三角帆蚌谷胱甘肽硫转移酶基因克隆表达及其类胡萝卜素转运功能分析  

Cloning and expression of glutathione S-transferase pi gene and analysis of carotenoid transport function in Hyriopsis cumingii

作　　者：颜玲 钟婧妍 袁永斌 张瑶[1] 胡宏辉 陆婷婷 白志毅[1,2,3] YAN Ling;ZHONG Jingyan;YUAN Yongbin;ZHANG Yao;HU Honghui;LU Tingting;BAI Zhiyi(Key Laboratory of Freshwater Aquatic Genetic Resources,Ministry of Agriculture and Rural Affairs,Shanghai Ocean University,Shanghai 201306,China;Shanghai Collaborative Innovation Center of Aquatic Animal Breeding and Green Aquaculture,Shanghai Ocean University,Shanghai 201306,China;Shanghai Aquaculture Engineering Technology Research Center,Shanghai Ocean University,Shanghai 201306,China)

机构地区：[1]上海海洋大学,农业农村部淡水水产种质资源重点实验室,上海201306 [2]上海海洋大学,上海市水产动物良种创新与绿色养殖协同中心,上海201306 [3]上海海洋大学,上海水产养殖工程技术研究中心,上海201306

出　　处：《水产学报》2024年第8期79-88,共10页Journal of Fisheries of China

基　　金：国家重点研发计划(2022YFD2400105);国家现代农业产业技术体系(CARS-49);国家自然科学基金(31872565)。

摘　　要：为了阐明谷胱甘肽硫转移酶Pi类基因(HcGSTP1)在三角帆蚌类胡萝卜素转运中的作用,并探讨该基因表达与三角帆蚌壳色的相关性。本实验克隆并鉴定了三角帆蚌HcGSTP1基因,对其进行了序列特征和进化分析,通过实时荧光定量PCR(qRT-PCR)和原位杂交(ISH)技术检测了HcGSTP1基因在三角帆蚌中的表达及定位情况,利用RNAi技术对其功能及作用机制进行了初步分析。结果显示,HcGSTP1基因的全长cDNA序列为1317 bp,其中开放阅读框(ORF)区为618 bp,编码205个氨基酸,包含一个GST-N-pi结构域和GST-C-Pi结构域。qRT-PCR结果显示,HcGSTP1基因在紫蚌肝胰腺和斧足中表达量极显著高于白蚌,且在紫蚌边缘膜和中央膜中表达量显著高于白蚌。原位杂交结果显示,HcGSTP1基因在外套膜的外褶、背膜区、腹膜区、部分中褶以及外褶与中褶连接处出现明显的阳性信号。RNAi技术结果显示,HcGSTP1基因在边缘膜中表达的干扰率达83.74%,同时发现边缘膜中总类胡萝卜素含量(TCC)降低了30.12%。上述实验结果初步证实HcGSTP1基因参与三角帆蚌类胡萝卜素的转运,进而可能会影响贝壳及珍珠呈色,为深入理解三角帆蚌类胡萝卜素转运及贝壳和珍珠颜色形成机制补充了分子依据。Hyriopsis cumingii,a pearl mussel endemic to China's freshwater waters,produces more than 70%of the country's freshwater pearls.However,the low quality of pearls is a crucial issue,among which color is one of the main factors affecting pearl quality.Carotenoids are the class of fat-soluble natural colorants,and many studies have shown that carotenoids metabolism significantly affects the color of aquatic objects,and pearl color is closely related to carotenoids.Carotenoids cannot be synthesized directly in most animals and must be ingested from food.The metabolism of carotenoids in shellfish is a complex process,and the mechanism of absorption,transport and cleavage in the body is regulated by multi-level and multi-level factors,involving many key genes.Carotenoids are important natural pigments,and it has been found that carotenoids metabolism is significantly related to shell color of shellfish.Glutathione S-transferase Pi(GSTP1),a carotenoid-binding protein,was used to elucidate the function of the HcGSTP1 gene in carotenoids transportation in H.cumingii and to explore the correlation between the expression of HcGSTP1 gene and shell color of H.cumingii.HcGSTP1 gene was cloned and identified in this study,its sequence characteristics and evolution were analyzed.The expression and localization of HcGSTP1 gene in H.cumingii were detected by qRT-PCR and in situ hybridization technology.The function and mechanism of HcGSTP1 gene were preliminarily analyzed by RNAi technology.The results showed that the full-length cDNA sequence of HcGSTP1 gene in H.cumingii was 1317 bp,of which opening reading frame(OFR)was 618 bp,encoding 205 amino acids and containing a GST-N-pi domain and a GST-C-Pi domain.The results of qRT-PCR showed that the expression level of HcGSTP1 gene in hepatopancreas and axetopods of purple mussel was significantly higher than that in the corresponding tissues of white mussel(P<0.01),and the expression level in the marginal membrane and central membrane of purple mussel was significantly higher than t

关 键 词：三角帆蚌 HcGSTP1基因 类胡萝卜素转运 

分 类 号：Q786[生物学—分子生物学] S966.23[农业科学—水产养殖]