BmFoxO基因调控家蚕寿命的分子机制研究

发布时间：2018-05-11 14:13

本文选题：家蚕 + 寿命　；参考：《西南大学》2017年博士论文

【摘要】：实验动物是现代生命科学研究的重要基础和强力支撑。随着科学技术的发展,越来越多的实验动物被应用到生命科学研究中。长久以来,益寿延年一直是人类追求的梦想,因此,衰老和寿命决定机制研究一直是生命科学领域的热点。目前应用于寿命研究的无脊椎动物模型主要包括秀丽隐杆线虫和黑腹果蝇,脊椎动物模型主要包括小鼠和猴子。由于衰老和寿命调控机制的复杂性,模式生物在该领域研究中的利用至关重要。其中,身体构造简单而器官分化却相对完整的无脊椎实验动物受到广大研究者的青睐。至今,少数的无脊椎动物模型并不能完全满足复杂的寿命机制研究的需要,新型实验动物的开发利用十分必要。数千年的驯养和蚕业发展赋予了家蚕独特的生物学特点及文化内涵。长达一个世纪收集和研究积淀了丰富的突变资源,以及率先开展了基因组和功能基因组研究,为家蚕的拓展利用提供了良好的基础。同时,家蚕也具备诸多作为寿命研究的实验动物的特征,如生命周期和个体大小适中、繁殖力强、发育阶段界限清晰、外源注射所导致的物理损伤较小、寿命可塑性较强及不存在伦理问题等。有鉴于此,本研究选择家蚕作为实验动物,鉴定重要寿命基因及分析基因调控家蚕寿命的机制,为寿命决定分子机制的阐释提供新的认知。主要获得了如下研究结果:1.家蚕寿命与BmFoxO基因表达量间的关系家蚕各品系的发育时长不同,寿命也存在差异。从家蚕基因库中选取一组品系进行寿命统计,并对其中的三个代表性品系(夏芳、Dazao-N、N4)进行寿命相关基因表达模式的调查,结果显示:夏芳的平均寿命和最大寿命均较长,Dazao-N次之,N4品系的平均寿命和最大寿命较短。鉴于Fox O在多个物种中被报道是调控寿命的关键因子,我们调查了这些品系中BmFoxO在不同发育时期的表达水平,发现在蛹10天(可能决定成虫寿命的时间点),BmFoxO的表达量为夏芳显著高于Dazao-N和N4,并且N4显著低于Dazao-N。同时,在被调查的其他发育时期,BmFoxO表达趋势与相应品系的寿命也呈现出高度一致。通过控制家蚕饮食摄入量,成功构建了家蚕饮食限制实验模型;寿命统计结果表明饮食限制组寿命明显长于对照组,并且BmFoxO表达量显著高于对照组。鉴于BmFoxO的表达与寿命显著相关,所以推测该基因发挥了调控家蚕寿命的功能。2.家蚕Fox基因家族的鉴定与进化分析Fox基因编码的是一个转录因子家族,包含23个亚家族(Fox A-Fox S)。在家蚕中利用隐马尔可夫模型对Fox基因家族成员进行全基因组的分析鉴定,结果发现家蚕中含有17个Fox家族成员。通过系统发生分析,将这17个Fox家族基因归类于13个Fox亚家族。对其所属的染色体位置进行分析发现这些成员分布于13条不同的染色体上。利用相同的隐马尔可夫模型识别红带袖蝶、帝王蝶、果蝇、小鼠和人类中的Fox基因家族成员,并利用这些序列构建系统发生树,结果表明不同物种中的直系同源基因间序列高度保守。同时,对家蚕中Fox家族基因的结构和蛋白质结构域特征分析,表明家蚕中Fox基因大多属于单外显子基因,蛋白均包含有保守的Fork head结构域。家蚕中Fox家族基因的GO注释表明,其编码产物除未参与核孔复合体的生物合成过程外,参与了生物体内的诸多其他生物学过程。对家蚕中的Fox家族成员进行选择压力分析,结果表明Fox家族蛋白的结构域受到强烈的纯化选择。在系统发生分析中,依据参考序列对家蚕Fox基因家族中的每个成员进行了命名;同时发现FoxK亚家族在家蚕中特异性缺失。3.BmFoxO在调控寿命中的功能探究为了探索BmFoxO基因在寿命调控过程中的作用,进行了BmFoxO的干涉实验。鉴于BmFoxO在家蚕羽化1天的血淋巴中表达量较高,选择此时期的血淋巴作为BmFoxO干涉的组织,设计并合成特异的BmFoxO双链RNA干涉片段(BmFoxO-dsRNA)。由于双链干涉发挥效应需要一定时间,利用家蚕外源注射技术将BmFoxO-dsRNA通过家蚕气孔注入家蚕蛹10天的血淋巴中。结果表明,BmFoxO-dsRNA能够显著降低BmFoxO的表达水平,进而显著缩短家蚕成虫期的寿命。为了探测BmFoxO的过表达能否延长家蚕成虫期寿命,构建了piggy Bac-IE1-BmFoxO-SV40过表达载体并将其导入蛹10天家蚕血淋巴中,BmFoxO的表达水平检测发现其在注射后42h被显著上调。寿命统计结果表明,与对照组相比,过表达BmFoxO能够显著延长家蚕成虫期的平均寿命和最大寿命。利用Crispr-Cas9技术对BmFoxO进行敲除,分子检测表明G1代家蚕中BmFoxO的基因组序列确实发生了编辑。后续研究需要确定其能否对G2代家蚕的寿命产生影响。4.BmFoxO的靶基因筛选及其对寿命的影响BmFoxO表达水平的升高能够显著延长家蚕个体寿命已经得到了证实,然而其延长寿命的分子机制仍需要进一步研究。由于BmFoxO属于转录因子,其在执行功能过程中需要通过调节下游靶基因的转录活性来实现,因此FoxO下游靶基因的筛查和鉴定将为FoxO调控寿命的机制解析提供清晰的方向。构建BmFoxO组成型过表达和干涉载体,转染家蚕Bm N-SWU1细胞,经抗生素筛选后,获得了较为稳定的BmFoxO组成型过表达和干涉的细胞系。免疫荧光和western实验检测发现,BmFoxO组成型过表达载体转染后表达的蛋白位于细胞核中,能够发挥调控靶基因的功能。定量检测确认组成型过表达和干涉细胞系中BmFoxO的表达水平,相比于对照组确实被上调和下调。就BmFoxO组成型过表达和干涉的细胞系取材提取RNA,进行表达谱测序。结合本小组唐冬梅对表达谱测序结果的分析所发现的符合正调控或负调控的差异表达基因共212个;我们结合果蝇Fox O靶基因的对比分析和多层筛选,最终选出家蚕和果蝇靶基因中共有的基因2个和家蚕中特有的基因5个。对筛选到的这7个基因的上游调控序列进行克隆,成功构建其中5个基因的双荧光素酶报告载体。双荧光素酶报告结果显示,5个筛选到的基因均受到BmFoxO的调控,其中BGIBMGA002356基因受到的调控最为强烈。为了探测BGIBMGA002356基因是否参与家蚕寿命的调控过程,在家蚕蛹10天对其进行了基因的双链干涉实验。结果表明干涉BGIBMGA002356基因后,其在羽化后12h的表达水平被显著下调,同时家蚕成虫期寿命被显著缩短。综上所述,我们证明了BmFoxO基因能够调控家蚕寿命,同时鉴定到FoxO下游的一个新的靶基因BGIBMGA002356,并通过实验证明了该靶基因受到BmFoxO的直接调控,且能够调控家蚕寿命。我们推测BGIBMGA002356可能参与了BmFoxO调控家蚕寿命的过程,这极有可能是FoxO调控寿命的一条新的路径。本研究不仅进一步丰富了我们对FoxO调控寿命机制的理解,同时也为利用家蚕作为寿命研究的实验动物提供了重要依据。
[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.

【学位授予单位】：西南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q963

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