Cloning And Analysis Of Glutamate Chloride Channel Gene From Bursaphelenchus Xylophilus

Pine Wilt Disease is a devastating disease caused by Bursaphelenchus xylophilus,but there is still no good control.The main control strategy is trunk injection with nematocides.The practice shows that the avermectin compounds have good prevention and treatment effect on Bursaphelenchus xylophilus,and it has been shown that the target of avermectin insecticides is glutamate-gated chloride channel(Glu Cl),but there is no study on whether the glutamate-gated chloride channel of pine wood nematode is a target of avermectin insecticides.Therefore,this study cloned the glutamate-gated chloride channel gene of Bursaphelenchus xylophilus and preliminarily verified its function,and obtained the following research results: 1 Gene cloning and expression pattern analysis of the glutamate-gated chloride channel gene of Bursaphelenchus xylophilus.1)We obtained 3 gene sequences of glutamate-gated chloride channel from the transcription group of Bursaphelenchus xylophilus,and 3 genes were obtained by designing specific primer clones,named Bx Clu Cl1?Bx Clu Cl2?Bx Clu Cl3 respectively.The similarity of 3 glutamate-gated chloride channel gene sequences with other nematodes was about 40%-70% after sequence alignment.2)The physicochemical properties,amino acid molecular weight and equal electricity point,and hydrophilicity were deduced according to the Glu Cl amino acid sequence of Bursaphelenchus xylophilus.The relative molecular weight of Bx Clu Cl1 amino acid is about 43.481 KD,the theoretical p I is 9.45,the hydrophilic average coefficient is-0.057;The relative molecular weight of Bx Clu Cl2 amino acid is about 53.307 KD,the theoretical p I is 8.61,the hydrophilic average coefficient is-0.204;The relative molecular weight of Bx Clu Cl3 amino acid is about 34.879 KD,the theoretical p I is 5.91,the hydrophilic average coefficient is 0.048.3)The coding protein structure domain,secondary structure and tertiary structure were speculated according to the amino acid sequence of Glu Cl.3 Glu Cl were composed of five polypeptide subunits.And each subunit has 4 hydrophobic transmembrane domains(TM),a longerextracellular domain(ECD)and an intracellular domain comprising M1-M4 and a large cytoplastic loop between M3 and M4.2 Expression of the gene of glutamate-gated chloride channel in the heterologous expression systems.3 Glu Cl genes were expressed in the CHO?HEK 293?COS-7 and Sf 9 cells.Under the stimulation of specific neurotransmitter L-glutamic acid,patch clamp technique was used to test the reaction of 3 Glu Cl gene expression products to L-glutamic acid,no reaction was found.Maybe the mammalian cell and insect cell expression system could not express Glu Cl gene or express the Non-functional Glu Cl gene.Or it may be necessary to express two or more genes simultaneously to induce glutamate-gated action.3 Glu Cl genes were expressed in Xenopus oocyte.Under the stimulation of the specific neurotransmitter L-glutamic acid,voltage clamp technique was used to test 3 Glu Cl gene expression products to L-glutamic acid.Preliminary results showed that Bx Clu Cl3 was active against L-glutamic acid,but Bx Clu Cl1 and Bx Clu Cl2 have no current response to L-glutamic acid.At present,Xenopus oocytes have been preliminarily used to express 3 Glu Cl genes,and voltage clamp techniques have been used to verify the function of 3 Glu Cl genes genes in Bursaphelenchus xylophilus,indicating that some cloned genes have functional responses.Our group will conduct further experiments on the 3 genes in the next work to verify its function,and we will ues this technique to study the affinity of different structures of avermectin compounds and chloride ion channels of Bursaphelenchus Xylophilus,and to further clarify the correlation of avermectin compounds activity and structure.