Application of glutamate dehydrogenase as target spot in pest control
A technology of glutamate dehydrogenase and pest control, applied in the biological field, can solve the problem of little understanding of the function of silkworm GDH
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Embodiment 1
[0065] Example 1GDH protein phylogenetic tree construction and protein sequence analysis
[0066] The evolutionary conservation of the homologous sequences of GDH proteins in 11 species was analyzed. These species include Lepidoptera: Bombyx mori (Bombyx mori), Wild silkworm (Bombyx mandarina), Spodoptera litura, Diamondback moth (Plutella xylostella), Cabbage caterpillar (Pieris rapae), Cotton bollworm (Helicoverpa armigera), Golden Phoenix Butterfly (Papilio machaon); Diptera: Drosophila melanogaster; Rodentia: Mus musculus (Mus musculus); Artiodactyla: Wild boar (Sus scrofa); Primates: Homo sapiens. The GDH protein phylogenetic tree was constructed using the ML method (Maximum Likelihood), tested by the bootstrap method (Bootstrap, 1000 replicates), the evolutionary distance was analyzed by Poisson correction, and the data was analyzed by MEGA7. The results of phylogenetic analysis of its protein sequence showed that the evolution was relatively conservative, and Lepidopte...
Embodiment 2
[0067] Example 2 Transgenic CRISPR / Cas9 system to knock out the silkworm glutamate dehydrogenase (GDH) gene
[0068] 1. Selection of target sites
[0069] GDH基因的CDS序列共1665bp,具体序列为:ATGCTGCATCTCAAGAATATCGCCAAGTCAGTGGTTCCGCCGCTCAAGAATTCTGTTCAAAATGAAGCCCTCAATACAATGTTCCGAATCATACCAGCTGGAGTGAATGTCTGCTGCCGCACATACGCTAGTCATGAGATTCCAGATAAGCTCAAAGATATTCCTACAAGTGCGAATCCGAAGTTCTTCCACATGGTAGAATATTTTTTCCACCGAGCCTGTCAAGTTGTCGAAGACAAGCTTGTTGAAGATTTGAAGTCAAGGACACCCATTGAAGAGAAGAAAAAGAAAGTAGCCGGTATTCTAAAACTTATGGAACCATGCGATCACATTCTTGAGATTCAATTTCCTCTGAGGCGCGATTCTGGCGATTACGAAATGATATTAGGCTATCGCGCACAACATTCCACACACAGGACTCCAACCAAAGGAGGTATTCGATTCTCAACGGACGTAACCAGAGATGAAGTTAAGGCGTTATCAGCTTTGATGACCTTCAAGTGCGCGTGCGTGGACGTGCCTTTCGGCGGTGCTAAGGCCGGTATCAAGATCAATCCCAAAGAATACTCCGAGCATGAACTGGAAAAGATCACTCGTCGTTTCACCCTTGAACTTGCCAAAAAAGGATTCATTGGGCCTGGCGTGGATGTCCCCGCTCCTGACATGGGTACCGGCGAACGAGAAATGTCTTGGATCGCCGATACTTATGCGAAGACCGTCGGTTTTCAAGACATCAACGCTCACGCCTGCGTCACTGGCAAACCTATTAACCAGGGTGGCATCCACGGCAGAGTTTCAGCCACGGGCAGGGGC...
Embodiment 3
[0135] Example 3 Phenotype of Bombyx mori GDH Gene Knockout (Knockout by CRISPR / Cas9 System)
[0136] (1) Increased mortality in the larval stage
[0137] Compared with the control group (GDH non-knockout, Wild type), the GDH knockout mutant (△BmGDH) is more susceptible to infection and death in the laboratory environment, such as Figure 7 As shown, the GDH knockout (△BmGDH) worms were more susceptible to viral softening disease than the control group, and the mortality rate in the third instar was as high as 58.9% ( Figure 8 ); Few individuals can survive to the fifth instar and wandering stage, so knocking out GDH in Lepidoptera insects will lead to a large number of larval deaths.
[0138] (2) Larval weight loss
[0139] Compared with the control group (GDH non-knockout, Wild type), the size of the GDH knockout mutant (△BmGDH) gradually increased from the third age ( Figure 9 ), significantly reduced body weight ( Figure 10 ).
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