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Cloning,Sequencing And Functional Expression Of Apolygus Lucorum Sodium Channel

Posted on:2019-10-30Degree:MasterType:Thesis
Country:ChinaCandidate:K ZhangFull Text:PDF
GTID:2393330548486220Subject:Agricultural Entomology and Pest Control
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In recent years,due to the large amount of BT transgenic cotton,the control of lepidoptera pests such as Helicoverpa armigera and Pectinophora gassypiella in the farmland has been clearly controlled,but it has also led to the development of dominant insect pests such as the Apolygus lucorum and Tetranychus cinnabarinus.Apolygus lucorum(Meyer-dür)belong to the genus semi-winged pests.It is difficult to control because of it's ability to fly short distances and small individuals in the field.Currently,the main prevention and control method for Apolygus lucorum is chemical control.If it is used for a long time,it will bring a series of drug resistance problems,which will make future prevention and control more difficult.Pyrethroid insecticides are gradually being used to control the damage of the Apolygus lucorum because of their numerous advantages.The voltage-gated sodium channel is the target of DDT and pyrethroid insecticides.It is of great significance to study the mechanism of the insect sodium ion channel in solving insect resistance.In this study,based on the brain transcriptome of Apolygus lucorum,bioinformatics analysis was combined with in vitro functional studies to clone the full length of the sodium channel gene(NCBI serial number: MF627838)and analyze the alternative splicing in the full-length sequence.The function of sodium channels was identified by two electrode voltage clamp technique in RNA editing and in Xenopus oocyte expression system.The main experimental results are as follows:(1)The full-length sodium channel gene Al Nav was cloned by RT-PCR,and the Al Nav1-1 open reading frame was 6015 bp,which can encode 2005 amino acids.Through the multiple alignment of Al Nav and Blattella germanica sodium channels(Bg Nav),Drosophila melanogaster sodium channels(Dm Nav)and Bombus impatiens sodium channels(Bi Nav),the amino acid sequences of Al Nav and Bg Nav were consistent of 82.10%,the identity with the Dm Nav para amino acid sequence was 75.32%,the identity with the Bi Nav amino acid sequence was 82.11%.Further phylogenetic tree analysis with other 54 insect sodium channels showed that Al Nav has high similarity to other species of insect sodium channels.After TMHMM analysis,the amino acid sequence of Al Nav includes four homologous domains I,II,III and IV.Each domain contains six transmembrane segments S1,S2,S3,S4,S5 and S6,and Al Nav is in I-IV.There are four amino acid residues D,E,K,and A between the S5 and S6 domains,and MFM exists between III and IV.Sequencing results of 102 Sodium channel clones were analyzed for multiple sequence alignments.Al Nav had 9 alternative splicing sites(j,n,o,a,p,b,s,q,t),35 RNA modifications in Al Nav,of which 18 were edited from A to I and 11 were edited from U to C.(2)The expression of Al Nav function was measured by Two-electrode voltage clamp technique through the Xenopus oocyte expression system.When the pulse is activated,significant inward currents are produced when Al Nav and Tip E are co-expressed.The Xenopus oocytes were clamped at-120 m V.From the curve of Al Nav1-1 steady-state activation voltage-dependent data fitting curves,the sodium channels depolarized from-80 m V to 60 m V,the step voltage was 5 m V,V1/ 2(half-activation voltage)is-33.5±1.3 m V and the K(slope)is 3.1±0.4 m V.First give a time of 200 ms pre-stimulation,and then give 20 ms of-10 m V test stimulation,from the steady-state inactivation voltage-dependent data fitting curves to see that the stimulation from-120 m V to 40 m V,the step voltage is 5m V,V1/2(Semi-deactivation voltage)-47.9 ± 1.9 m V,K(slope)is 4.7 ± 0.2 m V.The functional expression results showed that no inward current was recorded in the oocytes that had not been injected with c RNA;the injection of Al Nav alone could detect the current and the expression rate was slow;the Al Na V and Tip E co-expressed in the Apolygus lucorum and the expression rate was rapid.By with apparent sodium currents were tested to obtain 18 variants with obvious currents.The activation voltage dependence of sodium channels,the voltage dependence of inactivated sodium channels,and the slow inactivation voltage dependence were determined,respectively,demonstrating that Al Nav was Successful expression in the Xenopus oocyte system.
Keywords/Search Tags:Apolygus lucorum, voltage-gated sodium channel, Alternative splicing, RNA editing, Xenopus Oocyte, two electrode voltage clamp
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