hil-1基因通过饮食限制通路调节秀丽隐杆线虫寿命

H1 Linker Histone Gene Regulates Lifespan via Dietary Restriction Pathways in Caenorhabditis elegans

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。

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.