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Construction methods and application of insect-resistant gene of jerusalem artichoke and expression vector thereof

A technology of expression vector and plant expression vector, which is applied in the field of construction of Jerusalem artichoke insect resistance gene and its plant expression vector, and can solve the problems of inability to replace chemical insecticides, field instability, narrow insect resistance spectrum of Bt gene, lack of insect resistance genes, etc. question

Active Publication Date: 2016-10-26
YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, except for the Bt gene which is widely used in insect-resistant genetic engineering, insect-resistant genes that can be applied on a large scale are still very scarce.
Bt genes have high specificity to pests and safety to the environment, but Bt insecticides are prone to resistance, instability in the field, and a single Bt gene has a narrow spectrum of insect resistance, making it unable to replace chemical insecticides The search for new insect-resistant genes is the core and basis for the realization of insect pest biological control

Method used

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  • Construction methods and application of insect-resistant gene of jerusalem artichoke and expression vector thereof
  • Construction methods and application of insect-resistant gene of jerusalem artichoke and expression vector thereof
  • Construction methods and application of insect-resistant gene of jerusalem artichoke and expression vector thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Cloning of JaSPI gene from Jerusalem artichoke

[0045] 1. Jerusalem artichoke cDNA library construction

[0046] Take 50-100 mg of fresh Jerusalem artichoke tuber tissue, add liquid nitrogen, quickly and fully grind, homogenate, transfer to 1.5mL EP tube, and use Tiangen RNA extraction kit to extract total RNA.

[0047] Total RNA was identified by 1% agarose gel electrophoresis, and the product results are shown in figure 1 , there are obvious RNA bands in the figure and the two bands of 28S and 18S rRNA are clear and not diffused, indicating that the extracted total RNA is not degraded and the quality is high.

[0048]Using total RNA as a template, cDNA double strands were synthesized under the action of reverse transcriptase and polymerase according to the instructions of the cDNA library construction kit of Clontech Company.

[0049] 2. PCR method to obtain JaSPI gene fragment

[0050] According to the existing gene data of similar species in the NCBI database, u...

Embodiment 2

[0091] Construction of Plant Expression Vector pCAMBIA2301-JaSPI

[0092] Use restriction endonucleases XBaⅠ and SacⅠ to double-digest the PCR product of the restriction site of the JaSPI gene and the pCAMBIA2301 vector, respectively recover the JaSPI gene fragment and the pCAMBIA2301 vector, connect them at a ratio of 5:1, and pass PCR and restriction enzyme digestion Identification of recombinant plasmids.

[0093] 1. The enzyme digestion system is:

[0094]

[0095] 37°C overnight.

[0096] 2. Recover the JaSPI gene fragment and the pCAMBIA2301 vector according to the method of Example 1.

[0097] 3. Ligate the recovered JaSPI fragment with the pCAMBIA2301 expression vector overnight at 16°C. The ligation system is:

[0098]

[0099] 4. Transform the ligation product into Escherichia coli XL1-Blue, pick a single colony, inoculate it in a 20mL LB+Km Erlenmeyer flask, culture at a constant temperature of 37°C with shaking overnight, and use the bacterial liquid as a ...

Embodiment 3

[0102] Obtaining Transgenic Arabidopsis Plants and Analysis of Insect Resistance

[0103] 1. Transformation of plant expression vector into Agrobacterium

[0104] Utilize the freeze-thaw method to transform Agrobacterium EHA105 with the plant expression vector obtained in Example 2, obtain recombinant Agrobacterium, and obtain a positive transformant (containing the transformant of the JaSPI gene shown in SEQ ID NO: 1) through PCR identification, use for infecting Arabidopsis plants.

[0105] 2. Acquisition and identification of JaSPI transgenic Arabidopsis

[0106] Infect the inflorescence of the Arabidopsis plant to be transformed with the above-mentioned recombinant Agrobacterium, and harvest its seeds. The harvested seeds were planted on plates of MS solid medium (containing Km 60 μg / mL) to screen for T 0 generation of positive transgenic plants. Move the green seedlings into pots to grow, and wait for T 0 The green seedlings of the first generation grow up, and the s...

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Abstract

The invention specifically relates to construction methods and application of an insect-resistant gene of jerusalem artichoke and an expression vector thereof, belonging to the field of genetic engineering technology. The base sequence of the insect-resistant gene (JaSPI) of jerusalem artichoke is as shown in SEQ ID No. 1; or the insect-resistant gene (JaSPI) is a gene with a base sequence 85% or more homological to the base sequence as shown in SEQ ID No. 1. The amino acid sequence coded by JaSPI is as shown in SEQ ID No. 2. The JaSPI gene and the expression vector thereof can be applied to genetic transformation of plants for improvement of insect resistance and stress resistance of plants. Moreover, the JaSPI gene provided by the invention can be used for in-vitro expression, so a novel approach is provided for development of novel biological preparations like biopesticides and auxiliary Bt for co-prevention of agricultural insects.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a construction method and application of a Jerusalem artichoke insect-resistant gene and a plant expression vector thereof. Background technique [0002] Insect pests have always been an important factor restricting high, stable, and high-quality crops worldwide. For a long time, the use of chemical pesticides has been the main means of pest control in agricultural production. The long-term and large-scale application of chemical pesticides not only makes pests increasingly resistant to chemical pesticides, but also brings a series of social problems, such as the occurrence of human and animal poisoning accidents, pesticide residues in food, and environmental pollution. With the strengthening of human awareness of ecology and environmental protection, vigorously developing green agriculture has become a requirement of the new era, and insect-resistant genetic enginee...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/82C12N15/10A01H5/00
Inventor 杨少丽秦松任鹏鸿王义鹏王倩倩苏振
Owner YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
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