Hovenia trifoliate transcription factor ptrahl and its application in genetic improvement of plant cold resistance

A transcription factor and plant technology, applied in the field of plant genetic engineering, to reduce agricultural production costs and achieve environmental friendliness

Active Publication Date: 2022-05-27
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few reports on the cold resistance of AHL transcription factors, and the study of the mechanism of AHL protein's low temperature resistance is of great value for crop cold resistance breeding

Method used

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  • Hovenia trifoliate transcription factor ptrahl and its application in genetic improvement of plant cold resistance
  • Hovenia trifoliate transcription factor ptrahl and its application in genetic improvement of plant cold resistance
  • Hovenia trifoliate transcription factor ptrahl and its application in genetic improvement of plant cold resistance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Cloning of full-length cDNAs of Citrus aurantium PtrAHL14 and PtrAHL17 genes

[0031] Using Citrus citrus cDNA as a template, high-fidelity enzyme was used for amplification. The amplification primer sequences were: Forward primer for PtrAHL14 gene amplification: 5'-ATGGAACCAAATGATACGCAGC-3' and reverse amplification primer: 5'-CTAGTCTGCAATTTGGTCATAAGTC-3' ; Forward primer for amplification of PtrAHL17 gene: 5'-ATGAAAAGTGATTATGTAGTAGAACCC-3' and reverse primer for amplification: 5'-TCAATAAGGCGGTGGTGGGTGG-3';

[0032] AxyPrep-96 DNA gel recovery kit was used to purify and recover the amplified product. Using DNA seamless cloning technology, the purified product was ligated into pEASY-Blunt vector, and then the ligated product was transformed into DH5α competent cells, plated, shaken bacteria, and then a positive identification was performed. The positive clones were obtained and sent to Wuhan Qingke Biological Company for sequencing. According to the sequenci...

Embodiment 2

[0034] Example 2: Expression analysis of PtrAHL14 and PtrAHL17 under low temperature treatment

[0035] Take wild-type citrus aurantium seedlings that are 2 months old and have the same growth, and place them in a low-temperature incubator (HP400G-E, Ruihua, China) for low-temperature treatment (4°C), and the sampling time points are 0h, 6h, and 10h. , 12h, 24h, 48h, 72h. The leaves were collected at each time point and quickly placed in liquid nitrogen for freezing, and then placed in a -80°C refrigerator for later use in gene expression pattern analysis and immunoblotting experiments.

[0036] The low-temperature expression patterns of PtrAHL14 and PtrAHL17 genes were analyzed by real-time fluorescence quantitative PCR (qRT-PCR). AceQ qPCR SYBR Green Master Mix reagent was used for real-time fluorescence quantitative PCR, and the method was referred to the instructions. The prepared reaction system was reacted with QuantStudioTM7Flex Real-Time PCR fluorescence quantitative ...

Embodiment 3

[0039] Example 3: Analysis of PtrAHL14 and PtrAHL17 Subcellular Localization and Transcriptional Activation Activity

[0040] The ORF regions of PtrAHL14 and PtrAHL17 (without stop codon) were amplified and fused to the vector pEG104 (with YFP protein), and the expression was driven by the CaMV35S promoter. Afterwards, the control 35S:YFP+mCherry, 35S:PtrAHL14-YFP+mCherry, 35S:PtrAHL17-YFP+mCherry were transiently transformed into the leaf epidermal cells of N. benthamiana respectively, and the fluorescence of the control was observed by confocal microscopy and found that the fluorescence of the control filled the entire epidermal cells. , including the cytoplasm and nucleus, while the fluorescence of transformed 35S:PtrAHL14-YFP and 35S:PtrAHL17-YFP was detected only in the nuclear matrix ( image 3 In A), to confirm this, total nuclear and nuclear matrix proteins were extracted and analyzed by immunoblotting using PtrAHL14 and PtrAHL17 antibodies. The results showed that Pt...

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Abstract

The invention belongs to the field of plant genetic engineering and discloses the trifoliate transcription factor PtrAHL and its application in genetic improvement of plant cold resistance. PtrAHL The gene was derived from the extremely cold-resistant Trifoliate trifoliate ( Poncirus trifoliata ), two transcription factors isolated and cloned in ), named PtrAHL14 and PtrAHL17 , whose sequence is shown in SEQ ID NO.1 and SEQ ID NO.2. The two genes were respectively constructed as overexpression and interference vectors, and were respectively introduced into lemon and trifoliate orange through Agrobacterium-mediated genetic transformation, and the obtained transgenic plants were verified by biological functions, indicating that the cloned gene of the present invention PtrAHL14 and PtrAHL17 Genes have the function of controlling the cold resistance of plants. The development and utilization of the genetic resource is conducive to reducing the cost of agricultural production and realizing environmental friendliness.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, in particular to the transcription factor PtrAHL of Citrus aurantium and its application in the genetic improvement of plant cold resistance. Each gene was overexpressed in non-cold-resistant plants, and the obtained transgenic plants had significantly improved cold resistance. Background technique [0002] Low temperature stress adversely affects plant growth and development, greatly limiting plant geographic distribution and crop economic yield (Ding et al., 2020). Low temperature will affect the stability of cell membrane, reduce enzyme activity, generate reactive oxygen species, lead to disorder of cell metabolism, and ultimately delay plant growth, and even lead to plant death in severe cases (Barnes et al., 2016). Plants have evolved a complex set of adaptive mechanisms to sense cold signals and respond by triggering various signal transduction pathways that ultimately help them ma...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H6/78
CPCC07K14/415C12N15/8273
Inventor 刘继红巴沙尔张杨李春龙
Owner HUAZHONG AGRI UNIV
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