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Method for improving the low-temperature stress resistance of rice and plant expression vector used in method

A technology of low temperature resistance and high ability, applied in the field of methods and plant expression vectors, can solve the problems of complex resistance genes, limited practical applications, and unsatisfactory resistance levels, and achieve the effect of improving low temperature resistance

Inactive Publication Date: 2020-08-14
HUNAN PLANT PROTECTION INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although researchers have tried to find resistance resources in different ways, excavate related genes, and make effective use of them, they have only succeeded in drought resistance, salt-alkali resistance, etc., and found some resistance-related genes and resistant varieties, but because of the level of resistance Factors such as undesired or complex interactions of resistance genes limit their practical application. At present, there are very limited applications that can be applied to actual production on a large scale.

Method used

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  • Method for improving the low-temperature stress resistance of rice and plant expression vector used in method
  • Method for improving the low-temperature stress resistance of rice and plant expression vector used in method
  • Method for improving the low-temperature stress resistance of rice and plant expression vector used in method

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Effect test

Embodiment 1

[0031] Cloning of embodiment 1, 5-aminolevulinic acid synthase gene

[0032] 1) According to the sequence registration information in NCBI, design 5-ALAS gene-specific PCR amplification primers and introduce enzyme cutting sites. The primer sequences are as follows:

[0033] 5' primer: 5'-CCATGGATGGATTACACCAAGTTCTTC-3' italic part is the NcoI restriction site (for the construction of pCAMBIA1300:2×35S promoter:ALAS:poly(A) plant expression vector)

[0034] 3' primer: 5'-GGATCCTTATTCCGCAGCGAGCGGCTT-3' italic part is the BamHI restriction site (for the construction of pCAMBIA1300:2×35S promoter:ALAS:poly(A) plant expression vector)

[0035] 5' primer: 5'-CCCGGGATGGATTACACCAAGTTCTTC-3' italic part is the SmaI restriction site (for the construction of pCAMBIA1300: Ubiquitin promoter: ALAS: Ubiquitin terminater plant expression vector)

[0036] 3' primer: 5'-GAGCTCTTTATTCCGCAGCGAGCGGCTT-3' italic part is the SacI restriction site (for the construction of pCAMBIA1300: Ubiquitin pro...

Embodiment 2

[0041] Embodiment 2, have the construction of constitutive expression promoter 2 * 35S promoter or Ubiquitin promoter and 5-aminolevulinic acid synthase gene (being 5-ALAS gene) plant expression vector

[0042] Among them, the construction strategy process of the plant expression vector pCAMBIA1300:2×35S:ALAS:poly(A) is as follows figure 2 shown. The construction strategy of the plant expression vector pCAMBIA1300: Ubiquitin promoter: ALAS: Ubiquitin terminater is as follows image 3 shown.

[0043] 1) Construction of the intermediate vector pBsK:2×35S:ALAS:poly(A)

[0044] The pGEM-T-Easy:ALAS recombinant plasmid and the pBsK plasmid were double-digested with restriction endonucleases NcoI and BamHI, respectively. The 5-ALAS fragment excised from the pGEM-T-Easy:ALAS recombinant plasmid and the large fragment after pBsK double digestion were recovered by electrophoresis and gel slicing. The recovered fragment was transformed with T4 DNA ligase at a ratio of 1:3 (molar ra...

Embodiment 3

[0052] Example 3: Plant expression vector pCAMBIA1300:2×35S:ALAS:poly(A) and pCAMBIA1300:Ubiquitin promoter:ALAS:Ubiquitin terminater were introduced into Agrobacterium

[0053] Competent cells of Agrobacterium EHA105 were prepared, and the above-constructed plant expression vectors pCAMBIA1300:2×35S:ALAS:poly(A) and pCAMBIA1300:Ubiquitin promoter:ALAS:Ubiquitin terminator were introduced into Agrobacterium EHA105 by heat shock transformation method, Transformants were selected on medium plates plus rifampicin and kanamycin. Agrobacterium competent cells taken out from -70°C were placed on ice for about 5 minutes. After they were thawed, 1 μl of plasmid was added to mix with Agrobacterium competent cells, placed on -20°C ice water for 30 minutes, and quickly transferred to a 37°C water bath. Heat shock for 30min, add 800μl liquid SOC medium, recover at 28℃, 200rpm for 3-5hrs, take 200μl coated with 50μg / ml kanamycin, 50μg / ml rifampicin on the YEP plate, culture in the dark at ...

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Abstract

The invention discloses a method for improving the low-temperature stress resistance of rice and a plant expression vector used in the method. The method is characterized in that a 5-aminolevulinic acid synthetase gene is introduced and expressed in a plant, so that the low-temperature stress resistance of the transgenic plant is improved. The invention also provides a method for preparing transgenic rice with the improved low-temperature stress resistance, which comprises the following steps of transforming the plant by using the plant expression vector containing the 5-aminolevulinic acid synthetase gene, and performing screening to obtain the transgenic plant with the improved low-temperature stress resistance. Experimental results show that the low-temperature stress resistance of eachstrain of the 5-ALAS transgenic rice is greatly enhanced, so that the method has the popularization and application value in the cultivation of low-temperature stress-resistant varieties such as cold-resistant varieties.

Description

technical field [0001] The invention belongs to the field of biotechnology and plant breeding, and specifically relates to a method for improving the low temperature stress resistance of plants, especially rice, and a plant expression vector thereof. Background technique [0002] 5-aminolevulinic acid (5-ALA) is a plant physiologically active substance with multiple effects, and is a green biodegradable insecticide. However, due to the complex chemical synthesis process of pure 5-ALA, it is not suitable for the requirements of industrial production; while the biosynthesis has made progress, and the yield has been improved, but it is far from the requirements of industrial production. In addition, 5-ALA is expensive in the market and has poor stability in the natural state, so it cannot be used on a large scale in production. According to reports (Meller&Gassman, Biosynthesis of-amino-levulinic acid: Two pathways superior plants. Plant Science Letters, 1982, 26 (1): 23-29) t...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N15/82A01H5/00A01H6/46
CPCC12N9/1029C12N15/8273C12Y203/01037
Inventor 谭新球张德咏刘勇欧阳超刘思珍陈岳李成刚张卓成飞雪张松柏朱春晖
Owner HUNAN PLANT PROTECTION INST
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