美国白蛾自噬基因HcAtg8的鉴定及其对沉默HcV-ATPase A的响应 Click here to download the full text Citation：王薄毓,王晓洁,韩莹,王海,赵丹,王倩,姚亚林,陆秀君.美国白蛾自噬基因HcAtg8的鉴定及其对沉默HcV-ATPase A的响应.Journal of Plant Protection,2026,53(2):459-466 Hits: Download times: Author Name Affiliation E-mail Wang Boyu  College of Plant Protection, Hebei Agricultural University, Baoding 071001, Hebei Province, China    Wang Xiaojie  College of Plant Protection, Hebei Agricultural University, Baoding 071001, Hebei Province, China    Han Ying  College of Plant Protection, Hebei Agricultural University, Baoding 071001, Hebei Province, China    Wang Hai  College of Plant Protection, Hebei Agricultural University, Baoding 071001, Hebei Province, China    Zhao Dan  College of Plant Protection, Hebei Agricultural University, Baoding 071001, Hebei Province, China    Wang Qian  College of Plant Protection, Hebei Agricultural University, Baoding 071001, Hebei Province, China    Yao Yalin  College of Plant Protection, Hebei Agricultural University, Baoding 071001, Hebei Province, China  yaoyalin@hebau.edu.cn  Lu Xiujun  College of Plant Protection, Hebei Agricultural University, Baoding 071001, Hebei Province, China  luxiujun@hebau.edu.cn  中文摘要:为明确美国白蛾Hyphantria cunea调控细胞能量代谢的V型质子ATP酶催化亚基A(V-type proton ATPase catalytic subunit A,V-ATPase A)基因HcV-ATPase A与自噬相关基因（autophagyrelated gene,Atg）HcAtg8的级联效应，采用PCR技术克隆HcAtg8基因，分析该基因的生物学信息及系统进化关系，利用原核表达系统体外表达HcAtg8，并进行Western blot检测，通过实时荧光定量PCR(quantitative real-time PCR, qRT-PCR)技术分析美国白蛾不同龄期及不同肠道部位中HcAtg8基因的表达情况，以及沉默HcV-ATPase A后中肠自噬相关组织的病理学变化及HcAtg8基因的表达情况。结果显示：克隆得到的美国白蛾自噬基因HcAtg8编码区为354 bp，编码117个氨基酸，HcAtg8蛋白和家蚕Bombyx mori BmAtg8聚在一个分支，亲缘关系较近。构建得到p MAL-c2X-HcAtg8重组表达质粒，体外原核表达得到大小为56.4 kD的HcAtg8蛋白，诱导8 h后表达量最高。美国白蛾HcAtg8基因在不同龄期和不同肠道部位均有表达，在蛹中和中肠的表达量最高。沉默HcV-ATPase A后，处理组自噬溶酶体数量为11.0个，显著高于对照组的3.5个，自噬溶酶体数量上调3.1倍，处理组脂滴数量为73.8个，显著高于对照组的12.5个，脂滴数量上调5.9倍；沉默HcVATPase A后溶酶体酸性环境改变，导致自噬小体及相关代谢产物积累；qRT-PCR结果显示自噬相关基因HcAtg8在24 h时的表达量为对照的6.1倍，在48 h时表达量最高，为对照的375.2倍；在72 h时该基因表达量降低，为对照的2.5倍，与组织切片的结果相符。表明通过沉默HcVATPase A导致了HcAtg8的高表达以及自噬小体和脂滴的积累，破坏了中肠细胞的稳态，导致细胞功能障碍或者死亡。 中文关键词:美国白蛾  自噬相关基因  基因克隆表达  沉默HcV-ATPase A  中肠组织变化   Identification of the autophagy gene HcAtg8 in fall webworm Hyphantria cunea and its response to HcV-ATPase A silencing Author Name Affiliation E-mail Wang Boyu  College of Plant Protection, Hebei Agricultural University, Baoding 071001, Hebei Province, China    Wang Xiaojie  College of Plant Protection, Hebei Agricultural University, Baoding 071001, Hebei Province, China    Han Ying  College of Plant Protection, Hebei Agricultural University, Baoding 071001, Hebei Province, China    Wang Hai  College of Plant Protection, Hebei Agricultural University, Baoding 071001, Hebei Province, China    Zhao Dan  College of Plant Protection, Hebei Agricultural University, Baoding 071001, Hebei Province, China    Wang Qian  College of Plant Protection, Hebei Agricultural University, Baoding 071001, Hebei Province, China    Yao Yalin  College of Plant Protection, Hebei Agricultural University, Baoding 071001, Hebei Province, China  yaoyalin@hebau.edu.cn  Lu Xiujun  College of Plant Protection, Hebei Agricultural University, Baoding 071001, Hebei Province, China  luxiujun@hebau.edu.cn  Abstract:To elucidate the cascade effect between the V-type proton ATPase catalytic subunit A(V-ATPase A), a key regulator of cellular energy metabolism, and the autophagy-related gene HcAtg8 in the fall webworm Hyphantria cunea, the HcAtg8 gene was cloned by PCR. The bioinformatic characteristics and phylogenetic relationships were analyzed. The HcAtg8 protein was expressed in vitro using a prokaryotic expression system and detected via Western blot. Quantitative real-time PCR(qRT-PCR) was used to analyze the expression of HcAtg8 in different developmental stages and tissues(foregut, midgut, and hindgut). Additionally, after silencing HcV-ATPase A, histopathological changes in midgut tissues related to autophagy and the expression of HcAtg8 were examined. The results showed that the cloned coding region of HcAtg8 was 354 bp in length, encoding 117 amino acids. Phylogenetic analysis indicated that HcAtg8 clustered closely with BmAtg8 from Bombyx mori, suggesting a close evolutionary relationship. A recombinant expression vector pMAL-c2X-HcAtg8 was constructed, and a 56.4 kD HcAtg8 protein was obtained by prokaryotic expression in vitro, with the highest expression level observed after 8 h of induction. HcAtg8 was expressed across all developmental stages and gut tissues, with the highest expression observed in pupae. Among gut tissues, expression was highest in the midgut. After silencing HcV-ATPase A, the number of autophagosomes in the treated group was 11.0, significantly higher than that in the control group(3.5), representing a 3.1-fold increase. The number of lipid droplets in the treated group was 73.8, significantly higher than that in the control group(12.5), representing a 5.9-fold increase. Silencing HcV-ATPase A disrupted the acidic environment of lysosomes, leading to the accumulation of autophagosomes and related metabolites. qRT-PCR results showed that the expression level of HcAtg8 was 6.1-fold higher than that of the control at 24 h, peaked at 375.2-fold at 48 h, and subsequently decreased to 2.5-fold at 72 h, consistent with the histological observations. These results indicate that silencing HcV-ATPase A leads to the upregulation of HcAtg8 expression and the accumulation of autophagosomes and lipid droplets, thereby disrupting midgut cellular homeostasis, and resulting in cellular dysfunction or death. keywords:Hyphantria cunea  autophagy-related gene  gene cloning and expression  HcV-ATPase A silencing  midgut tissue changes View Full Text  View/Add Comment  Download reader

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