【摘要】 目的 揭示H1连接子组蛋白基因（H1 linker histone gene,hil-1）的生理学功能，以及其调控线虫寿命的分子机制。方法 以秀丽隐杆线虫为模式生物，采用RNA干扰菌喂食、hil-1（gk229）突变体回交纯化以及过表达质粒显微注射技术来敲降、敲除以及过表达hil-1基因，然后观察线虫存活寿命及产卵情况，通过热耐受实验、百草枯应激实验和重金属Cr⁶⁺应激实验评价hil-1（gk229）突变体的抗逆性，利用实时荧光定量PCR实验以及构建双突变体线虫进一步确定hil-1调控寿命所关联的信号通路和作用靶点。结果 与野生型N2线虫相比，RNA干扰后的线虫寿命以及hil-1（gk229）突变体线虫寿命明显缩短（P<0.001），而hil-1全身性过表达后线虫寿命延长（P<0.05）。与野生型N2线虫相比，hil-1（gk229）突变体线虫对热压力和氧化压力的耐受性明显降低（P<0.001,P<0.05），而对重金属的耐受能力无差异（P>0.05）。并且，相比于野生型N2线虫，hil-1（gk229）突变体线虫的发育周期缩短（P<0.001），产卵提前（P<0.001），但产卵总量没有显著变化（P>0.05）。给eat-2（ad465）突变体线虫喂食hil-1 RNA干扰菌后，hil-1表达下调不影响eat-2（ad465）突变体线虫的寿命（P>0.05）。相较于野生型N2线虫，hil-1（gk229）突变体线虫中daf-16表达水平明显下调（P<0.001），其下游基因mtl-1和ctl-1表达也下调（P<0.05,P<0.001）。与daf-2（e1370）突变体相比，daf-2（e1370）;hil-1（gk229）双突变体线虫的寿命无明显变化（P>0.05）；而与daf-16（mu 86）突变体相比，daf-16（mu86）;hil-1（gk229）双突变线虫的寿命明显缩短（P<0.001）。与对照组相比，在表皮和肠道中RNA干扰hil-1基因表达后线虫寿命明显缩短（P<0.001）。结论 hil-1基因缺失明显缩短线虫寿命，同时使线虫对热压力和氧化压力的耐受性降低。hil-1基因可能通过饮食限制信号通路调控秀丽隐杆线虫的寿命，且该作用主要在表皮和肠道。更多还原

【Abstract】 Objective To reveal the physiological function of H1 linker histone gene （hil-1） and its molecular mechanism for regulating the lifespan in Caenorhabditis elegans （C.elegans）.Methods C.elegans was used as a model organism and hil-1 gene was knock-down,knock-out and over-expressed via RNA interference technology,hil-1（gk229） mutants backcross purification and microinjection technology.Then the survival and oviposition of C.elegans were observed.Physiological tests including heat shock test,paraquat stress test and heavy metal Cr⁶⁺stress test were conducted to evaluate the stress resistance of hil-1 mutants.After constructing a dual mutant nematode,real-time fluorescence quantitative PCR（RT-qPCR） was used to further identify the signaling pathways and target sites associated with hil-1 gene regulatory lifespan.Results Compared with wild-type N2 worms,the lifespan of C.elegans of RNA interference and hil-1（gk229） mutants were significantly shortened （P<0.001）,while overexpression of hil-1 in the whole body increased lifespan （P<0.05）.The tolerance of hil-1（gk229） mutants to heat stress and oxidative stress was significantly decreased （P<0.001,P<0.05）,but the tolerance to heavy metals was not different compared to wild-type N2 worms （P>0.05）.In addition,the developmental cycle of hil-1（gk229）mutants was shortened and the time of oviposition was advanced （P<0.001）,but there was no significant change in total number of oviposition （P>0.05）.After feeding hil-1 RNA interference bacteria to eat-2（ad465） mutants,the down-regulation of hil-1 expression did not affect the lifespan of eat-2（ad465）mutants （P>0.05）.Compared with wild-type N2 worms,the expression level of daf-16 in hil-1（gk229） mutants was significantly down-regulated （P<0.001）,and the expressions of downstream genes,mtl-1 and ctl-1,were also down-regulated （P<0.05,P<0.001）.Compared with daf-2（e1370） mutants,the lifespan of daf-2（e1370）;hil-1（gk229） mutants did not shortened （P>0.05）.Compared with daf-16（mu86） mutants,the lifespan of daf-16（mu86）;hil-1（gk229） mutants was significantly shortened （P<0.001）.The knockdown of hil-1 via RNA interference technology,specifically in epidermis and intestine,was sufficient for lifespan reduction （P<0.001）.Conclusion The deletion of hil-1 gene significantly shortened the lifespan of C.elegans and decreased the tolerance to heat and oxidative stress.The hil-1 gene regulates the lifespan of C.elegans via dietary restriction pathway and acts mostly in epidermis and intestine.更多还原