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A Sorbet cold resistance transcription factor pubhlh and its application

A technology of transcription factors and transgenic plants, which is applied in the field of PubHLH, a cold-resistant transcription factor of Sorbet and its application, bHLH family transcription factor PubHLH, can solve the problems of spending a lot of creative labor, reduce agricultural production costs, improve cold resistance, and realize Environmentally friendly effect

Active Publication Date: 2016-05-04
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It can be seen that although the identification of bHLH genes is relatively easy, it takes a lot of creative labor to predict the function of bHLH genes in different species

Method used

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  • A Sorbet cold resistance transcription factor pubhlh and its application
  • A Sorbet cold resistance transcription factor pubhlh and its application
  • A Sorbet cold resistance transcription factor pubhlh and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0028]Embodiment 1 PubHLH gene cloning and expression analysis

[0029] RNA was extracted from sorbus leaves under low-temperature treatment at 4°C and reverse-transcribed, and the resulting first-strand cDNA was used to amplify the full-length PubHLH gene. Amplified gene PubHLH primer pair is: Forward primer: PubHLHForward, 5'-TT GCTGCC ATGCTGCCGAGGCTGAACGGTGGTG-3' (SEQ ID NO: 3); reverse primer: PubHLHReverse, 5'-GG GGTACC CTACACCATGCCATGGAACCCG-3' (SEQ ID NO: 4). The 50 μl reaction system included 100 ng cDNA, 1× buffer (TransStartFastPfuBuffer), 10 mMdNTP, 1UTaq polymerase (TransStartFastPfuDNA Polymerase) (the aforementioned buffer and Taq polymerase were purchased from TRANS), and 1.0 μM of the above primers. The PCR reaction was completed on the Roche480 (Applied Biosystem) amplification instrument according to the following procedures: 95°C, 1 minute, 95°C denaturation for 20 seconds, 58°C annealing for 20 seconds, 72°C extension for 60 seconds, 40 cycles; after the...

Embodiment 2

[0031] Example 2 PubHLH Gene Subcellular Localization, Transcription Activation Analysis

[0032] Since the PubHLH gene has a nuclear localization signal (NLS), the present invention utilizes onion epidermis to study the subcellular localization of the PubHLH gene. The entire ORF reading frame of the PubHLH gene was amplified by RT-PCR, and two restriction sites, NcoI and SpeI, were added to the two ends of the amplification primers. Its amplification primer is (forward primer PubHLH-F1: 5'- CCATGG ATGCTGCCGAGGCTGAACGGTGGTG-3' (SEQ ID NO: 11); reverse primer PubHLH-R1: 5'-GG ACTAGT CACCATGCCATGGAACCCGATCAA-3' (SEQ ID NO: 12), firstly digest the amplified product. At the same time, pCAMBIA1302 was digested with NcoI and SpeI, and the product was recovered and ligated to obtain the pCAMBIA1302-PubHLH-GFP recombinant vector, which was transformed into Agrobacterium EHA105. Agrobacterium infection of onion epidermis is carried out as follows: (1) draw a plate, pick a single c...

Embodiment 3

[0033] Embodiment 3 Plant Transformation Overexpression Vector Construction

[0034] According to the analysis of the multiple cloning site of pMV vector (patent application number 201210258254.4) and the restriction site on the coding region sequence of PubHLH gene, Xhol and KpnI were selected as endonucleases. First, the clone of the PubHLH gene will be used as a template for PCR amplification (amplification primer pair forward primer: PubHLHForward, 5'-TT GCTGCC ATGCTGCCGAGGCTGAACGGTGGTG-3' (SEQ ID NO: 3); reverse primer: PubHLHReverse, 5'-GG GGTACC CTACACCATGCCATGGAACCCG-3' (SEQ ID NO: 4), followed by digestion of the PCR product together with the pMV vector at 37°C, purification and recovery after digestion for 3-4 hours. In the ligation reaction system, the ratio of PubHLH gene to vector pMV is 3:1, the total reaction volume is 10 μl, 1 μl of 10×T4 ligation buffer, 1 μl of T4 ligase, and ligated at 16°C for 13-16 hours. The ligation product was transformed into Escher...

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Abstract

The invention discloses a sorb cold-resistant transcription factor PubHLH and an application thereof. The nucleotide sequence of sorb bHLH transcription factor gene PubHLH is represented by SEQ ID NO: 1 in a sequence table; 409-2043bp position of the sequence represented by SEQ ID NO: 1 is a coding area of the gene; an amino acid sequence coded by the coding area is represented by SEQ ID NO: 1. A new bHLH gene PubHLH is cloned in the sorb; an agrobacterium-mediated genetic transformation method is used for transforming the gene to tobacco and pear; cold resistance ability of the obtained transgenic plant is obviously improved; biological functional verification shows that the cloned PubHLH gene has cold resistance regulation function. The sorb bHLH transcription factor gene PubHLH provided by the invention can be applied to improvement of plant cold resistance or construction of cold-resistant transgenic plant.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, relates to a sorrel cold-resistant transcription factor PubHLH and its application, in particular to a bHLH (basicchelix-loop-helix, basic helix-loop-helix) family transcription factor cloned from sorrel (Poncirustrifoliata) PubHLH, and then the gene was introduced into tobacco and pear respectively, and the cold resistance of the obtained transgenic plants was obviously improved. Background technique [0002] Plants are often subjected to a series of environmental stresses, including drought, high temperature, low temperature, salinity, etc. Among them, low temperature is one of the most important factors that affect the growth and yield of crops and limit the geographical distribution of plants. During the long-term evolution process, plants have formed a complex set of mechanisms to cope with and adapt to these abiotic adversities. In the past two decades, scientists at home and abroa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/11C12N15/82C12N1/21C07K14/415A01H5/00
Inventor 张绍铃黄小三靳丛齐开杰
Owner NANJING AGRICULTURAL UNIVERSITY
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