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Cold-resistant gene PtrMYC2 of trifoliate orange and application of cold-resistant gene PtrMYC2 in cold-resistant genetic improvement of plants

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

Active Publication Date: 2020-08-21
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 cold resistance involving MYC transcription factors

Method used

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  • Cold-resistant gene PtrMYC2 of trifoliate orange and application of cold-resistant gene PtrMYC2 in cold-resistant genetic improvement of plants
  • Cold-resistant gene PtrMYC2 of trifoliate orange and application of cold-resistant gene PtrMYC2 in cold-resistant genetic improvement of plants
  • Cold-resistant gene PtrMYC2 of trifoliate orange and application of cold-resistant gene PtrMYC2 in cold-resistant genetic improvement of plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Cloning of full-length cDNA of PtrMYC2 gene in trifoliate orange and construction of overexpression vector pBI121

[0034]Using Hovenia trifoliate cDNA as a template, high-fidelity enzymes were used to amplify. The amplification system is shown in Table 1, and the amplification program is shown in Table 2. The primers used were pBI121-MYC2 overexpression primers: F: 5'-GAGAACACGGGGGACTCTAGAATGACGGACTACCGGTTACCTTC-3' and R: 5'-ATAAGGGACTGACCACCCGGGTTATTGGGTATCTCCAACTTTGGC-3'.

[0035] Table 1 Gene amplification system

[0036]

[0037] Table 2 Gene amplification PCR program

[0038]

[0039]

[0040] AxyPrep-96 DNA Gel Recovery Kit (Axygene, USA) was used to purify and recover the amplified product. Using DNA seamless cloning technology, the purified product was connected to the linearized overexpression vector pBI121, and then the ligated product was transformed into DH5α Competent cells were plated, shaken, and then positively identified. After the positive...

Embodiment 2

[0047] Genetic Transformation and Positive Identification of Tobacco

[0048] 1) Strain preparation: Take out the preserved Agrobacterium transformed into the pBI121-PtrMYC2 vector from -80°C, absorb a small amount of Agrobacterium liquid with a pipette tip, and place it in MS liquid medium without antibiotics, at 28°C, 200r / Min culture until the OD600 value reaches 0.6-0.8 for infection;

[0049] 2) Explant preparation: select the sterile tobacco with good growth, take the largest 2-3 leaves, remove the main vein and leaf edge, cut into 0.5cm 2 Put the left and right size cubes into a sterile triangular flask with a small amount of MS liquid medium for infection;

[0050] 3) Infection and co-cultivation: Pour the bacterial solution cultivated in the first step into the Erlenmeyer flask containing the explants, infect for 10 minutes, and shake gently during the infecting process. After infestation, blot dry the bacterium liquid that the explant carries with sterilized filte...

Embodiment 3

[0066] Analysis of cold resistance of PtrMYC2

[0067] Potted transgenic tobacco and wild-type tobacco (WT) at 30d seedling age were used for identification of low temperature resistance. Before low temperature treatment, there was no obvious phenotypic difference between tobacco overexpressing PtrMYC2 gene and wild-type tobacco, but after 12 hours of treatment at -2°C, the wild-type was more severely injured than the transgenic line, and most of the leaves were water-stained In the transgenic line, only part of the tobacco was water-soaked ( Figure 5 Middle A). Survival rate was counted after recovery, transgenic plants had higher survival rate ( Figure 5 Middle B), where the #2 line was 84%, the #4 line was 89%, and the survival rate of the wild type plant was only 8%. Conductivity measurement found that the relative conductivity of wild-type tobacco was higher after low temperature treatment ( Figure 5 Middle C), indicating that more severe cell membrane damage occur...

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Abstract

The invention belongs to the field of plant genetic engineering, and discloses a cold-resistant gene PtrMYC2 of trifoliate orange and an application of the cold-resistant gene PtrMYC2 in cold-resistant genetic improvement of plants. The PtrMYC2 gene is an MYC family transcription factor which is separated and cloned from Poncirusiferous trifoliate, and the sequence of the MYC family transcriptionfactor is shown as SEQ ID NO.1. An overexpression vector and a TRV2 vector are constructed by the gene, the gene can be respectively introduced into tobacco and trifoliate orange through agrobacteriumtumefaciens mediated genetic transformation, and biological function verification shows that the cloned PtrMYC2 gene has the function of improving the cold resistance of plants. The discovery of thegene provides a new gene resource for plant stress-resistant molecular design breeding, and provides a new genetic resource for implementing green agriculture and water-saving agriculture, and the development and utilization of the genetic resource are beneficial to reducing the agricultural production cost and realizing environmental friendliness.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, specifically relates to a transcription regulator PtrMYC2 isolated and cloned from Citrus trifoliate (Poncirustrifoliata), and also relates to the application of the gene in genetic improvement of plant cold resistance, overexpressing the gene in tobacco and Citrus trifoliata, The cold resistance of the obtained transgenic plants is obviously improved. Background technique [0002] Due to the immovable nature of the plant itself, it must constantly monitor changes in the surrounding environment and respond to environmental changes. In different environments, plants are subjected to different environmental stresses, including biotic stress and abiotic stress. Among them, biotic stress mainly includes diseases and insect pests, while abiotic stress includes other physical stresses such as low temperature, high temperature, high salinity, drought, and insufficient light. After long-term nat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H6/82A01H6/78
CPCC07K14/415C12N15/8273
Inventor 刘继红明如宏黄小三
Owner HUAZHONG AGRI UNIV
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