Method for improving insect resistance of plan and plant expression vector

An ability and plant technology, applied in the method and its plant expression vector to improve the field of plant insect resistance, can solve the problems of poor stability, high price, difficult research and application of 5-ALA, and achieve the goal of reducing use and production costs. Effect

Active Publication Date: 2020-07-28
HUNAN PLANT PROTECTION INST
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

However, due to the complex chemical synthesis process of 5-ALA pure product, it is not suitable for industrial production requirements; and biosynthesis has made progress, and the output has been improved, but it is far from the industrial production requirements.
In addition, 5-ALA is expensive in the market, and its stability is poor 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) there are two synthesis pathways of C4 and C5 in plants, but C5 The synthesis pathway is the main route, and 5-ALA does not accumulate in cells due to the inhibition of biochemical feedback regulation, which brings certain difficulties to the research and application of plant-derived 5-ALA

Method used

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  • Method for improving insect resistance of plan and plant expression vector
  • Method for improving insect resistance of plan and plant expression vector
  • Method for improving insect resistance of plan and plant expression vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Cloning of embodiment 1, 5-aminolevulinic acid synthase gene

[0030] 1) According to the sequence registration information in NCBI, design 5-ALAS gene-specific PCR amplification primers and introduce restriction sites. The primer sequence is as follows: 5'primer: 5'-GGTACCATGGATTACACCAAGTTCTTC-3'The part in italics is the KpnI restriction site point

[0031] 3' primer: 5'-GGATCCTTATTCCGCAGCGAGCGGCTT-3' italic part is BamHI restriction site

[0032] 2) Preparation of the photosynthetic bacterium Rhodoblastus acidophilus PSB-8 genome: take the photosynthetic bacterium Rhodoblastus acidophilus PSB-8 strain, inoculate the liquid medium at a ratio of 1:100, 30°C, 2500LX light Cultured for 7 days. Take 10ml of bacterial liquid, centrifuge at room temperature at 12000rpm for 5min to collect bacterial cells. After dissolving with 500 μl TE buffer, add 30 μl 10% SDS and 3 μl proteinase K, mix well, and incubate at 37°C for 1 hour. Add 100 μl of 5M NaCl, mix well, add 80 μl ...

Embodiment 2

[0035] Embodiment 2, the construction of the plant expression vector of phloem-specific promoter rolC and 5-aminolevulinic acid synthase gene

[0036] The construction strategy of the plant expression vector pCAMBIA1300 with phloem-specific promoter (rolC promoter) and 5-aminolevulinic acid synthase gene (ie 5-ALAS gene): rolC promoter: ALAS: rolc teminator is as follows figure 2 shown. The specific experimental steps are described as follows.

[0037] 1) Construction of the intermediate vector pCAMBIA1300:ALAS: the pGEM-T-Easy:ALAS recombinant plasmid and the pCAMBIA1300 plasmid (purchased from Cambia institute from Australia) were double-digested with restriction enzymes KpnI and BamHI, respectively. The 5-ALAS fragment excised from the pGEM-T-Easy:ALAS recombinant plasmid and the large fragment after pCAMBIA1300 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 ratio), an...

Embodiment 3

[0042] Example 3. Plant expression vector pCAMBIA1300: rolC promoter: ALAS: rolc teminator introduced into Agrobacterium

[0043] Competent cells of Agrobacterium EHA105 were prepared, and the above-constructed plant expression vector pCAMBIA1300:rolC promoter:ALAS:rolc teminator was introduced into Agrobacterium EHA105 by the heat shock transformation method, and the transformation was screened using the culture medium of rifampicin and kanamycin son. Put the Agrobacterium competent cells taken out at -70°C on ice for about 5 minutes. After thawing, add 1 μl of plasmid and mix with the Agrobacterium competent cells, let stand on ice water at -20°C for 30 minutes, and quickly transfer to 37°C Heat shock in water bath for 30min, add 800μl liquid SOC medium, recover at 28℃, 200rpm for 3-5hrs, take 200μl evenly coated on YEP plate containing 50μg / ml kanamycin and 50μg / ml rifampicin, incubate in dark at 28℃ for 48hrs , Perform colony PCR screening to obtain positive transformants...

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Abstract

The invention discloses a method for improving insect resistance of a plant. The method is characterized in that a 5-aminolevulinic acid synthetase gene is introduced and expressed into the plant to obtain the insect resistance of a transgenic plant. The invention also provides a method for preparing the transgenic plant with improved insect resistance, wherein the method comprises the following steps of transforming the plant by using the plant expression vector containing the 5-aminolevulinic acid synthetase gene, and carrying out screening to obtain the transgenic plant with improved insectresistance. Experimental results show that each strain of the 5-ALAS transgenic rice shows enhanced resistance response to the rice planthopper, so that the method has relatively high popularizationand application values.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for improving plant resistance to insects and a plant expression vector thereof. Background technique [0002] Crop production is usually harmed by piercing-sucking mouthparts pests (rice planthoppers, aphids, whiteflies, leafhoppers, etc.), resulting in varying degrees of production reduction or product quality decline, accompanied by the spread of plant virus diseases, ranging from The loss is 20%-40%, and the weight is 100%. At present, chemical pesticides are mainly used for prevention and control, but they cause environmental and agricultural product pollution while controlling pests, leading to the rapid evolution of pest resistance, especially the long-term large-scale single planting of high-yield and high-quality crop varieties, changes in climate and planting patterns, provide pests with Sufficient food sources are lost, resulting in frequent occurrenc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/54A01H5/00A01H6/46A01H6/82
CPCC12N9/1029C12N15/8286C12Y203/01037
Inventor 谭新球刘勇张德咏张卓陈岳李成刚成飞雪张松柏朱春晖欧阳超刘思珍
Owner HUNAN PLANT PROTECTION INST
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