基于RNAi技术解析美国白蛾HcAnk1和HcAnk2基因功能及对HcNPV的敏感性

doi:10.12302/j.issn.1000-2006.202203049

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南京林业大学学报（自然科学版） ›› 2024, Vol. 48 ›› Issue (3): 181-190.doi: 10.12302/j.issn.1000-2006.202203049

基于RNAi技术解析美国白蛾HcAnk1和HcAnk2基因功能及对HcNPV的敏感性

彭萌萌¹(), 吴红渠¹^,²(), 张佳雯¹, 闫丽琼¹, 曹传旺¹, 孙丽丽¹^,^*()

1.东北林业大学森林生态系统可持续经营教育部重点实验室,黑龙江哈尔滨 150040
2.湖北省生物农药工程研究中心,湖北武汉 430070

收稿日期:2022-03-24 修回日期:2022-09-05 出版日期:2024-05-30 发布日期:2024-06-14
通讯作者: *孙丽丽(sunlili@nefu.edu.cn),副教授。
作者简介:彭萌萌(1522020351@qq.com),负责试验设计与初稿撰写;吴红渠(139711877@qq.com),负责部分实验操作与数据分析。
基金资助:
国家自然科学基金面上项目(32171791);东北林业大学本科生创新项目(202010225031);黑龙江省优秀青年基金(YQ2021C007)

HcAnk1 and HcAnk2 genes function and HcNPV susceptibility of Hyphantria cunea based on RNAi

PENG Mengmeng¹(), WU Hongqu¹^,²(), ZHANG Jiawen¹, YAN Liqiong¹, CAO Chuanwang¹, SUN Lili¹^,^*()

1. Key Laboratory of Sustainable Forest Ecosystem Management-Ministry of Education, Northeast Forestry University, Harbin 150040, China
2. Hubei Biopesticide Engineering Research Center, Wuhan 430070, China

Received:2022-03-24 Revised:2022-09-05 Online:2024-05-30 Published:2024-06-14
Supported by:
National Natural Science Foundation of China(32171791);National College Students’ Innovation and Entrepreneurship Training Program(202010225031);Natural Science Foundation of Heilongjiang(YQ2021C007)

摘要/Abstract

摘要：

【目的】锚定蛋白是连接细胞骨架与质膜的衔接蛋白家族。但昆虫中关于锚定蛋白基因功能研究甚少。笔者克隆美国白蛾(Hyphantria cunea)锚定蛋白基因,阐明该基因在其各发育期和组织中的表达特征及对美国白蛾核型多角体病毒(Hyphantria cunea nucleopolyhedrosisvirus, HcNPV)的敏感性,为进一步开发HcNPV增效剂提供理论依据。【方法】通过转录组文库筛选出Ankyrin1(HcAnk1)和Ankyrin2(HcAnk2)基因,利用生物信息学分析基因特性,采用qRT-PCR检测不同发育阶段、不同组织以及不同HcNPV浓度胁迫下HcAnks的表达量,通过RNAi技术研究HcAnks基因沉默后美国白蛾对HcNPV的敏感性。【结果】 HcAnk1和HcAnk2基因开放阅读框依次为1 392和1 866 bp,分别编码463和621个氨基酸,蛋白质分子质量分别为59.18和69.19 ku,理论等电点分别为5.74 和 8.66;进化树分析显示HcAnk1与粉纹夜蛾(Trichoplusia ni)亲缘关系最近而聚为一类。HcAnks在各个发育阶段均有表达,卵期表达量最高,6龄表达量最低;HcAnks分别在中肠和后肠中表达量最高;在卵巢和精巢中未检测到HcAnk1,而HcAnk2主要在中肠和精巢中表达。在不同浓度HcNPV胁迫下,HcAnks转录水平表现为低浓度诱导,高浓度抑制。沉默HcAnks美国白蛾4龄幼虫的相对生长率(RGR)和食物转化效率(ECD)显著降低。此外,沉默HcAnks的美国白蛾4龄幼虫对HcNPV敏感性显著增加。【结论】 HcAnks在抵抗HcNPV侵染过程中发挥重要作用,HcAnks可作为HcNPV增效剂,用于美国白蛾无公害防治。

关键词: 美国白蛾, 锚定蛋白, 美国白蛾核型多角体病毒, RNAi

Abstract:

【Objective】 Ankyrin is a family of adaptor proteins that connect the cytoskeleton to the plasma membrane. However, the functions of insect ankyrin are poorly understood. In this study, the Ankyrin genes of Hyphantria cunea were cloned and their expression characteristics were determined in different developmental stages and tissues of H. cunea. The mortality rate of H. cunea was measured under the stress of exposure to nucleopolyhedrosis virus, which provided a theoretical basis for further developing synergists for NPV-based control strategies. 【Method】 The Ankyrin1 (HcAnk1) and Ankyrin2 (HcAnk2) genes were screened by a transcriptome library, and the characteristics of the two Ankyrin genes were determined using bioinformatics. Using qRT-PCR, the expression of HcAnk1 and HcAnk2 genes were determined at different developmental stages and in different tissues, and under different HcNPV concentrations. The survival rate of H. cunea larvae under HcNPV stress was investigated after the gene silencing of HcAnk1 and HcAnk2 using an RNAi technique. 【Result】 The open reading frames of the HcAnk1 and HcAnk2 genes were 1392 and 1866 bp, encoding 463 and 621 amino acids, respectively. The molecular weights of the HcAnk1 and HcAnk2 proteins were 59.18 and 69.19 ku, respectively, and the theoretical isoelectric points were 5.74 and 8.66, respectively. A phylogenetic analysis showed that HcAnk1 was closely related to Trichoplusia ni and was clustered into one group. HcAnk1 and HcAnk2 were expressed at all developmental stages, with the highest expression in the egg stage and the lowest expression in the sixth instar larva. The highest expression of HcAnk1 and HcAnk2 was observed in the midgut and hindgut, respectively, but HcAnk1 was not detected in the ovary or testis, while HcAnk2 was mainly expressed in the midgut and testis. In tests with different levels of HcNPV stress, the transcription levels of HcAnk1 and HcAnk2 were induced at low concentrations and inhibited at high concentrations. The relative growth rate and food conversion efficiency (ECD) were significantly decreased after silencing HcAnks in H. cunea larvae. Additionally, the H. cunea with HcAnks silencing were significantly less resistant to HcNPV. 【Conclusion】 HcAnk1 and HcAnk2 play an important role in the resistance to HcNPV. HcAnk1 and HcAnk2 can be used as HcNPV synergists for the pollution-free control of H. cunea.

Key words: Hyphantria cunea, Ankyrins, Hyphantria cunea nucleopolyhedrovirus, RNAi

中图分类号:

S763.3

彭萌萌,吴红渠,张佳雯,等. 基于RNAi技术解析美国白蛾HcAnk1和HcAnk2基因功能及对HcNPV的敏感性[J]. 南京林业大学学报（自然科学版）, 2024, 48(3): 181-190.

PENG Mengmeng, WU Hongqu, ZHANG Jiawen, YAN Liqiong, CAO Chuanwang, SUN Lili. HcAnk1 and HcAnk2 genes function and HcNPV susceptibility of Hyphantria cunea based on RNAi[J].Journal of Nanjing Forestry University (Natural Science Edition), 2024, 48(3): 181-190.DOI: 10.12302/j.issn.1000-2006.202203049.

图/表 7

Table 1

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Table 2

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gene 基因	forward primer sequences 正向引物序列	reverse primer sequences 反向引物序列	primer usage 引物用途
dsHcAnk1	TAATACGACTCACTATAGG GATGCCTTCAGAAGTGATATCAG	TAATACGACTCACTATAGG GAAAAGTGAGCAGCAACTCCA	RNAi
dsHcAnk2	TAATACGACTCACTATAGGG CAGAAAACTATGACATGATTACAT	TAATACGACTCACTATAGGG CAACATCGCCTATTTCTTCG
dsGFP	TAATACGACTCACTATAGGG GGAGAAGAACTTTTCACTGG	TAATACGACTCACTATAGGG AGTTGAACGGATCCATCTTC	RT-qPCR
HcAnk1	CGTGGCTCCTAGCGCTGAGG	GGAGTCTTAGCACGGTGCATTAG
HcAnk2	ATGAATCCATCCTTGGAAGACA	GCAGTGGTTTTGTTTAGAAGATC

treatment 处理	relative growth ratio (RGR) 相对生长率	relative consumption rate (RCR) 相对取食量	efficiency of ingested food (ECI) 食物利用率	efficiency of conversion of food (ECD) 食物转化率
dsGFP	74.40±0.62 a	5.92±0.71 a	12.78±1.73 a	13.56±1.84 a
dsHcAnk1	48.33±0.68 c	4.80±0.13 b	10.09±0.22 a	10.40±0.34 b
dsHcAnk2	60.14±2.53 b	7.84±1.06 a	7.81±1.09 b	8.19±0.95c

基于RNAi技术解析美国白蛾HcAnk1和HcAnk2基因功能及对HcNPV的敏感性

HcAnk1 and HcAnk2 genes function and HcNPV susceptibility of Hyphantria cunea based on RNAi

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