本文選題：家蠶 + 壽命��； 參考：《西南大學(xué)》2017年博士論文

【摘要】：實驗動物是現(xiàn)代生命科學(xué)研究的重要基礎(chǔ)和強(qiáng)力支撐。隨著科學(xué)技術(shù)的發(fā)展,越來越多的實驗動物被應(yīng)用到生命科學(xué)研究中。長久以來,益壽延年一直是人類追求的夢想,因此,衰老和壽命決定機(jī)制研究一直是生命科學(xué)領(lǐng)域的熱點。目前應(yīng)用于壽命研究的無脊椎動物模型主要包括秀麗隱桿線蟲和黑腹果蠅,脊椎動物模型主要包括小鼠和猴子。由于衰老和壽命調(diào)控機(jī)制的復(fù)雜性,模式生物在該領(lǐng)域研究中的利用至關(guān)重要。其中,身體構(gòu)造簡單而器官分化卻相對完整的無脊椎實驗動物受到廣大研究者的青睞。至今,少數(shù)的無脊椎動物模型并不能完全滿足復(fù)雜的壽命機(jī)制研究的需要,新型實驗動物的開發(fā)利用十分必要。數(shù)千年的馴養(yǎng)和蠶業(yè)發(fā)展賦予了家蠶獨特的生物學(xué)特點及文化內(nèi)涵。長達(dá)一個世紀(jì)收集和研究積淀了豐富的突變資源,以及率先開展了基因組和功能基因組研究,為家蠶的拓展利用提供了良好的基礎(chǔ)。同時,家蠶也具備諸多作為壽命研究的實驗動物的特征,如生命周期和個體大小適中、繁殖力強(qiáng)、發(fā)育階段界限清晰、外源注射所導(dǎo)致的物理損傷較小、壽命可塑性較強(qiáng)及不存在倫理問題等。有鑒于此,本研究選擇家蠶作為實驗動物,鑒定重要壽命基因及分析基因調(diào)控家蠶壽命的機(jī)制,為壽命決定分子機(jī)制的闡釋提供新的認(rèn)知。主要獲得了如下研究結(jié)果:1.家蠶壽命與BmFoxO基因表達(dá)量間的關(guān)系家蠶各品系的發(fā)育時長不同,壽命也存在差異。從家蠶基因庫中選取一組品系進(jìn)行壽命統(tǒng)計,并對其中的三個代表性品系(夏芳、Dazao-N、N4)進(jìn)行壽命相關(guān)基因表達(dá)模式的調(diào)查,結(jié)果顯示:夏芳的平均壽命和最大壽命均較長,Dazao-N次之,N4品系的平均壽命和最大壽命較短。鑒于Fox O在多個物種中被報道是調(diào)控壽命的關(guān)鍵因子,我們調(diào)查了這些品系中BmFoxO在不同發(fā)育時期的表達(dá)水平,發(fā)現(xiàn)在蛹10天(可能決定成蟲壽命的時間點),BmFoxO的表達(dá)量為夏芳顯著高于Dazao-N和N4,并且N4顯著低于Dazao-N。同時,在被調(diào)查的其他發(fā)育時期,BmFoxO表達(dá)趨勢與相應(yīng)品系的壽命也呈現(xiàn)出高度一致。通過控制家蠶飲食攝入量,成功構(gòu)建了家蠶飲食限制實驗?zāi)Ｐ?壽命統(tǒng)計結(jié)果表明飲食限制組壽命明顯長于對照組,并且BmFoxO表達(dá)量顯著高于對照組。鑒于BmFoxO的表達(dá)與壽命顯著相關(guān),所以推測該基因發(fā)揮了調(diào)控家蠶壽命的功能。2.家蠶Fox基因家族的鑒定與進(jìn)化分析Fox基因編碼的是一個轉(zhuǎn)錄因子家族,包含23個亞家族(Fox A-Fox S)。在家蠶中利用隱馬爾可夫模型對Fox基因家族成員進(jìn)行全基因組的分析鑒定,結(jié)果發(fā)現(xiàn)家蠶中含有17個Fox家族成員。通過系統(tǒng)發(fā)生分析,將這17個Fox家族基因歸類于13個Fox亞家族。對其所屬的染色體位置進(jìn)行分析發(fā)現(xiàn)這些成員分布于13條不同的染色體上。利用相同的隱馬爾可夫模型識別紅帶袖蝶、帝王蝶、果蠅、小鼠和人類中的Fox基因家族成員,并利用這些序列構(gòu)建系統(tǒng)發(fā)生樹,結(jié)果表明不同物種中的直系同源基因間序列高度保守。同時,對家蠶中Fox家族基因的結(jié)構(gòu)和蛋白質(zhì)結(jié)構(gòu)域特征分析,表明家蠶中Fox基因大多屬于單外顯子基因,蛋白均包含有保守的Fork head結(jié)構(gòu)域。家蠶中Fox家族基因的GO注釋表明,其編碼產(chǎn)物除未參與核孔復(fù)合體的生物合成過程外,參與了生物體內(nèi)的諸多其他生物學(xué)過程。對家蠶中的Fox家族成員進(jìn)行選擇壓力分析,結(jié)果表明Fox家族蛋白的結(jié)構(gòu)域受到強(qiáng)烈的純化選擇。在系統(tǒng)發(fā)生分析中,依據(jù)參考序列對家蠶Fox基因家族中的每個成員進(jìn)行了命名;同時發(fā)現(xiàn)FoxK亞家族在家蠶中特異性缺失。3.BmFoxO在調(diào)控壽命中的功能探究為了探索BmFoxO基因在壽命調(diào)控過程中的作用,進(jìn)行了BmFoxO的干涉實驗。鑒于BmFoxO在家蠶羽化1天的血淋巴中表達(dá)量較高,選擇此時期的血淋巴作為BmFoxO干涉的組織,設(shè)計并合成特異的BmFoxO雙鏈RNA干涉片段(BmFoxO-dsRNA)。由于雙鏈干涉發(fā)揮效應(yīng)需要一定時間,利用家蠶外源注射技術(shù)將BmFoxO-dsRNA通過家蠶氣孔注入家蠶蛹10天的血淋巴中。結(jié)果表明,BmFoxO-dsRNA能夠顯著降低BmFoxO的表達(dá)水平,進(jìn)而顯著縮短家蠶成蟲期的壽命。為了探測BmFoxO的過表達(dá)能否延長家蠶成蟲期壽命,構(gòu)建了piggy Bac-IE1-BmFoxO-SV40過表達(dá)載體并將其導(dǎo)入蛹10天家蠶血淋巴中,BmFoxO的表達(dá)水平檢測發(fā)現(xiàn)其在注射后42h被顯著上調(diào)。壽命統(tǒng)計結(jié)果表明,與對照組相比,過表達(dá)BmFoxO能夠顯著延長家蠶成蟲期的平均壽命和最大壽命。利用Crispr-Cas9技術(shù)對BmFoxO進(jìn)行敲除,分子檢測表明G1代家蠶中BmFoxO的基因組序列確實發(fā)生了編輯。后續(xù)研究需要確定其能否對G2代家蠶的壽命產(chǎn)生影響。4.BmFoxO的靶基因篩選及其對壽命的影響B(tài)mFoxO表達(dá)水平的升高能夠顯著延長家蠶個體壽命已經(jīng)得到了證實,然而其延長壽命的分子機(jī)制仍需要進(jìn)一步研究。由于BmFoxO屬于轉(zhuǎn)錄因子,其在執(zhí)行功能過程中需要通過調(diào)節(jié)下游靶基因的轉(zhuǎn)錄活性來實現(xiàn),因此FoxO下游靶基因的篩查和鑒定將為FoxO調(diào)控壽命的機(jī)制解析提供清晰的方向。構(gòu)建BmFoxO組成型過表達(dá)和干涉載體,轉(zhuǎn)染家蠶Bm N-SWU1細(xì)胞,經(jīng)抗生素篩選后,獲得了較為穩(wěn)定的BmFoxO組成型過表達(dá)和干涉的細(xì)胞系。免疫熒光和western實驗檢測發(fā)現(xiàn),BmFoxO組成型過表達(dá)載體轉(zhuǎn)染后表達(dá)的蛋白位于細(xì)胞核中,能夠發(fā)揮調(diào)控靶基因的功能。定量檢測確認(rèn)組成型過表達(dá)和干涉細(xì)胞系中BmFoxO的表達(dá)水平,相比于對照組確實被上調(diào)和下調(diào)。就BmFoxO組成型過表達(dá)和干涉的細(xì)胞系取材提取RNA,進(jìn)行表達(dá)譜測序。結(jié)合本小組唐冬梅對表達(dá)譜測序結(jié)果的分析所發(fā)現(xiàn)的符合正調(diào)控或負(fù)調(diào)控的差異表達(dá)基因共212個;我們結(jié)合果蠅Fox O靶基因的對比分析和多層篩選,最終選出家蠶和果蠅靶基因中共有的基因2個和家蠶中特有的基因5個。對篩選到的這7個基因的上游調(diào)控序列進(jìn)行克隆,成功構(gòu)建其中5個基因的雙熒光素酶報告載體。雙熒光素酶報告結(jié)果顯示,5個篩選到的基因均受到BmFoxO的調(diào)控,其中BGIBMGA002356基因受到的調(diào)控最為強(qiáng)烈。為了探測BGIBMGA002356基因是否參與家蠶壽命的調(diào)控過程,在家蠶蛹10天對其進(jìn)行了基因的雙鏈干涉實驗。結(jié)果表明干涉BGIBMGA002356基因后,其在羽化后12h的表達(dá)水平被顯著下調(diào),同時家蠶成蟲期壽命被顯著縮短。綜上所述,我們證明了BmFoxO基因能夠調(diào)控家蠶壽命,同時鑒定到FoxO下游的一個新的靶基因BGIBMGA002356,并通過實驗證明了該靶基因受到BmFoxO的直接調(diào)控,且能夠調(diào)控家蠶壽命。我們推測BGIBMGA002356可能參與了BmFoxO調(diào)控家蠶壽命的過程,這極有可能是FoxO調(diào)控壽命的一條新的路徑。本研究不僅進(jìn)一步豐富了我們對FoxO調(diào)控壽命機(jī)制的理解,同時也為利用家蠶作為壽命研究的實驗動物提供了重要依據(jù)。
[Abstract]:Experimental animals are the important foundation and strong support of modern life science research. With the development of science and technology, more and more experimental animals have been applied to the research of life science. For a long time, it has been a dream of human pursuit. Therefore, the research of aging and life determining machine system has always been a hot spot in the field of life science. The invertebrate models used for life study mainly include Caenorhabditis elegans and Drosophila melanogaster, and vertebrate models mainly include mice and monkeys. Because of the complexity of aging and life regulation mechanisms, the use of model organisms in this field is crucial. A few invertebrate models are not fully satisfied with the needs of the complex life mechanism research. The development and utilization of new experimental animals are very necessary. Thousands of years of domestication and silkworm development endowed the silkworm with unique biological characteristics and cultural connotations. The collection and research have accumulated rich mutation resources, and first carried out the genome and functional genome research, which provided a good foundation for the development and utilization of the silkworm. At the same time, the silkworm has many characteristics of the experimental animals, such as the life cycle and the small size of the individual, the strong fecundity, the clear boundaries of the development stage, and the external development. The physical damage caused by the injection is smaller, the life life is more plastic and there is no ethical problem. In this case, this study chose the silkworm as an experimental animal to identify the important life span gene and the mechanism of the analysis gene regulation of the life of silkworm, and provide new cognition for the interpretation of the molecular mechanism of life determination. The main results are as follows: 1. The relationship between the life of silkworm and the expression of BmFoxO gene was different. The life span of each strain of silkworm was different. A group of strains were selected from the silkworm gene bank for life statistics, and the three representative lines (Xia Fang, Dazao-N, N4) were investigated for the life Xiang Guanji expression pattern. The results showed that the average life of Xia Fang was the average life of the silkworm. Life and maximum life span are longer, Dazao-N times, the average life span and maximum life span of the N4 strain are shorter. Given that Fox O is reported to be a key factor in regulating life in many species, we investigated the level of BmFoxO expression at different developmental stages in these strains, and found that the 10 day of the pupa (the time point for the life of the adult), the table of BmFoxO The amount of summer fragrance was significantly higher than that of Dazao-N and N4, and the N4 was significantly lower than that of Dazao-N.. In the other developmental period of the investigation, the expression trend of BmFoxO was also highly consistent with the life of the corresponding strain. By controlling the dietary intake of silkworm, the dietary restriction model of silkworm was successfully constructed. The life statistics showed that the diet restricted group longevity. The expression of BmFoxO was significantly higher than that in the control group, and the expression of BmFoxO was significantly higher than that in the control group. In view of the significant correlation between the expression and life span of the silkworm, the gene played a role in the identification and evolution of the.2. family of the silkworm, silkworm, silkworm, silkworm, Bombyx mori Fox gene family, and the Fox gene encoded a transcription factor family, including 23 subfamilies (Fox A-Fox S). In the silkworm, the whole genome of the Fox gene family was analyzed by the hidden Markov model. The results showed that there were 17 Fox family members in the silkworm. Through phylogenetic analysis, the 17 Fox family genes were classified into 13 Fox subfamilies. On the same chromosomes, the same hidden Markov model is used to identify the Fox gene family members of the red band sleeves, monarch butterflies, Drosophila, mice and humans, and use these sequences to construct a phylogenetic tree. The results show that the sequence of direct homologous genes in different species is highly conserved. At the same time, the structure and protein of the Fox family genes in the silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm and silkworm The analysis of the qualitative domain characteristics shows that most of the Fox genes in the silkworm belong to the mono exon gene and the protein contains the conservative Fork head domain. The GO annotation of the Fox family gene in the silkworm showed that the encoding product was not involved in the biosynthesis process of the nucleus pore complex and participated in many other biological processes in the raw silkworm. The Fox family members selected pressure analysis. The results showed that the domain of the Fox family protein was strongly purified. In the phylogenetic analysis, each member of the Fox gene family of the silkworm was named according to the reference sequence, and the specific deletion of the FoxK subfamily in the silkworm, the function of.3.BmFoxO in the regulation of the life span of the silkworm. In order to explore the role of the BmFoxO gene in the life regulation process, the BmFoxO interference experiment was carried out. In view of the high expression of BmFoxO in the 1 day blood drenching of the silkworm, the haemolymph of this period was selected as the tissue of BmFoxO interference, and the specific BmFoxO double stranded RNA interference fragment (BmFoxO-dsRNA) was designed and synthesized. The volatiles needed a certain time, using the Bombyx mori injection technique to inject BmFoxO-dsRNA through the silkworm blowhole into the blood Bazhong for 10 days. The results showed that BmFoxO-dsRNA could significantly reduce the expression level of BmFoxO and significantly shorten the life span of the silkworm adult period. The over expression vector of piggy Bac-IE1-BmFoxO-SV40 was constructed and introduced into the silkworm haemolymph of the pupae for 10 days. The expression level of BmFoxO showed that the 42h was significantly up-regulated after the injection. The results of life expectancy showed that the overexpression of BmFoxO could significantly prolong the life span and the maximum life span of the silkworm adult period compared with the control group. Crispr-Cas9 BmFoxO was knocked out by technology, and molecular detection showed that the genome sequence of BmFoxO in the G1 silkworm had indeed been edited. Subsequent studies need to determine whether the target gene screening for the life span of the silkworm of the G2 generation and its impact on the life span of the silkworm, and the increase of the expression level of BmFoxO can significantly prolong the life of the silkworm individual. It is confirmed that the molecular mechanism of its extended life needs further study. Since BmFoxO belongs to the transcription factor, it needs to be realized by regulating the transcriptional activity of the downstream target gene in the execution function process. Therefore, the screening and identification of the target gene of the downstream FoxO will provide a clear direction for the mechanism analysis of FoxO regulation life. Construction of B MFoxO type overexpression and interference carrier, transfected with Bm N-SWU1 cells of silkworm, obtained a more stable BmFoxO component over expression and interference cell lines. Immunofluorescence and Western test found that the protein expressed in the BmFoxO component overexpression vector was located in the nucleus and could regulate the target group. Function. Quantitative detection confirmed the expression level of BmFoxO in the component overexpression and interference cell lines. Compared to the control group, it was actually up and down. The cell lines of the BmFoxO components overexpressed and interfered were extracted and sequenced, and the expression spectrum was sequenced. The results of Tang Dongmei's analysis of the results of Tang Dongmei's sequence analysis of Fu Hezheng There are 212 differentially expressed genes regulated or negatively regulated; we combine the comparative analysis of the Fox O target gene of Drosophila melanogaster and multilayer screening, and finally select 2 genes of the silkworm and Drosophila target genes and 5 specific genes in the silkworm. The upstream regulation sequence of the selected 7 genes is cloned, and 5 genes are successfully constructed. The results of the double Luciferase Report showed that all the 5 screened genes were regulated by BmFoxO, and the BGIBMGA002356 gene was most regulated. In order to detect whether the BGIBMGA002356 gene was involved in the regulation of the life of the silkworm, the double chain interference experiment of the gene was carried out at the home of the silkworm chrysalis for 10 days. After the interference of BGIBMGA002356 gene, the expression level of 12h was significantly reduced after the emergence of the 12h, while the life span of the adult silkworm was shortened significantly. In summary, we proved that the BmFoxO gene could regulate the life of the silkworm, and also identified a new target gene BGIBMGA002356 in the downstream of the FoxO, and the target gene was proved to be BmFox by the experiment. The direct regulation of O and the ability to regulate the life of silkworm, we speculate that BGIBMGA002356 may be involved in the process of BmFoxO regulation of the life of silkworm, which is very likely to be a new path for the regulatory life of FoxO. This study not only further enriched our understanding of the mechanism of FoxO's regulatory life, but also for the use of silkworm as a life study. Animal testing provides an important basis.

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：Q963

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1 宋江波;BmFoxO基因調(diào)控家蠶壽命的分子機(jī)制研究[D];西南大學(xué);2017年

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本文編號：1874289