白背飞虱几丁质合成酶1基因的结构及特性研究

doi:10.13560/j.cnki.biotech.bull.1985.2017-0569

摘要/Abstract

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（SfCHS1）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SfCHS1基因的结构特性,采用定量PCR技术研究SfCHS1基因的时空表达特性,最后利用显微注射RNAi方法研究SfCHS1基因的RNAi效果。通过转录组测序获得的SfCHS1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 kD,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SfCHS1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,SfCHS1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,SfCHS1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,SfCHS1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SfCHS1基因的转录水平,并导致白背飞虱的死亡。研究结果说明,SfCHS1基因具有两个可变外显子结构,SfCHS1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SfCHS1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。

关键词: 白背飞虱, 几丁质合成酶1, 可变剪切, 表达特性, RNA干扰

Abstract: 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（SfCHS1）,transcriptome sequencing combined with PCR amplification technology were employed to study the structure characteristic of SfCHS1 gene,then quantitative PCR technology to study the spatio-temporal expression characteristics of of SfCHS1 gene,and the method of micro injection of RNAi to study the RNAi efficiency of SfCHS1 gene. The SfCHS1 cDNA contained an open reading frame of 4 719 bp and encoded 1 572 amino acids with the putative molecular weight of 180.6 kD. Homology analysis indicated that SfCHS1 included 6 N-glycosylation sites and 16 transmembrane helixes. In addition,2 transcripts（CHS1a and CHS1b）resulting from exclusively alternative splicing were identified in S. furcifera by PCR amplification and sequencing.Phylogenetic analysis suggested that SfCHS1 shared 97% identity with the known CHS1（Laodelphgax striatellus and Nilaparvata lugens）. The analysis of temporal expression characteristics showed that SfCHS1 was most highly expressed in the first day of every instar. SfCHS1 was mainly expressed in epidermis and then in trachea,a little in gut. Efficient silencing of the SfCHS1 gene through micro injection of RNAi led to rapid and significant reduction levels of SfCHS1 mRNA and resulted in S. furcifera deaths. This study suggests that SfCHS1cDNA has two variants（SfCHS1a and SfCHS1b）,and SfCHS1 is highly expressed in chitin biosynthesis stages and tissues. RNAi injection-based significantly reduces the expression level of SfCHS1 gene and causes their high-rate deaths.

Key words: Sogota furcifera, chitin synthase 1, alternative splicing, expression character, RNAi

陈静, 张道伟, 钱正敏. 白背飞虱几丁质合成酶1基因的结构及特性研究[J]. 生物技术通报, 2018, 34(1): 195-201.

CHEN Jing, ZHANG Dao-wei, QIAN Zheng-min. Structure and Characteristics Analysis of Chitin Synthase 1 from Sogota furcifera[J]. Biotechnology Bulletin, 2018, 34(1): 195-201.

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