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Method for improving plant drought resisting cold resisting capability by utilizing poncirus trifoliata argininedecarboxylase gene PtADC

A gene and cold-resistant technology, applied in the field of plant genetic engineering, can solve the problems of unseen gene cloning and functional verification reports, unseen PtADC gene transformation plants resistant to abiotic stress, etc.

Inactive Publication Date: 2010-10-06
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008]Although many studies have shown that the ADC gene has been cloned in some plants, there is no report on the cloning and functional verification of the gene in Citrus trifoliate, let alone its application Literature reports on the improvement of resistance to abiotic stress in plants transformed with PtADC gene of Hovenia trifoliate

Method used

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  • Method for improving plant drought resisting cold resisting capability by utilizing poncirus trifoliata argininedecarboxylase gene PtADC
  • Method for improving plant drought resisting cold resisting capability by utilizing poncirus trifoliata argininedecarboxylase gene PtADC
  • Method for improving plant drought resisting cold resisting capability by utilizing poncirus trifoliata argininedecarboxylase gene PtADC

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1: Isolation and cloning of PtADC gene

[0034] Search the citrus EST database (www.harvest.ucr.edu, Harvest: Citrus ver.0.51) with arginine decarboxylase as the keyword, and obtain 26 similar sequences, and use CAP3 (common software) for the 26 similar sequences that were initially selected Form a complete sequence, use the sequence as a template to design primers with Primer Premier 5.0 software, and use the commonly used RT-PCR method (refer to J. Sambrook, EF Fritsch, T Mani Artis, Huang Peitang, Wang Jiaxi, etc. Translated, Molecular Cloning Experiment Guide (Third Edition), Science Press, 2002 Edition) the full length of the extended sequence. RNA was extracted and reverse transcribed from leaves of Fructus Fructus Fructus under low temperature stress at 4°C, and the obtained first-strand cDNA was used to amplify the full-length sequence. The RNA extraction method refers to the instruction manual of the Trizol kit (purchased from Invitrogen Company, op...

Embodiment 2

[0035] Embodiment 2, plant transformation vector construction

[0036] According to the pMV vector (plant binary transformation vector pBI121 with the excised GUS gene, this vector was donated by Professor Ye Zhibiao, School of Horticulture and Forestry, Huazhong Agricultural University, see figure 2 Shown) the multiple cloning site and the coding region sequence of the PtADC gene, the forward and reverse primers for amplifying the entire coding region of the PtADC gene were designed using the Primer Premier 5.0 software according to the general principle of primer design. The 5' ends of the forward and reverse primers used were respectively added with Xho I and Kpn I restriction sites (restriction site sequences are underlined, as follows), and three protections were added outside the recognition sequence of the restriction sites Base, in order to facilitate the smooth progress of the enzyme cleavage reaction. The DNA sequence of the primer pair is as follows:

[0037] For...

Embodiment 3

[0040] Embodiment 3, plant genetic transformation

[0041] Apply the freeze-thaw method (referring to J. Sambrook, etc., translated by Huang Peitang, Molecular Cloning Experiment Guide (Third Edition), Science Press, 2002 Edition) to introduce the pMV-PtADC vector into Agrobacterium EHA105, and the transformation characteristics Stable and homozygous T-DNA of Arabidopsis thaliana was inserted into the AtADC1 mutant adc1-1 (gifted by the Shinozaki Research Group of RIKEN), and the positive plants were selfed for three generations after genetic transformation to obtain homozygous transgenic lines.

[0042] Specifically, in this example, the genetic transformation steps of Arabidopsis thaliana mediated by Agrobacterium tumefaciens are as follows:

[0043] 1. Culture of Arabidopsis

[0044] The Arabidopsis thaliana seeds after vernalization at 4°C for 2 days were sowed in a fully moistened substrate (by weight: nutrient soil: vermiculite = 1:1), and cultivated in a greenhouse (22...

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Abstract

The invention belongs to the plant genetic engineering field. The invention in particular relates to the citrus fruit tree genetic engineering technical field. Drought resistant cold resistant argininedecarboxylase gene PtADC is obtained by separation and cloning from citrus fruit tree poncirus trifoliata, the code sequence thereof is shown as 85-2340 bits of SEQ ID NO: 1. The gene is introduced into a model plant Arabidopsis or tobacco to carry out biological function verification and genetic transformation, thus obtaining a transgenic plant. The transgenic plant has obvious drought resistant cold resistant capability. The gene of the invention is hopeful to be further applied in citrus fruit trees.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering. It specifically involves isolating and cloning the trifoliate arginine decarboxylase gene PtADC from Poncirus trifoliata, and then introducing the gene into a model plant (Arabidopsis thaliana) for biological function verification, and the drought and cold resistance of the transgenic plants obtained Has improved significantly. Background technique [0002] Abiotic stress (such as drought, low temperature, salinity, etc.) affects the growth and development, geographical distribution and production capacity of plants, which is a severe challenge to agricultural production and a bottleneck for agricultural development in many regions. Taking drought as an example, according to statistics, the total area of ​​arid regions in the world accounts for more than 40% of the land area, and the annual loss of crop production due to water stress in the world is almost equal to the sum of the losses ...

Claims

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Application Information

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IPC IPC(8): C12N15/60C12N9/88C12N15/11A01H5/00
Inventor 刘继红王静孙培培张庆福
Owner HUAZHONG AGRI UNIV
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