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Application of pyrus betulaefolia Pb4RMYB gene and encoded protein thereof in enhancement of plant salt tolerance

A gene-encoded, salt-tolerant technology, applied in applications, plant peptides, plant products, etc., can solve problems such as less research on fruit trees, and achieve the effect of improving salt tolerance

Inactive Publication Date: 2018-09-14
SHANDONG INST OF POMOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on MYB mainly focuses on a few model plants such as Arabidopsis thaliana, rice, soybean, etc., but less research on fruit trees

Method used

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  • Application of pyrus betulaefolia Pb4RMYB gene and encoded protein thereof in enhancement of plant salt tolerance
  • Application of pyrus betulaefolia Pb4RMYB gene and encoded protein thereof in enhancement of plant salt tolerance
  • Application of pyrus betulaefolia Pb4RMYB gene and encoded protein thereof in enhancement of plant salt tolerance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1: Cloning of Du pear Pb4RMYB gene

[0033]The pear seedlings with 6-8 true leaves were used as test materials, the total RNA was extracted and reverse transcribed, and the obtained first-strand cDNA was used to amplify the Pb4RMYB gene. Utilize the improved rapid CTAB method (CTAB extraction buffer includes: 2% CTAB, 2.5% PVP, 100mM Tris-HCl (pH 8.0), 25mM EDTA (pH 8.0), 2M NaCl, 0.05% spermidine, 2% β-mercaptoethanol ) to extract total RNA, take 1 μg RNA sample, incubate with 1 U DNase I (purchased from TaKaRa Company) at 37° C. for 30 min, then add 1 μL EDTA (25 mM) and incubate at 65° C. for 10 min.

[0034] The first strand of cDNA was synthesized using PrimeScript II 1st Strand cDNA Synthesis Kit (TaKaRa code: D6210). The amplification primers were: forward primer Pb4RMYB-F1: 5'-ATGTCTCCTCCCTCCGATGACG-3'; reverse primer Pb4RMYB-R1: 5'-TTAACCACATTGACGCCGCCTCTTCG-3'. 25μL PCR reaction system includes: 1×PCR buffer (purchased from TakaRa company), 2.5mM ...

Embodiment 2

[0038] Example 2: qRT-PCR analysis of Pb4RMYB response to salt stress in Duli pear

[0039] The extraction of pear total RNA, the method for cDNA synthesis are the same as in Example 1. Pear tubulin (AB239681) was used as a control, and the primer sequences were as follows: forward primer TUB-F: 5'-TGGGCTTTGCTCCTCTTAC-3', reverse primer TUB-R: 5'-CCTTCGTGCTCATCTTACC-3'. Use Primer Premier 5.0 to design a gene-specific qRT-PCR primer pair within the open reading frame of the Pb4RMYB gene. The primer sequence is as follows: forward primer Pb4RMYB-F2: 5'-TGTATCTCCCGGGTCGTTCT-3', reverse primer Pb4RMYB-R2: 5' -TTGATACCGTGCCAAGCACT-3'.

[0040] SYBR Green kit (purchased from TaKaRa Company) was used for qRT-PCR. 20 μL PCR amplification system contains: 10 μL 2×SYBR Premix Ex Taq, 0.2 μM upstream and downstream primers and 100 ng template cDNA. 96-well plate (Axygen) and high-transmittance parafilm (Axygen) for qRT-PCR were used, and the fluorescence quantitative PCR instrument i...

Embodiment 3

[0042] Example 3: Subcellular localization of the Pb4RMYB gene

[0043] The subcellular localization of Pb4RMYB gene was studied by tobacco transient expression system, and the expression vector used was pCAMBIA1302, which contained GFP gene. Utilize RT-PCR to amplify the whole ORF of the Pb4RMYB gene, the ORF amplification primers are Pb4RMYB-F1 and Pb4RMYB-R1 in Example 1, and respectively add Add Nco I and Spe I two restriction sites to obtain amplification primers with restriction sites: Forward primer Pb4RMYB-F3: 5'-C CCATGG ATGTCTCCTCCCTCCGATGACG-3', reverse primer Pb4RMYB-R3: 5'-GG ACTAGT TTAACCACATTGACGCCGCCTCTTCG-3'. The underline is the enzyme cutting site, CCATGG is the Nco I restriction site, ACTAGT It is the Spe I restriction site.

[0044] First, the amplified product was connected to the pMD19-T vector to obtain the recombinant vector PMD19-Pb4RMYB. At the same time, pCAMBIA1302 and PMD19-Pb4RMYB were digested with Nco I and Spe I, and the products we...

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Abstract

The invention relates to application of a pyrus betulaefolia Pb4RMYB gene and encoded protein thereof in enhancement of plant salt tolerance. The nucleotide sequence of the MYB transcription factor Pb4RMYB separated from pyrus betulaefolia is shown as SEQ ID No.1, and the encoded amino acid sequence is shown as SEQ ID NO.2. The Pb4RMYB gene is converted into arabidopsis thaliana according to an agrobacterium-mediated genetic transformation method. According to biological function verification, the obtained transgenic plant is overexpressed by the Pb4RMYB gene, and has remarkably higher tolerance to high-salinity adversity stress than wild type control plants. The pyrus betulaefolia Pb4RMYB gene has great significance in research on salt-tolerant molecules mechanism of plants, in addition,has an important function in cultivation of salt-tolerant fruit tree stocks or varieties, thereby providing high possibility for cultivation of new stress-resistant fruit tree varieties and being significant for growth of fruit trees.

Description

technical field [0001] The invention relates to the application of Pb4RMYB gene and its coded protein of pear pear in improving the salt tolerance of plants, and belongs to the field of biotechnology. Background technique [0002] With the intensification of industrial pollution, the development of irrigated agriculture, and the improper use of chemical fertilizers, the secondary salinization of soil has become increasingly serious. It has become a worldwide problem affecting agricultural production. Nearly 20% of the world's arable land and nearly half of the irrigated land are Threatened by varying degrees of salt damage. For fruit tree production, soil salinization has become one of the main factors limiting the production and distribution of fruit trees. The main obstacle to the growth and development of fruit trees in saline-alkali land is excessively high salinity concentration. Due to the weak leaching effect of saline-alkali land, a large number of salt ions will g...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H6/20
CPCC07K14/415C12N15/8273
Inventor 冉昆孙晓莉王少敏魏树伟董肖昌
Owner SHANDONG INST OF POMOLOGY
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