Pyrus betulaefolia cold-resistant factor PbrMYB5 and application thereof

A transcription factor and gene technology, which is applied to the cold-resistant transcription factor PbrMYB5 of Du pear and its application field, can solve the problems of lack of cold-resistant genes, and achieve the effects of increasing VC content, improving cold resistance, and realizing sustainable agricultural development.

Active Publication Date: 2018-09-04
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the transcription factors that have been studied more in fruit trees include ICE1, bHLH, ABF, WRKY, NAC, ABF, etc., but there is still a lack of cold resistance genes or transcription factors suitable for pears.

Method used

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  • Pyrus betulaefolia cold-resistant factor PbrMYB5 and application thereof
  • Pyrus betulaefolia cold-resistant factor PbrMYB5 and application thereof
  • Pyrus betulaefolia cold-resistant factor PbrMYB5 and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The technical process of the present invention is as figure 1 shown

[0033] The PbrMYB5 gene was cloned, the subcellular localization of the gene was analyzed, the expression pattern analysis under different stress conditions, and the overexpression vector was constructed for tobacco transformation and the silencing vector was transformed for Qiuzi pear, and the positive plants were identified at the molecular level. Tobacco and pear seedlings were subjected to low temperature stress to observe the phenotype and identify the resistance of transformed plants from the physiological level.

Embodiment 2

[0035] Cloning of full-length cDNA of PbrMYB5 gene in Du pear

[0036] Using the MYB cis-element in the PbrDHAR2 promoter of the pear dehydroascorbate reductase gene PbrDHAR2 as bait, screen the yeast one-hybrid library, screen the nucleotide sequence of the gene PbrMYB5, submit the sequence to the pear genome database for BLAST, and select the score The highest nucleotide sequence and submit to Pfam for validation of conserved protein domains. A pair of specific primers for amplifying the sequence was designed using Primer Premier 5.0. The primer pair for amplifying the full-length gene of PbrMYB5 is:

[0037] Forward primer 1: 5'-ATGAGGAACCCATCGCCTTCGTCGA-3' (SEQ ID NO.3), reverse primer 1: 5'-CTCCGTTGGATCATTAACCCCTTTGGCG-3' (SEQ ID NO.4); use RT-PCR method to amplify its full length. The detailed steps are as follows: 1 μg of Du pear RNA was treated with 1 U of DNase I at 37°C for 30 minutes and immediately placed on ice, and 1 μL of 50mM EDTA was added and treated at 65...

Embodiment 3

[0040] qRT-PCR analysis of PbrMYB5 gene under different stress conditions

[0041] In order to analyze the response pattern of PbrMYB5 gene to low temperature, dehydration and high salt in Du pear, the expression pattern of PbrMYB5 gene was analyzed using Real-time PCR technology. The RNA was extracted by the CTAB method, and the synthesis of the first strand of DNA was carried out according to the operation manual of the TOYOBO reverse transcription kit. In the 20μL reaction system, there are: 10ul 2×Mix, 0.1ul cDNA, 5μL primer (using ubiqutin as the internal reference primer, the length is 208), 4.9ul water. The program of quantitative PCR is shown in Table 1:

[0042] Table 1 Quantitative PCR program

[0043]

[0044]

[0045] figure 2 It is a schematic diagram of expression of PbMYB5 gene under low temperature, dehydration and salt stress. in: figure 2 A is that the gene of the present invention is processed at 4 degrees in Du pear seedlings (not transgenic), ...

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Abstract

The invention relates to a pyrus betulaefolia cold-resistant factor PbrMYB5 and the application thereof and belongs to the technical field of plant genetic engineering. The amino acid sequence of thecold-resistant factor PbrMYB5 provided by the invention is of SEQ ID NO.1 as shown in the specification. By adopting the cold-resistant factor provided by the invention, the cold resistance of a plantcan be remarkably improved, novel germplasm resources can be provided for plant molecular breeding, novel genetic resources can be provided for green agriculture and modern agriculture, the agricultural production risks can be reduced, and sustainable development of agriculture can be achieved.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to the cold-resistant transcription factor PbrMYB5 of pear pear and its application. Background technique [0002] Pear is one of the three major fruits in my country, and its industry has very important economic value in the national economy (Zhang Shaoling et al., 2010). Over the past decades, my country's pear industry has developed rapidly, but it often encounters abiotic adversity stress, especially low temperature and freezing damage. Low temperature not only limits the cultivation area of ​​pears, but also reduces the yield and quality of pears due to periodic low temperature chilling damage and freezing damage, causing huge economic losses to the pear industry and seriously restricting its development. For example: In the winter of 2002-2003, Korla fragrant pear trees suffered severe frost damage. The city's pear tree damage area is 3866.60hm 2 , the dir...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00
CPCC07K14/415C12N15/8273
Inventor 黄小三邢才华刘月顾冰洁林泽崑董慧珍赵梁怡高俊芝李凌张绍铃胡轼
Owner NANJING AGRICULTURAL UNIVERSITY
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