白背飞虱几丁质合成酶1基因的结构及特性研究
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摘要
几丁质合成酶1(CHS1)是昆虫几丁质合成的关键酶,在昆虫几丁质合成的组织中发挥着重要作用。为探索白背飞虱Sogota furcifera几丁质合成酶1(Sf CHS1)的基因结构特征及其功能,利用转录组测序结合PCR扩增技术,研究了Sf CHS1基因的结构特性,采用定量PCR技术研究Sf CHS1基因的时空表达特性,最后利用显微注射RNAi方法研究Sf CHS1基因的RNAi效果。通过转录组测序获得的Sf CHS1基因开放阅读框为4 719 bp,编码1572个氨基酸,预测蛋白分子量为180.6 k D,预测有6个糖基化位点和16个跨膜螺旋。PCR扩增及测序结果表明Sf CHS1基因存在两个可变剪切,产生两个转录本(CHS1a和CHS1b)。通过系统进化树分析,Sf CHS1和灰飞虱Laodelphgax striatellus及褐飞虱Nilaparvata lugens的CHS1同源性最高,为97%。时间表达特异性分析结果表明,Sf CHS1基因在4龄期第1天,5龄期第1天,成虫第1天即几丁质合成时的表达量最高。组织特异性检测结果表明,Sf CHS1基因主要在白背飞虱的表皮中表达,其次为气管,在肠中也有少量表达。显微注射RNAi的结果表明该方法能显著降低Sf CHS1基因的转录水平,并导致白背飞虱的死亡。研究结果说明,Sf CHS1基因具有两个可变外显子结构,Sf CHS1基因在几丁质合成阶段及几丁质含量高的组织表达量较高,沉默Sf CHS1基因的表达会对白背飞虱产生致死的表型,出现较高的死亡率。
Chitin synthase 1(CHS1)is a crucial enzyme in chitin synthesis and plays a key role in the chitin formation in insect tissues.In order to explore the gene structure and function of Sogota furcifera chitin synthase 1(Sf CHS1),transcriptome sequencing combined withPCR amplification technology were employed to study the structure characteristic of Sf CHS1 gene,then quantitative PCR technology to studythe spatio-temporal expression characteristics of of Sf CHS1 gene,and the method of micro injection of RNAi to study the RNAi efficiency ofSf CHS1 gene. The Sf CHS1 c DNA contained an open reading frame of 4 719 bp and encoded 1 572 amino acids with the putative molecularweight of 180.6 k D. Homology analysis indicated that Sf CHS1 included 6 N-glycosylation sites and 16 transmembrane helixes. In addition,2 transcripts(CHS1 a and CHS1 b)resulting from exclusively alternative splicing were identified in S. furcifera by PCR amplification andsequencing. Phylogenetic analysis suggested that Sf CHS1 shared 97% identity with the known CHS1(Laodelphgax striatellus and Nilaparvata lugens). The analysis of temporal expression characteristics showed that Sf CHS1 was most highly expressed in the first day of every instar.Sf CHS1 was mainly expressed in epidermis and then in trachea,a little in gut. Efficient silencing of the Sf CHS1 gene through micro injection ofRNAi led to rapid and significant reduction levels of Sf CHS1 m RNA and resulted in S. furcifera deaths. This study suggests that Sf CHS1 c DNA has two variants(Sf CHS1 a and Sf CHS1 b),and Sf CHS1 is highly expressed in chitin biosynthesis stages and tissues. RNAi injection-basedsignificantly reduces the expression level of Sf CHS1 gene and causes their high-rate deaths.
Chitin synthase 1(CHS1)is a crucial enzyme in chitin synthesis and plays a key role in the chitin formation in insect tissues.In order to explore the gene structure and function of Sogota furcifera chitin synthase 1(Sf CHS1),transcriptome sequencing combined withPCR amplification technology were employed to study the structure characteristic of Sf CHS1 gene,then quantitative PCR technology to studythe spatio-temporal expression characteristics of of Sf CHS1 gene,and the method of micro injection of RNAi to study the RNAi efficiency ofSf CHS1 gene. The Sf CHS1 c DNA contained an open reading frame of 4 719 bp and encoded 1 572 amino acids with the putative molecularweight of 180.6 k D. Homology analysis indicated that Sf CHS1 included 6 N-glycosylation sites and 16 transmembrane helixes. In addition,2 transcripts(CHS1 a and CHS1 b)resulting from exclusively alternative splicing were identified in S. furcifera by PCR amplification andsequencing. Phylogenetic analysis suggested that Sf CHS1 shared 97% identity with the known CHS1(Laodelphgax striatellus and Nilaparvata lugens). The analysis of temporal expression characteristics showed that Sf CHS1 was most highly expressed in the first day of every instar.Sf CHS1 was mainly expressed in epidermis and then in trachea,a little in gut. Efficient silencing of the Sf CHS1 gene through micro injection ofRNAi led to rapid and significant reduction levels of Sf CHS1 m RNA and resulted in S. furcifera deaths. This study suggests that Sf CHS1 c DNA has two variants(Sf CHS1 a and Sf CHS1 b),and Sf CHS1 is highly expressed in chitin biosynthesis stages and tissues. RNAi injection-basedsignificantly reduces the expression level of Sf CHS1 gene and causes their high-rate deaths.
引文
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[13]Wang Y,Fan HW,Huang HJ,et al.Chitin synthase 1 gene andits two alternative splicing variants from two sap-sucking insects,Nilaparvata lugens and Laodelphax striatellus(Hemiptera:Delphacidae)[J].Insect Biochem Mol Biol,2012,42(9):637-646.
[14]Li T,Chen J,Fan X,et al.Micro RNA and ds RNA targeting chitinsynthase A reveal a great potential for pest management of thehemipteran insect Nilaparvata lugens[J].Pest ManagementScience.2017,73(7):1529-1537.
[15]Yang WJ,Xu KK,Cong L,et al.Identification,m RNA expression,and functional analysis of chitin synthase 1 gene and its twoalternative splicing variants in oriental fruit fly,Bactrocera dorsalis[J].Int J Biol Sci,2013,9(4):331-342.
[16]Zhang X,Zhu KY.Biochemical characterization of chitin synthaseactivity and inhibition in the African malaria mosquito,Anopheles gambiae[J].Insect Science,2013,20(2):158-166.
[17]Zhuo W,Fang Y,Kong L,et al.Chitin synthase A:a novelepidermal development regulation gene in the larvae of Bombyx mori[J].Mol Biol Rep,2014,41(7):4177-4186.
[18]Chen J,Zhang DW,Yao Q,et al.Feeding-based RNA interferenceof a trehalose phosphate synthase gene in the brown planthopper,Nilaparvata lugens[J].Insect Mol Biol,2010,19(6):777-786.
[19]Zha W,Peng X,Chen R,et al Knockdown of midgut genes byds RNA-transgenic plant-mediated RNA interference in thehemipteran insect Nilaparvata lugens[J].PLo S One,2011,6(5):e20504.
[2]Merzendorfer H.The cellular basis of chitin synthesis in fungiand insects:common principles and differences[J].EuropeanJournal of Cell Biology,2011,90(9):759.
[3]Merzendorfer H.Insect chitin synthases:a review[J].Journal ofComparative Physiology B,2006,176(1):1-15.
[4]Au-Young J,Robbins PW.Isolation of a chitin synthase gene(CHS1)from Candida albicans by expression in Saccharomyces cerevisiae[J].Molecular Microbiology,1990,4(2):197-207.
[5]Yarden O,Yanofsky C.Chitin synthase plays a major role in cell wallbiogenesis in Neurospora crassa[J].Genes Dev,1991,5:2420-2430.
[6]Arakane Y,Zhu YC,Kramer KJ,et al.Characterization of two chitinsynthase genes of the red flour beetle,Tribolium castaneum,andalternate exon usage in one of the genes during development[J].Insect Biochemistry and Molecular Biology,2004,34:291-304.
[7]Arakane Y,Muthukrishnan S,Kramer KJ,et al.The Tribolium chitinsynthase genes Tc CHS1 and Tc CHS2 are specialized for synthesisof epidermal cuticle and midgut peritrophic matrix[J].InsectMolecular Biology,2005,14:453-463.
[8]Tellam R,Vuocolo T,Johnson SE,et al.Insect chitin synthase c DNAsequence,gene organization and expression[J].European Journalof Biochemistry,2000,267:6025-6043.
[9]Zhang J,Liu X,Zhang J,et al.Silencing of two alternative splicingderived m RNA variants of chitin synthase 1 gene by RNAi is lethalto the oriental migratory locust,Locusta migratoria manilensis(Meyen)[J].Insect Biochem Mol Biol,2010,40(11):824-833.
[10]沈君辉,尚金梅,刘光杰.中国的白背飞虱研究概况[J].中国水稻科学,2003,17:7-22.
[11]周国辉,温锦君,蔡德江等.呼肠孤病毒科斐济病毒属一新种:南方水稻黑条矮缩病毒[J].科学通报,2008,53(20):2500-2508.
[12]翟保平,周国辉,陶小荣等.稻飞虱暴发与南方水稻黑条矮缩病流行的宏观规律和微观机制[J].应用昆虫学报,2011,48(3):480-487.
[13]Wang Y,Fan HW,Huang HJ,et al.Chitin synthase 1 gene andits two alternative splicing variants from two sap-sucking insects,Nilaparvata lugens and Laodelphax striatellus(Hemiptera:Delphacidae)[J].Insect Biochem Mol Biol,2012,42(9):637-646.
[14]Li T,Chen J,Fan X,et al.Micro RNA and ds RNA targeting chitinsynthase A reveal a great potential for pest management of thehemipteran insect Nilaparvata lugens[J].Pest ManagementScience.2017,73(7):1529-1537.
[15]Yang WJ,Xu KK,Cong L,et al.Identification,m RNA expression,and functional analysis of chitin synthase 1 gene and its twoalternative splicing variants in oriental fruit fly,Bactrocera dorsalis[J].Int J Biol Sci,2013,9(4):331-342.
[16]Zhang X,Zhu KY.Biochemical characterization of chitin synthaseactivity and inhibition in the African malaria mosquito,Anopheles gambiae[J].Insect Science,2013,20(2):158-166.
[17]Zhuo W,Fang Y,Kong L,et al.Chitin synthase A:a novelepidermal development regulation gene in the larvae of Bombyx mori[J].Mol Biol Rep,2014,41(7):4177-4186.
[18]Chen J,Zhang DW,Yao Q,et al.Feeding-based RNA interferenceof a trehalose phosphate synthase gene in the brown planthopper,Nilaparvata lugens[J].Insect Mol Biol,2010,19(6):777-786.
[19]Zha W,Peng X,Chen R,et al Knockdown of midgut genes byds RNA-transgenic plant-mediated RNA interference in thehemipteran insect Nilaparvata lugens[J].PLo S One,2011,6(5):e20504.