【作者】 王晓；

【导师】 许永玉；

【作者基本信息】 山东农业大学 ， 农业昆虫与害虫防治， 2021， 博士

【摘要】 中华通草蛉属于光周期敏感型昆虫,以成虫进行兼性滞育越冬,研究其滞育调控机制对其越冬保护及生防利用具有重要意义。滞育是昆虫在长期进化过程中为了躲避不利于其生存的环境而形成的一种适应机制。已有研究表明,胰岛素信号通路在昆虫滞育过程中扮演了重要作用,但关于胰岛素信号通路在生殖滞育过程中的研究还比较少。因此,本文以中华通草蛉为研究对象,围绕胰岛素信号通路在中华通草蛉生殖滞育过程中的生物学功能开展研究,主要结果如下:1.中华通草蛉有效内参基因是Arp3。本研究通过Ge Norm,Norm Finder,Bestkeeper,ΔCT method四种算法评估10个候选内参基因(Tub1,Arpc5,EF1a,128up,Rp S5,Rp S26e,GAPDH,Arp3,actin和α-Tub)在中华通草蛉生殖和滞育雌成虫不同发育阶段、组织中的表达稳定性。结果显示,在雌成虫不同发育阶段,Arp3和Rp S5是最稳定的基因组合;在雌成虫不同组织中,Arp3和Tub1是最稳定的基因组合。进一步通过q RT-PCR验证Vg和Vg R的表达水平,发现Arp3适合在雌成虫不同发育阶段及不同组织中作为内参基因。2.研究发现长短光周期处理对中华通草蛉卵巢发育、脂质积累及相关基因表达水平产生不同的影响。首先,通过组织形态学观察发现,室外种群生殖型和室内长光周期诱导生殖型中华通草蛉体色均为翠绿色,卵巢发育成熟,有少量脂肪体分散包裹卵巢等组织;室外种群滞育型和室内短光周期诱导滞育型中华通草蛉体背均出现典型的滞育斑,卵巢发育受阻,整个腹部充斥着大量紧凑的脂肪体组织。室外种群中生殖型中华通草蛉总脂含量和甘油三酯含量显著低于滞育型中华通草蛉;同样地,室内长光周期诱导生殖型中华通草蛉总脂含量和甘油三酯含量显著低于室内短光周期诱导滞育型中华通草蛉。长短光周期处理能影响卵巢发育和脂质代谢相关基因的表达,InR1、InR2、Akt、Kr-h1、Vg R、Vg和Fas2基因在生殖过程中的表达水平显著高于其在滞育过程中的表达水平,Akh R、Fas1、Clka、Z9b、Ec R和Ilp7基因在生殖过程中的表达水平显著低于其在滞育过程中的表达水平。3.研究发现外源胰岛素和胰岛素受体抑制剂Linsitinib影响中华通草蛉生殖和滞育。短光周期诱导的中华通草蛉滞育雌成虫注射外源牛胰岛素后,促进了中华通草蛉解除滞育,雌成虫未出现滞育斑,卵巢发育成熟,并有卵粒生成,而脂肪体含量减少。对长光周期诱导的中华通草蛉生殖雌成虫注射胰岛素受体抑制剂后,中华通草蛉生殖发育受阻,雌成虫卵巢未发育,并且脂肪体发育缓慢,有较多丝状物。4.研究发现InR1在中华通草蛉生殖和滞育过程中发挥重要作用。首先,利用RACE技术克隆InR1基因全长序列并进行生物信息学分析,发现InR1基因全长共5677 bp,CDS区4344 bp,该蛋白有配体结合域、半胱氨酸富集区、纤连蛋白III型结构域、酪氨酸激酶催化结构域,属于胰岛素受体家族成员之一。q RT-PCR研究发现,与长光周期相比,短光周期处理能显著下调InR1在卵巢、脂肪体和头中的表达水平(p<0.05),显著上调其在中肠中的表达水平(p<0.01)。为了研究长短光周期对InR1蛋白表达的影响,本研究制备了InR1多克隆抗体,该抗体能特异识别InR1蛋白。Western blot结果显示,InR1蛋白在生殖型中华通草蛉的脂肪体和头中的表达量较高,其次是卵巢和胸,中肠内的表达量最低。与长光周期相比,短光周期下中华通草蛉腹部InR1蛋白的表达水平显著降低。进一步利用RNAi技术研究InR1基因在中华通草蛉生殖和滞育中的功能。腹腔注射ds InR1后,InR1基因的干扰效率高达78%,InR1蛋白表达水平显著降低。长光周期下沉默InR1基因导致中华通草蛉卵巢发育停滞,脂肪体组织量增多且脂滴变大,甘油三酯含量显著升高。q RT-PCR研究发现,沉默InR1基因导致长光周期中Akt、Krh1和Vg的表达水平显著下调,并达到与短光周期相似的表达水平。5.Akt等DEGs是中华通草蛉生殖和滞育中InR1调控的下游候选基因。首先,对初羽化的中华通草蛉分别注射ds InR1和ds GFP,长光周期下处理8 d后,提取整虫RNA并进行了RNA-Seq分析和差异基因筛选。L vs S比较组中发现1693个DEGs,其中900个DEGs表达水平显著上调,793个DEGs表达水平显著下调。ds InR1 vs ds GFP比较组中发现2291个DEGs,其中1379个DEGs表达水平显著上调,912个DEGs表达水平显著下调。利用q RT-PCR技术随机验证了10个筛选出的DEGs,结果显示,RNA-Seq分析结果与q RT-PCR结果高度一致,证明RNA-Seq结果具有较高的准确性。ds InR1 vs ds GFP比较组和L vs S比较组之间有731个DEGs重叠,其中352个DEGs表达水平显著上调,372个DEGs表达水平显著下调。结合KEGG分析结果,筛选出6个生殖调控相关的DEGs(Akt,Pk N,Skp2,Cyc B3,BTr C和Aurk A)和8个脂质代谢相关的DEGs(FadΔ11、Ech A、Ec I、Ugts(2A3,1-9)、AR、Gpdh和Cbr)。综上所述,短光周期处理通过下调InR1的m RNA和蛋白表达水平抑制中华通草蛉的卵巢发育。长光周期下InR1沉默导致中华通草蛉卵巢发育停滞、脂质积累增多。Akt、Pk N和Skp2等14个DEGs是中华通草蛉生殖和滞育过程中InR1调控的下游候选基因。更多还原

【Abstract】 Chrysoperla sinica belongs to the photoperiod sensitive insect,which enters diapause for overwintering in adult stage,and is an important natural enemy for biological control.It is of great significance to study the diapause regulation mechanism of C.sinica for its overwintering protection and biocontrol utilization.Diapause is an adaptive mechanism for insects to avoid the environment that is not conducive to their survival in the long-term evolution process.Studies have shown that the insulin signaling pathway plays an important role in the process of insect diapause,but there are still few studies on the insulin signaling pathway in the process of reproductive diapause.Therefore,this study took C.sinica as the research object,and carried out research on the biological function of the insulin signaling pathway in the reproductive and diapause process of C.sinica.This article will start from the following aspects:1.Arp3 is an effective reference gene for research on reproduction and diapause of C.sinica.In this study,we evaluated the expression stability of 10 candidate reference genes(Tub1,Arpc5,EF1 a,128up,Rp S5,Rp S26 e,GAPDH,Arp3,Actin and α-Tub)in adults under diapause and non-diapause induction conditions using four statistical algorithms including Ge Norm,Norm Finder,Bestkeeper,ΔCT method.Results showed that Arp3 and Rp S5 were the most stable reference gene in different developmental stages of female adults,Arp3 and Tub1 were the most stable reference genes in different tissues of female adults.Meanwhile,the expression levels of Vg and Vg R were verified by q RT-PCR,it was found that Arp3 is suitable as an reference gene in different developmental stages and different tissues of female adults.2.The effects of long and short photoperiod treatments on ovarian development,lipid accumulation and related gene expression levels of C.sinica are different.Firstly,we observed and compared the tissue morphological characteristics of the ovaries and fat bodies of the wildtype and the Photoperiod-inducible C.sinica.Results showed that the reproductive and diapause characteristics of the wild-type and the photoperiod-inducible C.sinica were the same.During the reproductive period,C.sinica has emerald green body surface,mature ovaries,and a small amount of fat body scattered to cover ovaries and other tissues.During the diapause period,C.sinica has yellow body surface with typical diapause spots on both sides of the yellow longitudinal band on the back of the body.Ovarian development is blocked,and the entire abdomen is filled with a lot of compact fat body tissue.Meanwhile,we detected the total lipid and triglyceride content of wild-type and photoperiod-inducible C.sinica.Results showed that the total lipid contents of wild-type and photoperiod-induced C.sinica during the diapause period were significantly higher than that of the reproductive period.The triglyceride contents of wild-type and photoperiod-inducible C.sinica during the diapause period were significantly higher than that of the reproductive period.The expression results of genes related to ovarian development and lipid metabolism,such as InR1,also showed that the gene expression of C.sinica induced by photoperiod is generally consistent with the gene expression of wild-type C.sinica.The expression levels of InR1,InR2,Akt,Kr-h1,Vg R,Vg and Fas2 genes during the reproductive period were significantly higher than those during the diapause process.The expression levels of Akh R,Fas1,Clka,Z9 b,Ec R and Ilp7 genes during the diapause process were significantly higher than those during the reproductive period.3.Exogenous insulin and insulin receptor inhibitor(Linsitinib)can affect the reproduction and diapause of C.sinica.In this study,we injected exogenous bovine insulin on the diapause female adults of C.sinica induced by short photoperiod.After the injection,bovine insulin promoted the elimination of diapause of C.sinica whose female adults form emerald green body surface,mature ovaries,oocytes and less fat body tissue.Meanwhile,we injected insulin receptor inhibitors on the reproductive female adults of C.sinica induced by long photoperiod.After the injection,insulin receptor inhibitors inhibited the reproduction of C.sinica,which the ovaries were not developed,and the fat body developed slowly.4.InR1 plays an important role in the reproductive and diapause processes of C.sinica.First,the full-length sequence of the InR1 gene was cloned by RACE technology and analyzed by bioinformatics.The result found that the full-length of InR1 is 5677 bp,and the CDS region is 4344 bp.The protein has Recep＿L＿domain,Furin-like domain,FN3 domain and Tyr Kc domain,belongs to one of the members of the insulin receptor family.The q RT-PCR results showed that compared with the long photoperiod,the short photoperiod treatment can significantly down-regulate the expression level of InR1 in the ovary,fat body and head(p <0.05),and significantly up-regulate its expression level in the midgut(p < 0.01).In order to study the effect of long and short photoperiods on InR1 protein expression,we prepared InR1 polyclonal antibody,which can specifically recognize InR1 protein.Western blot results showed that the expression of InR1 protein was higher in fat body and head of the reproductive C.sinica,followed by ovary and thorax,and the lowest expression in the midgut.Compared with the long photoperiod,the expression level of InR1 protein in the abdomen of C.sinica was significantly reduced under the short photoperiod.The function of InR1 gene in the reproduction and diapause of C.sinica was further studied by RNAi technology.After the injection of ds InR1,the interference efficiency of InR1 gene reached 78%,and the expression level of InR1 protein was significantly reduced.The ovarian development of C.sinica was inhibited,the amount of fat body tissue increased,and the lipid droplets became larger.There was no difference in total lipid content,but triglyceride content was significantly up-regulated.The expression levels of Akt,Kr-h1 and Vg genes were significantly down-regulated.5.14 DEGs such as Akt are the downstream candidate genes regulated by InR1 in reproduction and diapause of C.sinica.In this study,ds InR1 and ds GFP were injected into C.sinica under long photoperiod.After 8 days,RNA was extracted and sequenced.Differentially expressed genes(DEGs)were screened by RNA-seq analysis.1693 DEGs were found in the L vs S group,of which the expression level of 900 DEGs were significantly up-regulated,and the expression level of 793 DEGs were significantly down-regulated.2291 DEGs were found in the ds InR1 vs ds GFP group,of which the expression level of 1379 DEGs was significantly upregulated,and the expression level of 912 DEGs was significantly down-regulated.Ten randomly selected DEGs were verified by q RT-PCR technology.The results showed that the RNA-Seq analysis results were highly consistent with the q RT-PCR results,which proved that the RNA-Seq results have high accuracy.It was identified that the number of DEGs shared by the ds InR1 vs ds GFP group and the L vs S group was 731,including 352 up-regulated DEGs and 372 down-regulated DEGs.Combined with the results of KEGG analysis,this study focused on 6 DEGs(Akt,Pk N,Skp2,Cyc B3,BTr C and Aurk A)related to reproductive regulation and 8 DEGs(FadΔ11,Ech A,Ec I,Ugts(2A3,1-9),AR,Gpdh,and Cbr)related to lipid metabolism pathways.In summary,short photoperiod treatment inhibits the ovarian development of C.sinica by down-regulating the m RNA and protein expression levels of InR1.After silencing InR1 of C.sinica under long photoperiod,ovarian development is blocked and lipid accumulation increases.The 14 DEGs,including Akt,Pk N and Skp2,are downstream candidate genes regulated by InR1 during the reproduction and diapause of C.sinica.更多还原