The invention also discloses application of GrpE protein and coding gene thereof as molecular targets in cultivation of resistant plants
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A technology of transgenic plants and genes, applied in the application field of cultivating resistant plants, can solve problems such as lack of target genes
Active Publication Date: 2021-03-12
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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Problems solved by technology
At present, the control measures for sucking pests are still dominated by pesticides. Therefore, in the face of the relative lack of target genes, the...
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Embodiment 1
[0056] Embodiment 1, construction of recombinant plasmid
[0057] The target sequence regions of amiRGPa and amiRGPb are shown in figure 1 A.
[0058] The schematic diagrams of plasmid GP1, plasmid GP2, plasmid GP3 and plasmid GP4 are shown in figure 1 the B.
[0059] 1. Construction of GrpE gene silencing plant expression vector (GrpE-RNAi)
[0060] 1. Prepare the double-stranded DNA molecule shown in sequence 2 of the sequence listing.
[0061] 2. Using the DNA molecule in step 1 as a template, a primer pair consisting of GPa-Eco and GPa-kpn is used for PCR amplification, and the PCR amplification product is recovered.
[0064] 3. Take the PCR amplification product obtained in step 2, perform double digestion with restriction endonucleases EcoR I and Kpn I, and recover the digested product.
[0065] 4. Using the DNA molecule in step 1 as a templat...
Embodiment 2
[0081] Embodiment 2, preparation and identification of transgenic rice
[0082] 1. Preparation of transgenic rice
[0083] 1. Introduce the recombinant plasmid into Agrobacterium EHA105 to obtain the recombinant Agrobacterium.
[0084] 2. Co-cultivate the mature embryo callus of recombinant Agrobacterium and rice Nipponbare, then carry out resistance selection (the resistance selection adopts hygromycin at a concentration of 50 mg / L), then carry out pre-differentiation, differentiation, and rooting successively to obtain T 0 generation of regenerated plants.
[0085] 3. Take T 0 Plants are regenerated, selfed and seeds harvested.
[0086] 4. Take the seeds obtained in step 3 and culture them in a medium containing 50 mg / L hygromycin to screen for T. 1 Substitute plants.
[0087] 5. Cultivate normal growing T 1 Transgenic plants were generated, selfed and seeds were harvested.
[0088] 6. Take the seeds obtained in step 5 and culture them in a medium containing 50 mg / L ...
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Abstract
The invention discloses application of GrpE protein and a coding gene thereof as molecular targets in cultivation of resistant plants. The invention provides an application of a nucleic acid moleculeor a recombinant vector for inhibiting GrpE gene expression in preparation of a transgenic plant for resisting piercing-sucking pests. The invention also provides an application of the nucleic acid molecule or recombinant vector for inhibiting GrpE gene expression in preparation of stripe disease resistant plants. The application is achieved by inhibiting piercing-sucking pests as a propagation medium. In the process of researching the interaction of the rice laodelphax striatellus-RSV, a new gene GrpE is explored from the rice laodelphax striatellus, the rice laodelphax striatellus GrpE or the homologous gene thereof can be used as a molecular target for resistance breeding of crops for resisting piercing-sucking pests, and the expression of the GrpE or the homologous gene thereof in insects is inhibited through RNAi technology; therefore, transgenic plants with resistance to piercing-sucking pests such as laodelphax striatellus are obtained.
Description
technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to the application of GrpE protein and its coding gene as a molecular target in cultivating resistant plants. Background technique [0002] Plant pests are an important factor that endangers the yield and quality of crops. With the large-scale planting of a single crop species and the impact of global warming, the harm of crop pests is becoming more and more serious. At present, the effective measures to control pests are still based on the application of chemical pesticides, which have disadvantages such as pesticide residues and environmental pollution, while ideal biological control has many limitations in practical applications. [0003] With the rapid development of modern biotechnology, transgenic insect-resistant plants (such as cotton, poplar, etc.) have been planted on a large scale in China, and insect-resistant genes have partially replaced conventional pesticides,...
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