花绒寄甲成虫3条Mthl基因及Mth-like 2-5干扰效果研究
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摘要
筛选花绒寄甲成虫高表达的mthl基因,注射目标基因dsRNA,以确定干扰效果并解析其功能。合成花绒寄甲成虫目标基因mthl保守序列的dsRNA,分别注射0.3、1.2μg/μL和4.8μg/μL的dsRNA,利用实时荧光定量PCR技术检测目标基因的表达量,记录处理组虫体的体重、存活率和雌虫产卵情况。从花绒寄甲转录组数据库中分别鉴定出Mthlike11、Mth-like 2-4、Mth-like2-5基因。注射目标基因Mth-like 2-5的长度约400bp的dsRNA,对目标基因表达干扰效果显著,但干扰过程中雌雄虫均具有应激表达反应。其中0.3μg/μL低浓度dsRNA对目标基因干扰效果差,应激高表达反应明显;1.2μg/μL中等浓度dsRNA对目标基因有显著的干扰效果,应激高表达反应弱、引起的体重下降和死亡率较小,雌虫产卵期推迟;4.8μg/μL高浓度dsRNA干扰效果显著,引起的体重下降和死亡率较高,雌虫难以产卵。注射Mth-like 2-5dsRNA可影响Mth-like2-5基因的正常表达和转录,随dsRNA浓度增大和处理后时间的延长,目标基因表达量、雌雄成虫体重明显降低,死亡率增高,雌成虫产卵期推迟或难以产卵。
The high-expression mthl gene was screened and the target gene dsRNA was injected to determine the interference effect and analyze its function in Dastarcus helophoroides.Synthetic mthl gene target gene conservative sequence of dsRNA was injected in the adult of D.helophoroides with dosages of 0.3,1.2μg/μL and 4.8μg/μL dsRNA respectively.Real-time qPCR technique was used to detect the expression of target genes.The body weight of treatment group,survival rate,and the conditions of female egglaying were recorded.The results showed that,Mth-like 11,Mth-like 2-4 and Mth-like 2-5 genes were identified from the transcriptome database of D.helophoroides.The length of the target gene Mth-like 2-5 was about 400 bp dsRNA,which had a significant effect on the expression of the target gene.Low concentration dsRNA with 0.3μg/μL had poor interference effect on target genes and obvious high expression response to stress.Medium concentration dsRNA(1.2μg/μL)had significant interference effect on target genes with weak high expression response to stress,decreased weight and low mortality,and delayed spawning period of female.High concentration dsRNA(4.8μg/μL)had significant interference effect,resulting in weight loss and high mortality,and female was difficult to lay eggs.Injected Mth-like 2-5 dsRNA could affect the normal expression and transcription of the Mth-like 2-5 genes;with the increase of concentration of dsRNA and the extension of processing time,the target gene expression,male and female adult weight decreased,mortality rate increased,female adult spawning period delayed or was difficult to lay eggs.
The high-expression mthl gene was screened and the target gene dsRNA was injected to determine the interference effect and analyze its function in Dastarcus helophoroides.Synthetic mthl gene target gene conservative sequence of dsRNA was injected in the adult of D.helophoroides with dosages of 0.3,1.2μg/μL and 4.8μg/μL dsRNA respectively.Real-time qPCR technique was used to detect the expression of target genes.The body weight of treatment group,survival rate,and the conditions of female egglaying were recorded.The results showed that,Mth-like 11,Mth-like 2-4 and Mth-like 2-5 genes were identified from the transcriptome database of D.helophoroides.The length of the target gene Mth-like 2-5 was about 400 bp dsRNA,which had a significant effect on the expression of the target gene.Low concentration dsRNA with 0.3μg/μL had poor interference effect on target genes and obvious high expression response to stress.Medium concentration dsRNA(1.2μg/μL)had significant interference effect on target genes with weak high expression response to stress,decreased weight and low mortality,and delayed spawning period of female.High concentration dsRNA(4.8μg/μL)had significant interference effect,resulting in weight loss and high mortality,and female was difficult to lay eggs.Injected Mth-like 2-5 dsRNA could affect the normal expression and transcription of the Mth-like 2-5 genes;with the increase of concentration of dsRNA and the extension of processing time,the target gene expression,male and female adult weight decreased,mortality rate increased,female adult spawning period delayed or was difficult to lay eggs.
引文
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[2]WALLENFANG M R,NAYAK R,DINARDO S.Dynamics of the male germline stem cell population during aging of Drosophila melanogaster[J].Aging Cell,2006,5(4):297-304.
[3]PETROSYAN A,HSIEH I H,SABERI K.Age-dependent stability of sensorimotor functions in the life-extended Drosophila,mutant methuselah[J].Behavior Genetics,2007,37(4):585-594.
[4]LIN Y J,SEROUDE L,BENZER S.Extended life-span and stress resistance in the Drosophila mutant methuselah[J].Science,1998,282(5390):943-946.
[5]SONG W,RANJAN R,DAWSON-SCULLY K,et al.Presynaptic regulation of neurotransmission in Drosophila by the g protein-coupled receptor methuselah[J].Neuron,2002,36(1):105.
[6]PATEL M V,HALLAL D A,JONES J W,et al.Dramatic expansion and developmental expression diversification of the methuselah gene family during recent Drosophila evolution[J].Journal of Experimental Zoology Part B Molecular&Developmental Evolution,2012,318(5):368.
[7]LI C,ZHANG Y,YUN X,et al.Methuselah-like genes affect development,stress resistance,lifespan and reproduction in Tribolium castaneum[J].Insect Molecular Biology,2014,23(5):587.
[8]LI C,CHEN M,SANG M,et al.Comparative genomic analysis and evolution of family-B G protein-coupled receptors from six model insect species[J].Gene,2013,519(1):1.
[9]DE M A,JONES J W,FRIEDRICH M.Methuselah/Methuselah-like G protein-coupled receptors constitute an ancient metazoan gene family[J].Scientific Reports,2016,6(21801).
[10]FIRE A,XU S,MONTGOMERY M K,et al.Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J].Nature,1998,391(6669):806.
[11]BELLES X.Beyond drosophila:RNAi in vivo and functional genomics in insects[J].Annual Review of Entomology,2010,55(55):111.
[12]高晓飞,包晶晶,陈勇军,等.利用RNA干扰机制抑制猪繁殖与呼吸综合征病毒的增殖[J].中国病毒学,2006,21(3):226-230.GAO X F,BAO J J,CHEN Y J,et al.RNA interference inhibits replication of porcine reproductive and respiratory syndrome virus[J].Virologica Sinica,2006,21(3):226-230.(in Chinese)
[13]岳建苏.桔小实蝇钠离子通道基因和P450基因RNAi载体构建及对柑橘的转化[D].成都:西南大学,2014.
[14]BAUM JA,BOGAERT T,CLINTON W,et al.Control of coleopteran insect pests through RNA interference[J].Nat.Biotech.,2007,25(11):1322-1326.
[15]MAO Y B,CAI W J,WANG J W,et al.Silencing a cotton bollworm P450monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol[J].Nature Biotechnology,2007,25(11):1307-1313.
[16]HUNTER W,ELLIS J,ENGELSDORP D,et al.Large-scale field application of RNAi technology reducing israeli acute paralysis virus disease in honey bees(Apis mellifera,Hymenoptera:Apidae)[J].PLo S Pathog,2010,6(12):e1001160.
[17]MAO Y B,TAO X Y,XUE X Y,et al.Cotton plants expressing CYP6AE14,double-stranded RNA show enhanced resistance to bollworms[J].Transgenic Research,2011,20(3):665-673(9).
[18]秦瑞豪,李孟楼,张翌楠,等.花绒寄甲防治松褐天牛最佳释放条件的筛选[J].西北农林科技大学学报:自然科学版,2012,40(2):92-96.QIN R H,LI M L,ZHANG Y N,et al.Screening for the release conditions of Dastarcus helophoroides to control Monochamus alternatus[J].Journal of Northwest A&F University:Nat.Sci.Edi.,2012,40(2):92-96.(in Chinese)
[19]雷琼,李孟楼,杨忠歧.花绒坚甲的生物学特性研究[J].西北农林科技大学学报:自然科学版,2003,31(2):62-66.LEI Q,LI M L,YANG Z Q.A study on biological feature of Dastarcus longulus[J].Journal of Northwest Sci-Tech University of A&F:Nat.Sci.Ed.,2003,31(2):62-66.(in Chinese)
[20]李孟楼,李有忠,雷琼,等.释放花绒寄甲卵对光肩星天牛幼虫的防治效果[J].林业科学,2009,45(4):78-82.LI M L,LI Y Z,LEI Q,et al.Biocontrol of Asian longhorned bettle larva by relaasing eggs of Dastarcus helophoroides(Coleoptera:Bothrideridea)[J]Scientia Silvae Sinicae,2009,45(4):78-82.(in Chinese)
[21]李孟楼,王培新,马峰,等.花绒坚甲对光肩星天牛的寄生效果研究[J].西北农林科技大学学报:自然科学版,2007,35(6):152-162.LI M L,WANG P X,MA F,et al.Study on the parasitic efficiency of Dastarcus helophoroides on Anoplophora glabripennis[J].Journal of Northwest A&F University:Natural Science Edition,2007,35(6):152-162.(in Chinese)
[22]WANG H D,LI F F,HE C,et al.Molecular cloning and sequence analysis of novel Cytochrome P450cDNA fragments from Dastarcus helophoroides[J].Journal of Insect Science,2014,14(28):415-422.
[23]王海东.花绒寄甲P450基因的分子克隆与表达[D].杨陵:西北农林科技大学,2013.
[24]郭瑞坚.花绒寄甲抗氧化酶基因表达研究[D].杨陵:西北农林科技大学,2015.
[25]王化鹏,郝春凤,张正青,等.花绒寄甲methuselah-like基因的鉴定和表达[J].东北林业大学学报,2016,44(7):102-107.WANG H P,HAO C F,ZHANG Z Q,et al.Identification and expression of Methuselah-like gene in Dastarcus helophoroides[J].Journal of Northeast Forestry University,2016,44(7):102-107.(in Chinese)
[26]ZHANG W,SONG W,ZHANG Z,et al.Transcriptome analysis of Dastarcus helophoroides(Coleoptera:Bothrideridae)using illumina hiseq sequencing[J].Plos One,2014,9(9):e100673.
[27]ZHANG Z,WANG H,HAO C,et al.Identification,characterization and expression of Methuselah-like genes in Dastarcus helophoroides(Coleoptera:Bothrideridae)[J].Genes,2016,7(10):91.
[28]田宏刚.利用饲喂法RNAi研究昆虫生长发育相关基因的功能[D].中山:中山大学,2009.
[29]BULOW S,ZAMMATTEO N,REMACLE J.Gene-related RNAi transfection method:EP,US20050059019[P].2005.
[30]STPHANIE J P,LE T G,JOL B,et al.Gene knockdown by RNAi in the pea aphid Acyrthosiphon pisum[J].Bmc Biotechnology,2007,7(1):63.
[31]张燕.利用RNA干扰技术研究豌豆蚜唾液腺基因的功能[D].济南:山东农业大学,2012.
[32]CLAUDE R,CHRISTOPHIDES G K,JOL B,et al.Gene knockdown by RNAi in the pea aphid Acyrthosiphon pisum[J].Bmc Biotechnology,2007,7(1):63.
[33]宋旺,王小纪,郭瑞坚,等.花绒寄甲荧光定量PCR分析中内参基因的选择[J].西北农业学报,2015,24(2):156-161.SONG W,WANG X J,GUO R J,et al.Selection of reference genes for quantitative real-time PCR normalization in Dastarcus helophoroides[J].Acta Agriculturae Boreali-Occidentalis Sinica,2015,24(2):156-161.(in Chinese)
[34]SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))Method[J].Methods,2001,25(4):402-408.
[35]ZHANG R,GAO G,CHEN H.Silencing of the olfactory coreceptor gene in Dendroctonus armandi leads to EAG response declining to major host volatiles[J].Scientific Reports,2016,6:231-236.
[36]PAABY A B,SCHMIDT P S.Functional significance of allelic variation at methuselah,an aging gene in Drosophila[J].Plos One,2008,3(4):e1987.
[37]NICHOLSON R H,NICHOLSON A W.Molecular characterization of a mouse cDNA encoding dicer,a ribonuclease III ortholog involved in RNA interference.[J].Mammalian Genome,2002,13(2):67.
[38]WHYARD S,SINGH A D,WONG S.Ingested double-stranded RNAs can act as species-specific insecticides[J].Insect Biochemistry&Molecular Biology,2009,39(11):824-832.
[39]SALEH M C,RIJ R P V,HEKELE A,et al.The endocytic pathway mediates cell entry of dsRNA to induce RNAi silencing[J].Nature Cell Biology,2006,8(8):793-802.
[40]ZHUANG S,KELO L,NARDI J B,et al.Multipleα,subunits of integrin are involved in cell-mediated responses of the manduca,immune system[J].Developmental&Comparative Immunology,2008,32(4):365-379.
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