【目的】航天蚕Bombyx mori后代小茧突变体sc的幼虫发育缓慢,食桑量少,推测其生长发育相关通路基因受到基因突变的影响。前期研究表明,sc突变受一对位于家蚕第3连锁群的隐性基因控制,但其控制基因并未被鉴定。本研究拟通过比较分析sc与...【目的】航天蚕Bombyx mori后代小茧突变体sc的幼虫发育缓慢,食桑量少,推测其生长发育相关通路基因受到基因突变的影响。前期研究表明,sc突变受一对位于家蚕第3连锁群的隐性基因控制,但其控制基因并未被鉴定。本研究拟通过比较分析sc与航天蚕后代正常茧品系TG的转录组数据,为sc突变体相应基因的鉴定及分子机制的解析提供参考。【方法】分别取sc和TG 5龄第4天幼虫的头部和中肠组织进行转录组测序(RNA-seq),并通过比较转录组分析获得差异表达基因(differentially expressed genes,DEGs),对DEGs进行GO注释和KEGG富集分析。利用qRT-PCR验证随机选取的DEGs在sc和TG中的表达量。此外,利用qRT-PCR调查感兴趣基因在sc中的表达量。【结果】TG vs sc比较组头部检测到1528个DEGs,其中820个上调表达,708个下调表达;TG vs sc比较组中肠检测到1401个DEGs,其中683个上调表达,718个下调表达。GO分析表明头部和中肠DEGs在生物学过程中,大多数DEGs参与细胞过程、代谢过程、生物调节和刺激反应等;在分子功能中,大多数DEGs参与结合、催化活性、结构分子活性、转运蛋白活性和ATP依赖活性等。头部和中肠DEGs均涉及Hippo,Insulin和mTOR等与家蚕生长发育相关的信号通路。qRT-PCR分析显示,基因的表达趋势与转录组测序结果一致;与TG相比,sc中生长发育相关通路中BMSK0008105,BMSK0009907,BMSK0002689,BMSK0000286,BMSK0012340和BMSK00083629等关键基因差异表达。【结论】sc和TG中生长发育相关的信号通路关键基因的差异表达,通过影响生长发育过程中的能量代谢、器官发生和细胞生长、增殖及凋亡等生理过程,进而影响小茧突变体sc的体型发育。研究结果有助于阐明sc突变体形成的分子机制,并为家蚕体型调控研究积累实验数据。展开更多
The domesticated silkworm(Bombyx mori)has evolved a highly efficient nitrogen utilization system to support silk production.The silk glands play a pleiotropic role in sequestering nitrogen resources for silk synthesis...The domesticated silkworm(Bombyx mori)has evolved a highly efficient nitrogen utilization system to support silk production.The silk glands play a pleiotropic role in sequestering nitrogen resources for silk synthesis,mitigating aminoacidemia by assimilating free amino acids,and reallocating nitrogen during metamorphosis through programmed cell death.However,the specific functions of nitrogen metabolism-related genes in this process remain unclear.Using CRISPR/Cas9-based gene editing,mutations were generated in glutamine synthetase(GS),glutamate synthetase(GOGAT),asparagine synthetase(AS),glutamate dehydrogenase(GDH)and glutamate oxaloacetate transaminase 1(GOT1).Disruption of GS,GOGAT,and AS consistently reduced silkworm cocoon and pupal weight and significantly down-regulated silk protein gene transcription,whereas GOT1 mutation had no such effect.GOGAT mutants exhibited abnormally enlarged silk glands,whereas GS and AS mutants showed delayed programmed cell death in the silk glands.In contrast,GOT1 mutants displayed normal silk gland morphology but were consistently smaller.Disruption of GS,GOGAT,and AS led to more extensive transcriptional changes,including altered expression of transcription factors in the silk glands,compared with GOT1 mutants.Both GS and GOGAT mutants exhibited up-regulation of AS and GDH,while only GOGAT mutants displayed elevated AS enzymatic activity,suggesting that GOGAT may compete with AS for glutamine in the silk glands to support silk protein synthesis.AS mutants showed significantly elevated GOT activity and up-regulation of several metabolic pathways,indicating that AS may functionally interact with GOT in regulating both silk gland development and programmed cell death during metamorphosis.展开更多
文摘【目的】航天蚕Bombyx mori后代小茧突变体sc的幼虫发育缓慢,食桑量少,推测其生长发育相关通路基因受到基因突变的影响。前期研究表明,sc突变受一对位于家蚕第3连锁群的隐性基因控制,但其控制基因并未被鉴定。本研究拟通过比较分析sc与航天蚕后代正常茧品系TG的转录组数据,为sc突变体相应基因的鉴定及分子机制的解析提供参考。【方法】分别取sc和TG 5龄第4天幼虫的头部和中肠组织进行转录组测序(RNA-seq),并通过比较转录组分析获得差异表达基因(differentially expressed genes,DEGs),对DEGs进行GO注释和KEGG富集分析。利用qRT-PCR验证随机选取的DEGs在sc和TG中的表达量。此外,利用qRT-PCR调查感兴趣基因在sc中的表达量。【结果】TG vs sc比较组头部检测到1528个DEGs,其中820个上调表达,708个下调表达;TG vs sc比较组中肠检测到1401个DEGs,其中683个上调表达,718个下调表达。GO分析表明头部和中肠DEGs在生物学过程中,大多数DEGs参与细胞过程、代谢过程、生物调节和刺激反应等;在分子功能中,大多数DEGs参与结合、催化活性、结构分子活性、转运蛋白活性和ATP依赖活性等。头部和中肠DEGs均涉及Hippo,Insulin和mTOR等与家蚕生长发育相关的信号通路。qRT-PCR分析显示,基因的表达趋势与转录组测序结果一致;与TG相比,sc中生长发育相关通路中BMSK0008105,BMSK0009907,BMSK0002689,BMSK0000286,BMSK0012340和BMSK00083629等关键基因差异表达。【结论】sc和TG中生长发育相关的信号通路关键基因的差异表达,通过影响生长发育过程中的能量代谢、器官发生和细胞生长、增殖及凋亡等生理过程,进而影响小茧突变体sc的体型发育。研究结果有助于阐明sc突变体形成的分子机制,并为家蚕体型调控研究积累实验数据。
基金supported by the National Natural Science Foundation of China(32270458,32070411,2023A1515010657)State Key Laboratory of Resource Insects(SKLSGB-ORP202209)。
文摘The domesticated silkworm(Bombyx mori)has evolved a highly efficient nitrogen utilization system to support silk production.The silk glands play a pleiotropic role in sequestering nitrogen resources for silk synthesis,mitigating aminoacidemia by assimilating free amino acids,and reallocating nitrogen during metamorphosis through programmed cell death.However,the specific functions of nitrogen metabolism-related genes in this process remain unclear.Using CRISPR/Cas9-based gene editing,mutations were generated in glutamine synthetase(GS),glutamate synthetase(GOGAT),asparagine synthetase(AS),glutamate dehydrogenase(GDH)and glutamate oxaloacetate transaminase 1(GOT1).Disruption of GS,GOGAT,and AS consistently reduced silkworm cocoon and pupal weight and significantly down-regulated silk protein gene transcription,whereas GOT1 mutation had no such effect.GOGAT mutants exhibited abnormally enlarged silk glands,whereas GS and AS mutants showed delayed programmed cell death in the silk glands.In contrast,GOT1 mutants displayed normal silk gland morphology but were consistently smaller.Disruption of GS,GOGAT,and AS led to more extensive transcriptional changes,including altered expression of transcription factors in the silk glands,compared with GOT1 mutants.Both GS and GOGAT mutants exhibited up-regulation of AS and GDH,while only GOGAT mutants displayed elevated AS enzymatic activity,suggesting that GOGAT may compete with AS for glutamine in the silk glands to support silk protein synthesis.AS mutants showed significantly elevated GOT activity and up-regulation of several metabolic pathways,indicating that AS may functionally interact with GOT in regulating both silk gland development and programmed cell death during metamorphosis.
