二化螟Halloween家族基因Cyp302a1和Cyp315a1的克隆与时空表达谱分析

Molecular cloning of Cyp302a1 and Cyp315a1 and expression profiles of Halloween genes in Chilo suppressalis

[目的]Halloween家族基因参与合成蜕皮激素,调控昆虫发育、繁殖等重要生命过程。二化螟(Chilo suppressalis)是水稻的重要害虫,明确二化螟Halloween家族基因完整序列及表达谱特征,对后续开展二化螟生长发育等调控机制研究具有重要作用。[方法]利用反转录PCR(RT-PCR)和快速扩增cDNA末端(RACE)技术克隆二化螟Halloween家族基因成员Cyp302a1和Cyp315a1的完整转录本序列;通过多重比对分析序列保守结构特征;利用MEGA 7.0软件构建了分子系统进化树;利用实时荧光定量PCR(RT-qPCR)分析基因的时空表达谱。[结果]克隆获得二化螟Halloween家族2个基因CsCyp302a1和CsCyp315a1的全长cDNA序列。各基因均含有5个序列保守结构域:WxxxR(Helix-C)、GxE/DTT/S(Helix-I)、ExxR(Helix-K)、PxxFxPE/DRF(PERF motif)和PFxxGxRxCxG/A(heme-binding domain),并且与各自的基因家族成员聚为一支。各基因在不同发育时期和不同组织内的表达水平存在差异,其表达模式在不同基因间也存在差异。二化螟Cyp307a1相对表达量最高,Cyp315a1相对表达量最低。Cyp307a1在头部表达量高;Cyp306a1在中肠表达量高;Cyp302a1和Cyp315a1在头部和表皮中表达量高;Cyp314a1在脂肪体和表皮中表达量高。[结论]明确了二化螟Halloween家族2个基因序列特征和全部5个主要基因的时空表达谱特征。

[Objectives]Halloween genes are involved in the biosynthesis of ecdysone.The ecdysone plays an important role in regulation of the development and reproduction in insects.The stem rice borer,Chilo suppressalis,is one of the most destructive pests in rice plant.It is very important to understanding the characterizations and expression profiles of Halloween genes of C.suppressalis,which is helpful for uncovering the growth and development regulation mechanism in C.suppressalis.[Methods]The complete transcript sequence of Cyp302a1 and Cyp315a1,two members of Halloween genes in C.suppressalis were cloned by using reverse transcriptase polymerase chain reaction(RT-PCR)and rapid amplification of cDNA ends(RACE)methods.The conserved structures of the sequences were analyzed by multiple alignments.The molecular phylogenetic tree was constructed by MEGA 7.0 software.The expression profiles of Halloween genes at different developmental stages and in different tissues were detected by real-time quantitative PCR(RT-qPCR).[Results]The Halloween gene members,CsCyp302a1 and CsCyp315a1,were cloned from C.suppressalis.Each gene contained its own five conserved domains,WxxxR(Helix-C),GxE/DTT/S(Helix-I),ExxR(Helix-K),PxxFxPE/DRF(PERF motif),PFxxGxRxCxG/A(heme-binding domain).Both CsCyp302a1 and CsCyp315a1 clustered into a clade with their own family members.There were differences in the expression levels of each gene in different developmental stages and tissues.The expression patterns had differences between each gene.The relative expression level of Cyp307a1 in C.suppressalis was the highest and that of Cyp315a1 was the lowest.Besides,Cyp307a1 was highly expressed in the head,the expression level of Cyp306a1 was higher in the midgut,Cyp302a1 and Cyp315a1 were highly expressed in the head and integument,and the expression level of Cyp314a1 was higher in the fat body and integument.[Conclusions]The sequence characterizations of CsCyp302a1 and CsCyp315a1 and the temporal and spatial expression profiles of five Halloween genes of C.suppressalis were determined.