二化螟响应Bt杀虫蛋白Cry1Ca的小RNA转录组分析  

Transcriptome analysis of small RNAs in response to Bt insecticidal protein Cry1Ca in Asiatic rice borer Chilo suppressalis

作　　者：李真真 姚焯天 马康生 马伟华[1,2] 张伟[2,3] Li Zhenzhen;Yao Zhuotian;Ma Kangsheng;Ma Weihua;Zhang Wei(College of Plant Science and Technology,Huazhong Agricultural University,Wuhan 430070,Hubei Province,China;National Key Laboratory of Crop Genetic Improvement,Huazhong Agricultural University,Wuhan 430070,Hubei Province,China;College of Life Science and Technology,Huazhong Agricultural University,Wuhan 430070,Hubei Province,China)

机构地区：[1]华中农业大学植物科学技术学院,武汉430070 [2]华中农业大学作物遗传改良国家重点实验室,武汉430070 [3]华中农业大学生命科学技术学院,武汉430070

出　　处：《植物保护学报》2021年第6期1370-1379,共10页Journal of Plant Protection

基　　金：国家自然科学基金(31871957)。

摘　　要：为有效防控二化螟Chilo suppressalis,采用Illumina Solexa二代测序技术对Bt杀虫蛋白Cry1Ca处理后二化螟中肠内的小RNA序列进行测序,并对其差异表达谱进行注释与分析,并用实时荧光定量PCR技术对测序结果进行验证。结果显示,Cry1Ca对二化螟的亚致死量LC30为0.061μg/g。Cry1Ca处理和对照处理后转录组中小RNA分布峰值均为22 nt,被注释的已知小RNA比例很低,分别为7.2%和7.9%。Cry1Ca处理后,二化螟中肠内miRNA、snoRNA、snRNA、rRNA、piRNA数量均少于对照。与对照相比,Cry1Ca处理后二化螟中肠内共有358个小RNA表达上调,747个小RNA表达下调,其中有25个已知miRNA的表达量较高,其中3个已知miRNA表达量显著上调,22个已知miRNA表达量显著下调;在新预测的novel miRNA中,有23个上调表达,43个下调表达。KEGG分析结果显示,Cry1Ca诱导的miRNA影响了丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)通路,这个通路包含17个靶基因,其中11个靶基因可被多个miRNA调控,每个靶基因可被59~665个miRNA靶向调控,参与靶向调控miRNA的相对表达量变化倍数介于0.01倍~23289倍之间,表明miRNA在调控二化螟防御Bt杀虫蛋白Cry1Ca方面发挥着重要作用。实时荧光定量PCR的结果与测序结果一致,表明测序结果正确。In order to effectively control Asiatic rice borer Chilo suppressalis,the midgut small RNA transcriptome was sequenced by using high-throughput Illumina Solexa sequencing technology,which was compared and analyzed between the group fed with Bt insecticidal protein Cry1Ca and the control group without Cry1Ca treatment.The sequencing results were verified by quantitative real-time PCR.The results showed that the 30%lethal concentration(LC30)of Cry1Ca against C.suppressalis was 0.061μg/g.Most microRNA sequences were 22 nt in length.In the small RNA database,the proportions of annotated sequences were very low in both treatment and control groups,only 7.2%and 7.9%,respectively.After treated with Cry1Ca,the numbers of miRNA,snoRNA,snRNA,rRNA and piRNA were decreased.Compared with the control,358 miRNAs were up-regulated and 747 miRNAs were down-regulated in the treatment group.Twenty-five known miRNAs with the highest transcriptional level were selected for further analysis.The transcriptional levels of these 25 miRNAs were significantly different between the Cry1Ca-treated group and the control,among which,three miRNAs were significantly up-regulated and 22 were significantly down-regulated.Among the novel miRNAs,23 were upregulated and 43 were down-regulated.KEGG analysis indicated that miRNAs induced by Cry1Ca were involved in the mitogen-activated protein kinase pathway.This pathway contained 17 target genes,of which 11 were regulated by multiple miRNAs.The numbers of miRNAs regulating each target gene ranged from 59 to 665,and the relative expression levels of miRNAs varied from 0.01 times to 23289 times.These results suggested that miRNAs played an important role in regulation of defense against Bt insecticidal protein Cry1Ca in C.suppressalis.The results of quantitative real-time PCR were consistent with the sequencing results,suggesting an accurate sequencing result.

关 键 词：二化螟 杀虫蛋白Cry1Ca 测序 MIRNA 转录组 

分 类 号：S435.112.1[农业科学—农业昆虫与害虫防治]