / index. php) database (attached figure 1 ): The apple V-ATPase subunit gene MdVHA-B1 is located on chromosome 10 near the centromere; it consists of 14 exons and 13 introns.

example 3

[0110] Example 3: Response of MdVHA-B1 gene to salt stress

[0111] (1) Treat the tissue-cultured seedlings of Apple Gala with better growth condition with 100mM NaCl for 0h, 1h, 3h, 6h, 9h, 12h and 24h respectively, and then take samples respectively, fix them rapidly in liquid nitrogen, extract RNA respectively and reverse The cDNA of each sample was obtained by transcription.

[0112] (2) Design specific primers MdVHAB1(RT)-F, MdVHAB1(RT)-R and apple internal reference primers Md18S-F, Md18S-R in the 5' non-coding region of MdVHA-B1 gene.

[0113] Md18S-F: 5'-AAACGGCTACCACATCCA-3', its sequence is shown in SEQ.ID.NO.9;

[0114] Md18S-R: 5'-CACCAGACTTGCCCTCCA-3', its sequence is shown in SEQ.ID.NO.10;

[0115] MdVHAB1(RT)-F: 5'-TCTCTTTCTTCCGCTTGGCTC-3', its sequence is shown in SEQ.ID.NO.11;

[0116] MdVHAB1(RT)-R: 5'-ACAACCGGGTGACAAGCTAAC-3', its sequence is shown in SEQ.ID.NO.12;

[0117] (3) Use the cDNA obtained in step 1) of Example 3 as a template, and adjust these...

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Abstract

The invention relates to apple V-ATPase subunit gene MdVHA-B1S396A and stress-resistance application thereof. An MdVHA-B1 gene is separated from an apple; then, a serine residue at the 396 position is mutated into alanine through carrying out a PCR (Polymerase Chain Reaction) bridging method for two times so as to obtain the MdVHA-B1S396A gene; the expression level of transgenic apple healing can be further increased through over-expression of the gene in apple tree healing; and simultaneously, the adversity ability of transgenic apple healing, such as salinity resistance and the like, can be further increased.

Description

1. Technical field [0001] The present invention relates to an apple V-ATPase subunit gene MdVHA-B1S396A and its anti-stress application, in particular to the construction of a carrier of the apple V-ATPase subunit gene MdVHA-B1S396A and its ability to resist salt stress in transgenic apple callus Analysis and application, belonging to the field of molecular biology and biotechnology. 2. Background technology [0002] Plants are often attacked by various adverse environments and pests throughout their lives. During the long-term adaptation and evolution process, plants have formed a series of defense response mechanisms to resist damage from adverse environments. Among them, environmental stress mainly includes various biotic stress and abiotic stress. Biological stress mainly comes from insects, herbivores, and plant pathogens; there are many types of abiotic stress, including drought, salinity, extreme temperature, chemical pollution, and oxygen damage. In order to adapt ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82A01H5/00
Inventor 郝玉金胡大刚孙翠慧
Owner SHANDONG AGRICULTURAL UNIVERSITY
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