美国白蛾HcSID-1基因的克隆、表达模式分析及其在幼虫中的功能验证

doi:10.11707/j.1001-7488.20191006

摘要/Abstract

摘要：

目的: 克隆美国白蛾系统性RNA干扰缺失基因序列，对该基因进行序列特征分析和时空表达模式检测，并研究其在美国白蛾幼虫系统性RNA干扰中的功能。方法: 通过RT-PCR和RACE技术从美国白蛾幼虫中进行基因克隆，并进行生物信息学分析；利用qPCR技术检测该基因在美国白蛾不同发育阶段和幼虫不同组织中的相对表达量；利用RNAi和qPCR技术验证外源dsRNA对该基因的干扰效率，以及干扰该基因表达后对其他靶标基因干扰效率的影响。结果: 从美国白蛾幼虫中克隆得到一个美国白蛾系统性RNA干扰缺失基因并命名为HcSID-1（GenBank登陆号：MG696730）。该基因全长2 761 bp，开放阅读框（ORF）2 613 bp，编码870个氨基酸，预测蛋白分子量为97.08 kD，理论等电点（pI）6.81，有1个19 aa的信号肽。氨基酸序列分析表明，HcSID-1蛋白有1个长N末端（308 aa），序列中后段309—855位氨基酸之间具有典型的11个跨膜结构域；系统进化分析表明HcSID-1与鳞翅目昆虫同源性较高，和家蚕的BmSID-3亲缘关系最近；时空表达分析表明，HcSID-1在美国白蛾各发育阶段和幼虫各组织中均有表达，在幼虫中肠组织表达量最高。注射HcSID-1 dsRNA可显著降低该基因在转录水平的相对表达量，且该基因的下调能降低HcChi dsRNA在美国白蛾幼虫中的RNA干扰效率。结论: 获得具有典型家族特征的美国白蛾SID-1基因序列，该基因的下调能够影响其他靶标基因dsRNA的RNAi效率，表明该基因可能具有与其他SID基因在系统性RNAi过程中相同的功能。

关键词: 美国白蛾, 系统性RNA干扰缺失基因, 克隆表达, RNA干扰

Abstract:

Objective: SID gene encodes a transmembrane channel protein which participates in the transmembrane transport of RNAi signal such as dsRNA in many organisms. This study aimed to clone the SID-1 gene from Hyphantria cunea larva, and to analyze the gene sequence and expression profiles. Moreover, the gene function involved in systemic RNAi in H. cunea was explored. Method: The cDNA sequence of HcSID-1 gene was cloned from H. cunea larva using RT-PCR and RACE technique, and the putative amino acid sequence was analyzed by bioinformatics method. The temporal and spatial expression levels were detected by qPCR. The interference efficiency of exogenous dsRNA to HcSID-1 was validated by RNAi and qPCR technique. Furthermore, the interference efficiency to other target genes were detected when HcSID-1 expression was interfered simultaneously.Result: A systemic RNA interference defective (SID)gene named HcSID-1 (GenBank accession No.:MG696730)was cloned from H. cunea larva, and was 2 761 bp in length. The gene contained a 2 613 bp open reading frame (ORF), encoding 870 amino acid residues. The predicted molecular weight of the putative protein was 97.08 kD with the isoelectric point (pI)of 6.81. The protein contained a signal peptide which was 19 aa in length. Amino acid sequence analysis showed that the HcSID-1 protein had a long N-terminal with 308 aa, and 11 typical transmembrane domains between 309-855 amino acid residues. Phylogenetic analysis indicated that HcSID-1 had high similarity to SID genes of lepidopteron insects, and had the closest relationship with BmSID-3 of Bombyx mori. Temporal and spatial expression analysis showed that HcSID-1 expressed in all tested developmental stages and larva tissues, with a high expression level in the larva midgut. The expression level of HcSID-1 was able to be significantly decreased by HcSID-1 dsRNA injection in H. cunea larva. Meanwhile, the sensitivity to dsRNA of HcChi gene was reduced with the low expression level of HcSID-1 in H. cunea larva. Objective: A HcSID-1 gene obtained from H. cunea had the typical SID family characteristics. The down-regulation of HcSID-1 could affect RNA interference efficiency to other target genes, suggesting that this gene may have the same function similar to other SID genes in the systematic RNAi process.

Key words: Hyphantria cunea, SID-1, gene expression, RNA interference

中图分类号:

S758

王越,张苏芳,徐瑶,刘福,孔祥波,张真. 美国白蛾HcSID-1基因的克隆、表达模式分析及其在幼虫中的功能验证[J]. 林业科学, 2019, 55(10): 48-56.

Yue Wang,Sufang Zhang,Yao Xu,Fu Liu,Xiangbo Kong,Zhen Zhang. Cloning and Expression Profiles of HcSID-1 Gene and Its Function Verification in Hyphantria cunea Larvae[J]. Scientia Silvae Sinicae, 2019, 55(10): 48-56.

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引物 Primer name	引物序列 Primer sequence(5′ to 3′)	用途 Purpose
PxylSID-1F	GGCGACCAGGACMTGTGCTACTA	扩增HcSID-1初始片段Amplification of initial fragement of HcSID-1
PxylSID-1R	AGYACWGCYATCACGTACAT
3′GSP	GATTACGCCAAGCTTCGGCTCGCTCTCCGACTTCAAC	3′-RACE
5′GSP1	TAATAAGTACCCGACG	5′-RACE
5′GSP2	CGTGGTTGAAGTCGGAGA
5′GSP3	TGCGCGCACAGGAAGTTG
dsSID-1 F	CGTCGCAGGAAGATGAGGA	dsRNA合成 dsRNA synthesis
dsSID-1 T7F	TAATACGACTCACTATAGGCGTCGCAGGAAGATGAGGA
dsSID-1 R	GCATTGATGTCGGGGTGGT
dsSID-1 T7R	TAATACGACTCACTATAGGGCATTGATGTCGGGGTGGT
dsChi F	CACGCATCTCATCTACTCA
dsChi T7F	TAATACGACTCACTATAGGCACGCATCTCATCTACTCA
dsChi R	CGAACCTTTACCGACCCT
dsChi T7R	TAATACGACTCACTATAGGCGAACCTTTACCGACCCT

HcSID-1 qF	TCGCAGGAAGATGAGGAGAAA	定量PCR检测 Quantitative PCR detection
HcSID-1 qR	GTTAGGGCAGATGTGGTAGGC
HcChi qF	TCGGTCGTTCACTTTAGCAG
HcChi qR	TTTGTAAGCGTAGGGGCAT
HcActin qF	GGTTACTCTTTCACCACCACAG
HcActin qR	GGACTTCTCAAGGGAACTGC

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