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

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中文摘要：[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2 952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过量累积能够诱导大量表皮蛋白基因的显著上调表达,并使得其表皮结构特征发生改变.研究结果为后续探索黑色素前体对表皮结构基因的调控机制,以及理解昆虫表皮重要组分间的互作模式,对昆虫适应性状的影响提供了新的视角.

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

外文摘要：[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 2 952 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 comparison groups were significantly enriched to structural constituent of cuticle(GO:0042302).KEGG pathway enrichment analysis result indicated that the up-regulated DEGs in the dopa treatment group vs CK comparison group were significantly enriched to spliceosome pathway,and the down-regulated DEGs were significantly enriched to Toll-Imd and Hippo signaling pathways.The down-regulated DEGs in the dopamine treatment group vs CK comparison group were significantly enriched to autophagy pathway.No DEGs shared between these two comparison groups were significantly enriched to any pathways.In addition,65 significantly enriched up-regulated cuticular protein genes belonged to the CPR,CPF,TWDL,CPLC,and CPAP five families and distributed on three chromosomes,and some members were clustered.The expression levels detected by qPCR-PCR for 12 selected shared up-regulated cuticular protein genes in both comparison groups were consistent with the transcriptome data.[Conclusion]This omics-level study showed that the excessive accumulation of melanin in mosquito pupal integuments led to the significant up-regulation of a large number of cuticular protein genes and changes in cuticular structural features.These findings facilitate subsequent studies of the mechanisms that allow melanin precursors to regulate cuticular structural genes,and to understand the impacts of interactions between important components of the cuticle on the adaptive traits in insects,supplying a new perspective on the impact of insect adaptive traits.

外文关键词：

Anopheles sinensismelanizationmelanin precursorsintegumenttranscriptomecuticular protein genes

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、乔梁

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作者单位：

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

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

基金：

国家自然科学基金国家自然科学基金重庆市自然科学基金重庆市巴渝学者青年学者计划重庆市教委科技项目家蚕基因组生物学国家重点实验室开放基金重庆市留学人员回国创业创新支持计划重庆师范大学研究生科研创新项目

项目编号：

3177252731872262CSTB2022 NSCQ-MSX1355YS2019027KJZD-K202200507SKLSGB-ORP202113cx2022052CYS23399

出版年：

2024

DOI：

10.16380/j.kcxb.2024.03.003

昆虫学报

中国科学院动物研究所,中国昆虫学会

昆虫学报

CSTPCD北大核心

影响因子：0.756

ISSN：0454-6296

年,卷(期)：2024.67(3)

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