黑色素前体多巴和多巴胺培养后的中华按蚊蛹体壁结构特征及其转录组分析  

Structure characteristics and transcriptome analysis of the integument of Anopheles sinensis(Diptera:Culicidae)pupae after cultivation with melanin precursors dopa and dopamine

作　　者：向凯 邱品品 洪俊峰 凌瑕 周操 何树林 谢新元 蒋艳萍 王思艺 陈斌[1] 乔梁[1] XIANG Kai;QIU Pin-Pin;HONG Jun-Feng;LING Xia;ZHOU Cao;HE Shu-Lin;XIE Xin-Yuan;JIANG Yan-Ping;WANG Si-Yi;CHEN Bin;QIAO Liang(Chongqing Key Laboratory of Vector Insects,Institute of Entomology and Molecular Biology,Chongqing Normal University,Chongqing 401331,China)

机构地区：[1]重庆师范大学昆虫与分子生物学研究所,媒介昆虫重庆市重点实验室,重庆401331

出　　处：《昆虫学报》2024年第3期327-338,共12页Acta Entomologica Sinica

基　　金：国家自然科学基金项目(31772527,31872262);重庆市自然科学基金项目(CSTB2022 NSCQ-MSX1355);重庆市“巴渝学者青年学者”计划(YS2019027);重庆市教委科技项目(KJZD-K202200507);家蚕基因组生物学国家重点实验室开放课题(SKLSGB-ORP202113);重庆市留学人员回国创业创新支持计划(cx2022052);重庆师范大学研究生科研创新项目(CYS23399)。

摘　　要：【目的】筛选出促黑化条件下中华按蚊Anopheles sinensis蛹体壁中表达变化最显著的基因类群,并初步探究它们与黑色素前体累积及表皮结构特征间的相关性。【方法】在Grace’s昆虫细胞培养基中添加黑色素前体多巴(dopa)和多巴胺(dopamine),对化蛹20 min内中华按蚊体壁进行体外培养,对照组(CK)中不添加黑色素前体,培养16 h时利用体视显微镜观察黑色素前体处理组与对照组蛹体壁着色和表皮截面特征;通过Illumina测序平台对黑色素前体处理组与对照组蛹体壁进行转录组测序,对差异表达基因(different expression genes,DEGs)进行GO功能分类和KEGG通路富集分析;分析被显著富集的基因注释和染色体分布;利用qRT-PCR验证转录组数据。【结果】多巴和多巴胺处理组中华按蚊蛹体壁与表皮截面相比于CK明显黑化,且多巴处理组着色程度最深;多巴和多巴胺处理组中华按蚊蛹表皮相比于CK明显增厚,且增厚程度与黑化程度相关。多巴处理组vs CK与多巴胺处理组vs CK比较组的DEGs分别为2952和697个,且下调基因居多;这两类比较组间共有上调的DEGs有223个,共有下调的DEGs有347个。DEGs的GO功能注释结果表明,在上述比较组以及两比较组间共有的上调DEGs被显著富集到表皮结构组分(GO:0042302)。KEGG通路富集分析结果显示,多巴处理组vs CK比较组上调的DEGs被显著富集到剪切体通路,下调的DEGs被显著富集到Toll-Imd和Hippo信号通路;在多巴胺处理组vs CK比较组中下调DEGs被显著富集到自噬通路;这两比较组间共享的DEGs没有被显著被富集的通路;65个被富集的上调表皮蛋白基因来自于CPR,CPF,TWDL,CPLC和CPAP 5个家族,并分布于3条染色体,其中部分成员成簇分布。qRT-PCR结果显示,两比较组中所选择的共有上调的12个表皮蛋白基因的表达量与转录组数据一致。【结论】本研究在组学水平上证实了蚊虫体壁组织中黑色素的�【Aim】To identify gene groups with the most significant changes in the expression in integuments of Anopheles sinensis pupae under melanization-inducing conditions,and explore their correlations with melanin precursor accumulation and cuticular structural features.【Methods】Grace’s insect cell medium was supplemented with the melanin precursors dopa and dopamine.An.sinensis pupal integuments were subjected to in vitro cultivation within the first 20 min of pupation,and a control group(CK)was not treated with melanin precursors.At 16 h after cultivation,pupal integuments from the melanin precursor-treated groups and control group were observed to assess their coloration and cuticular cross-sectional features using a stereomicroscope.The integument transcriptome sequencing of the melanin precursor-treated and control groups was performed using the Illumina platform.GO functional classification and KEGG pathway enrichment analysis were performed on the differentially expressed genes(DEGs).The gene annotations and chromosome distributions of significantly enriched DEGs were analyzed.qRT-PCR was conducted to validate the transcriptome data.【Results】The integuments and cuticular cross-sections of pupae of An.sinensis treated with dopa and dopamine exhibited noticeable melanization compared with those of CK,and dopa treatment resulted in the deepest pigmentation.The cuticle exhibited significant thickening in the dopa and dopamine treatment groups compared with that of CK,and the degree of thickness was correlated with the degree of melanization.There were 2952 and 697 DEGs were identified from dopa treatment group vs CK and dopamine treatment group vs CK comparison groups,respectively,and the down-regulated genes predominated.The numbers of the shared up-regulated DEGs and down-regulated DEGs between these two comparison groups were 223 and 347,respectively.GO functional classification result showed that the shared up-regulated DEGs in the above comparison groups and the shared up-regulated DEGs in both com

关 键 词：中华按蚊 黑化 黑色素前体 体壁 转录组 表皮蛋白基因 

分 类 号：Q965[生物学—昆虫学]