A kind of anti-abiotic stress transcription factor pbrbhlh92 and its primers, encoded proteins and applications

A transcription factor, non-biological technology, applied in the field of plant genetic engineering, to achieve the effect of enhancing active oxygen scavenging ability, improving drought resistance performance, and less cell damage

Active Publication Date: 2020-09-01
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there have been no reports on the bHLH gene in pears

Method used

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  • A kind of anti-abiotic stress transcription factor pbrbhlh92 and its primers, encoded proteins and applications
  • A kind of anti-abiotic stress transcription factor pbrbhlh92 and its primers, encoded proteins and applications
  • A kind of anti-abiotic stress transcription factor pbrbhlh92 and its primers, encoded proteins and applications

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Experimental program
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preparation example Construction

[0039] In the present invention, the preparation method of the anti-abiotic stress transcription factor PbrbHLH92 preferably includes the following steps:

[0040] Using the pear cDNA as a template, PCR amplification is performed with the primer pair of the transcription factor PbrbHLH92 described in the above technical scheme to obtain the anti-abiotic stress transcription factor PbrbHLH92.

[0041] In the present invention, the method for preparing pear cDNA preferably comprises: extracting RNA from pear leaves, and reverse-transcribing the obtained RNA to obtain cDNA. The method for extracting RNA is not particularly limited in the present invention, and the method for extracting plant RNA conventionally used by those skilled in the art can be used.

[0042] In the present invention, the anti-abiotic stress transcription factor PbrbHLH92 is preferably derived from Pyrusbretschneideri, and the primer pair is designed according to the open reading frame of the gene PbrbHLH92....

Embodiment 1

[0066] Cloning and expression analysis of PbrbHLH92 gene

[0067] 1. RNA extraction

[0068] The research material, Du pear, was planted in the Pear Engineering Center of Nanjing Agricultural University, and its seedling age was 60 days. Select vigorously growing Du pear seedlings, randomly weigh 0.1g samples, and quickly freeze them with liquid nitrogen. RNA was extracted using the Total RNA Extraction Kit from Solebo, the specific method is as follows:

[0069] (1) Sample processing: Take 0.1g of fresh or -70°C frozen tissue and grind it in liquid nitrogen, add the powder to 1ml of lysate and mix well to obtain a homogenized sample;

[0070] (2) Place the treated sample at room temperature for 5 minutes, so that the nucleic acid protein complex is completely separated;

[0071] (3) Add 0.2 ml of chloroform to the homogenized sample after standing at room temperature, cover the tube cap, shake vigorously for 15 seconds, and place at room temperature for 3 to 5 minutes to o...

Embodiment 2

[0090] Example 2 qRT-PCR analysis of transcription factor PbrbHLH92 gene under different stress conditions

[0091] In order to analyze the effect of PbrbHLH92 gene on dehydration ( Figure 2A ), abscisic acid ( Figure 2B ) and salt ( Figure 2C ), the expression pattern of the PbrbHLH92 gene was analyzed using Real-time PCR technology.

[0092] The kit method was used to extract RNA (see Example 1 for the RNA extraction method). The synthesis of the first strand of cDNA was carried out according to the operation manual of TOYOBO Reverse Transcription Kit of Beijing Quanshijin Biotechnology Co., Ltd.

[0093] Quantitative PCR (qRT-PCR) detection of PbrbHLH92 gene expression was determined:

[0094] For quantitative PCR detection, the 20 μL reaction system contains: 10 μL 2×Mix, 0.1 μL cDNA, 5 μL primers (using ubiqutin as the internal reference primer, the length is 208 bp), 4.9 μL water.

[0095] The procedure of quantitative PCR is as follows:

[0096] Table 1 Quantit...

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Abstract

The invention provides an anti-abiotic-stress transcription factor PbrbHLH92 and belongs to the technical field of plant genetic engineering. The nucleotide sequence of the anti-abiotic-stress transcription factor PbrbHLH92 is as shown in SEQ ID No. 1. The transcription factor PbrbHLH92 capable of increasing plant abiotic stress resistance is screened from Pyrus betulaefolia, and protein encoded by the transcription factor PbrbHLH92 belongs to nucleoprotein. Experiments show that the anti-abiotic-stress transcription factor PbrbHLH92 plays an important role in plant drought and salt stress resistance, can increase plant sensitivity to abscisic acid and plays an important role in various abiotic stress resisting. The anti-abiotic-stress transcription factor PbrbHLH92 is transferred into tobacco or Pyrus betulaefolia, the active oxygen eliminating ability of the obtained transgenic tobacco or Pyrus betulaefolia can be effectively enhanced as compared with that of wild tobacco or Pyrus betulaefolia, and the drought resistance of the transgenic tobacco is increased effectively.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and in particular relates to an anti-abiotic stress transcription factor PbrbHLH92 and its primer, encoded protein and application. Background technique [0002] There are more than 30 species of Pyrus plants in the world, of which 13 species originate in my country. Wild pear species are widely distributed in my country, and pear trees are cultivated in all provinces of my country. The so-called Oriental pear is the general term for the cultivation of Chinese pears, and it is the most widely cultivated tree species in my country. The cultivated area and output of pear trees in my country rank first in the world. The layout and planning of pear dominant production areas have greatly promoted the development of my country's pear industry, but it is precisely because of the wide distribution that breeding of new varieties has become the most crucial factor in the development of the pear in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/11C07K14/415C12N15/82A01H5/00A01H6/82A01H6/74
Inventor 黄小三高浚芝刘月邢才华赵粱怡张绍铃王春孟金玉妍陶书田吴巨友吴俊谢智华
Owner NANJING AGRICULTURAL UNIVERSITY
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