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

Abstract

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

CLC Number:

S758

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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References 0

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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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Cloning and Expression Profiles of HcSID-1 Gene and Its Function Verification in Hyphantria cunea Larvae

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