家蚕卵壳基因鉴定、功能及其表达调控研究

发布时间：2018-06-05 02:39

本文选题：家蚕 + 卵壳基因　；参考：《西南大学》2017年博士论文

【摘要】：昆虫种类繁多、数量巨大,是地球生物多样性的重要贡献者。在长期的进化过程中,昆虫与人类形成了复杂而密切的关系。一方面,昆虫为人类提供了大量的物质产品,其中鳞翅目昆虫家蚕也创造了璀璨的蚕丝文化;另一方面,昆虫也给人类造成了重大的经济损失,其中直翅目昆虫蝗虫更是书写了一部人类农业社会的血泪史。昆虫数量多种类丰富的主要原因是惊人的产卵能力,大部分昆虫的产卵量高达数百颗。因此,对昆虫卵的研究将为有害昆虫的防治,有益昆虫产卵量的提高提供重要的理论基础。卵壳是昆虫卵细胞表面复杂的保护性结构,主要成分为卵壳蛋白,其编码基因为卵壳基因。本研究以鳞翅目家蚕为研究对象,关注了卵壳基因的功能与调控。首先,鉴定了家蚕卵壳基因并绘制了其时期表达模式图谱,利用LC-MS/MS,分析了卵壳的蛋白质组成;其次,在功能研究上,以卵壳基因突变体桂灰卵(Grk)为材料,分析了其可能的突变机理;最后,在表达调控上,以miRNA为研究切入点,重点分析了miRNA在卵壳基因表达调控中的角色。本研究获得的主要研究结果如下:1.卵壳基因的鉴定及进化分析家蚕卵壳基因是一个超基因家族,所有成员都位于2号染色体上的一段基因组区域内(卵壳基因位点)。卵壳基因经历了近40年的研究,近百个卵壳基因被鉴定。然而,高度的重复序列使卵壳基因位点测序非常困难,至今未被完整测序,这严重阻碍了卵壳基因全面、系统的研究。为此,本研究采用了对BAC测序的策略获得了卵壳基因位点全长序列。首先,分析了家蚕BAC文库中卵壳基因位点上200多个BAC的分布,筛选出7个能覆盖完整卵壳基因位点的BAC。通过对7个BAC的测序,拼接组装得到了871,711 bp的卵壳基因位点序列,利用FGENESH软件对卵壳基因位点序列进行基因预测,注释到127个卵壳基因和5个非卵壳基因,其中卵壳基因标注为BmCho-1~BmCho-127,包括36个早期、46个中期和45个后期卵壳基因。两个卵发育时期的cdna文库被用于进一步确认卵壳基因est的存在,反向证实了注释的卵壳基因。卵壳基因主要以基因对(genepair)的形式分布在基因组上。两个后期卵壳基因因编码区有多个终止密码子被成为假基因(pseudogene)。进化树分析结果显示同类型的卵壳基因聚成一簇,其中早期b类卵壳基因(earlyb)在进化树上分为两个分支,其中一支和中期b类卵壳基因(middleb)聚到一个大的分支上。卵壳基因的表达模式显示,这个早期b类卵壳基因分支中的7个基因有着中期卵壳基因相似的时期表达模式。进化树分析也揭示早期卵壳基因经历着较快的进化速度,后期卵壳基因在进化上相对保守。两个假基因bmcho-23和bmcho-26也经历了较快的进化速度。完整卵壳基因位点的鉴定是卵壳基因全面、系统研究的重要基础,本部分后面的研究都是以此为基础的。2.卵壳基因的表达特征分析为了分析卵壳基因在卵发育过程中的表达情况,我们构建了家蚕卵壳基因的精细表达模式图。首先,我们对蛹8天不同发育时期的卵进行了时期分段,因卵黄生成期(vitellogenesis)无卵壳基因表达,此时期的卵呈微黄色;卵壳生成期(choriogenesis)为卵壳基因次序表达的时期,此时因卵壳的形成使卵色发白发亮,这是卵黄与卵壳生成期分段的标志。根据卵壳基因表达的特征,将卵壳生成期细分为i~xi的11个发育时期。利用rt-pcr对鉴定的127个卵壳基因进行了时期表达模式图谱的构建,结果显示,早期和后期卵壳基因分别在卵壳生成期的早期(i~iii)和后期(ix~xi)表达,而中期卵壳基因则在整个卵壳生成期皆有表达。通过对家蚕cdna库搜索,鉴定到bmcho-1和bmcho-96在精巢中的转录本。卵泡上皮细胞和精巢中,bmcho-1的转录本有着相同的第二外显子序列,第一外显子则不同,bmcho-1的精巢转录本第一外显子位于卵泡上皮细胞转录本的内含子区域。精巢中bmcho-96的转录本有三个外显子,精巢和卵泡上皮细胞中转录本的编码区序列相同,预测将编码相同的蛋白质。在家蚕卵胚胎的cdna文库中,鉴定到bmcho-11的转录本,胚胎中的转录本编码区序列在5’端比卵泡上皮细胞中的转录本多27个碱基,预测多编码9个氨基酸。分析发现,几乎每个后期卵壳基因除了成熟的转录本之外,还有一个拼接中间体形式(splicingintermediate),这种结构包含5’utr、外显子、内含子、3’utr和多聚腺嘌呤序列。3.卵壳结构的蛋白质组分析通过lc-ms/ms质谱分析,在大造(p50)卵壳中鉴定到260个蛋白,包括88个卵壳蛋白、28个卵巢特异性蛋白(非卵壳蛋白)和144个其它类型的蛋白。家蚕卵壳基因主要以基因对(genepair)的形式分布在基因组上,每个基因对包含一个a类和一个b类基因,两个基因共用5’侧翼的启动子区域,a和b基因以相反的方向转录。在鉴定的卵壳蛋白中,基因对中b编码的蛋白更容易被鉴定到,这暗示双向启动子对b基因可能有更强的转录活性。卵壳中也鉴定到的一类卵巢特异的蛋白,编码这类蛋白的基因主要成簇地分布在2号、10号、15号和16号染色体上,但多数为功能未知的家蚕特异基因。在2号染色体的卵壳基因位点上游100kb的范围内分布着12个卵巢特异基因,其中的10个基因编码的蛋白在卵壳中被鉴定到,虽然它们的功能未知,但推测它们在卵壳基因的表达或卵壳结构形态构建中扮演着重要的作用。4.家蚕桂灰卵(grk)突变机理初探桂灰卵(grk)是家蚕卵壳基因突变导致的灰色卵突变体,突变基因位于卵壳基因位点内(2-7.2)。表现型为:同质型(grk/grk)卵为球形,受精率低;异质型(grk/+)卵形正常,卵色为灰色,受精率正常;正常型(+/+)卵色为深褐色。sem观察显示,grk三种基因型卵壳表面没有明显的差异,而它们的卵壳层和卵孔处有较大差异。在卵壳层上,grk正常型的卵壳层由平行于卵细胞的片层构成;而杂合型卵壳中间层的片层呈现垂直于卵细胞表面的表型;纯合型卵壳中间片层则出现了更大程度的破坏,几乎呈现紊乱的状态。在卵孔结构上,grk正常型卵孔被花瓣状斑纹围绕,内层的斑纹凸出卵壳表面,卵孔出有4个卵孔管口。杂合型卵孔周围的花瓣状斑纹明显比正常型小,且内层的斑纹凸出程度减小,卵孔管口个数正常;纯合型卵孔处的斑纹同样变小,内层的斑纹几乎无凸出的表型,卵孔管口仅为2个。grk正常型、杂合型和纯合型卵孔内层斑纹的花瓣数依次减少。通过对grk卵壳的观察,我们可以推断grk突变体在卵壳壳层结构发生的较大变化是导致其呈现灰色卵表型的基础,卵孔管口的减少则是卵受精率大大降低的原因。对卵壳结构蛋白提取时发现grk正常型、杂合型及纯合型卵壳结构在8m尿素/35mmdtt裂解液中溶解度依次降低,在此裂解液基础上增加dtt浓度,将增加grk突变体溶解度,但不能完全溶解。如果增加1%的sds,则能完全溶解grk突变体卵壳。这表明grk突变体卵壳结构的变化可能影响了卵壳蛋白的溶解。grk三种基因型卵壳的定量蛋白质组学分析显示,bmcho-20、bmcho-41、bmcho-57、bmcho-62、bmcho-73和bmcho-115在grk正常型、杂合型和纯合型卵壳中的含量呈依次增加,其中BmCho-20和BmCho-115为早期卵壳蛋白,BmCho-73为中期卵壳蛋白,BmCho-41、BmCho-57和BmCho-62为后期卵壳蛋白。富含半胱氨酸的后期卵壳蛋白主要分布在卵壳外层,其丰富的二硫健使卵壳形成坚硬的外层结构,因此,3个后期卵壳蛋白的上调增加了Grk突变体卵壳的硬度,使其更难溶解。同时,卵壳基因具有严格的时期特异性,如果Grk通过延长表达时间来上调卵壳蛋白,则将会扰乱卵壳形态构建的进程,造成卵壳结构的改变。另一方面,BmCho-17在正常型和杂合型卵壳中有表达,但在纯合型中丢失,且BmCho-17是中期卵壳蛋白,应该在卵壳中层构建中起作用。因此它在Grk纯合型卵壳中的丢失势必影响到中层卵壳的形态构建。5.miRNA对卵壳基因表达调控分析卵壳基因表达有着严格的组织和时期特异性,其表达调控研究是最早的模式基因之一。但已报道的调控因子仍不能完全清楚地解释数量庞大的卵壳基因的调控机制。为此,本研究分析了miRNA在卵壳基因表达调控中的角色,通过高通量测序,在家蚕蛹8天卵壳生成期的卵泡上皮细胞中鉴定到847个miRNA,其中包括399个已知miRNA和448个新鉴定的mi RNA。qPCR结果显示,33个mi RNA在卵泡上皮细胞特异表达。靶基因预测显示miRNA潜在的调控了所有类型的卵壳基因及其转录因子,分析也发现,在miRNA对卵壳基因的调控中,存在着一个miRNA基于相同的靶位点序列调控一类卵壳基因的模式。miRNA与卵壳蛋白质组整合分析发现,miRNA也参与了编码卵壳中其它结构蛋白的基因的调控。双荧光素酶实验表明,bantam-3p调控了家蚕卵壳基因转录因子BmC/EBP的表达。本部分研究结果表明,miRNA参与了卵壳基因的表达调控。
[Abstract]:Insects are an important contributor to the biodiversity of the earth. In the long period of evolution, insects and human beings have formed a complex and close relationship. On the one hand, insects provide a large amount of material products for human beings, in which the Lepidoptera silkworm also creates a bright silk culture; on the other hand, insects are also given to people. The class of Orthoptera insects locusts have written a history of blood and tears in a human agricultural society. The main reason for the abundance of insects is the ability to spawn, and the number of eggs of most insects is up to hundreds. Therefore, the study of insect eggs will be the prevention and control of harmful insects, which will benefit the eggs. The improvement of quantity provides an important theoretical basis. The egg shell is a complex protective structure on the surface of the egg cell of the insect. The main component is egg shell protein and its encoding gene is the egg shell gene. This study focuses on the function and regulation of the egg shell gene. First, the egg shell gene of the silkworm was determined and its expression was drawn. The pattern map, using LC-MS/MS, analyzed the protein composition of the egg shell; secondly, in the functional study, the mutation mechanism of the egg shell gene mutant cinnamon ash egg (Grk) was analyzed. Finally, in the expression and regulation, the role of miRNA in the regulation of the expression of the egg shell gene was analyzed with miRNA as the research entry point. The main results are as follows: 1. the identification and evolution of the egg shell gene, the egg shell gene of the silkworm is a supergene family. All the members are located in a section of the genome on chromosome 2 (eggshell gene loci). The egg shell gene has been studied for nearly 40 years, and nearly one hundred egg shell genes have been identified. However, the high repetition sequence makes the egg. The sequencing of the shell gene site is very difficult and has not been completely sequenced so far. This has seriously hindered the comprehensive and systematic study of the egg shell genes. Therefore, this study adopted the strategy of BAC sequencing to obtain the full length sequence of the egg shell gene loci. First, the distribution of more than 200 BAC on the egg shell gene loci in the BAC Library of the silkworm was analyzed, and 7 can be screened. The BAC. of the complete egg shell gene site was sequenced by 7 BAC, and the egg shell gene loci of 871711 BP were sequenced and assembled. The gene site sequence of the egg shell gene was predicted by FGENESH software, and 127 egg shell genes and 5 non egg shell genes were annotated. The egg shell gene was labeled as BmCho-1~BmCho-127, including 36 early and 46. The medium-term and 45 late egg shell genes. The cDNA library of the two egg development period was used to further confirm the existence of the egg shell gene EST, and the annotated eggshell gene was confirmed reverse. The egg shell gene is mainly distributed on the genome in the form of gene pair (genepair). The two later egg shell genes are false bases due to the number of terminating codons in the coding region. Pseudogene. Evolutionary tree analysis shows that the same type of egg shell gene is clustered into a cluster, in which the early B egg shell gene (earlyb) is divided into two branches in the evolutionary tree, of which one and medium-term B egg shell gene (middleb) is clustered into a large branch. The expression pattern of the egg shell gene shows that 7 of the early B egg shell gene branch is in the expression pattern. The evolutionary tree analysis also revealed that the early egg shell gene had a fast evolution speed and the later egg shell gene was relatively conservative in evolution. The two pseudogenes bmcho-23 and bmcho-26 also experienced a rapid evolutionary speed. The identification of the complete egg shell gene loci was the comprehensive egg shell gene. In order to analyze the expression of the egg shell gene in the process of egg development, we have constructed the fine expression pattern of the egg shell gene of the silkworm, which is based on the analysis of the expression characteristics of the egg shell gene in the egg development process. First, we have divided the eggs of the 8 days of the pupae at 8 different developmental stages. The egg of the egg yolk generation period (vitellogenesis) has no egg shell gene expression, the egg is yellowish in this period; the egg shell generation period (choriogenesis) is the time of the egg shell gene sequence expression, and the egg color brightens by the formation of the egg shell, which is the symbol of the egg yolk and the egg shell generation period. The 11 developmental stages of i~xi were subdivided into 127 egg shell genes identified by RT-PCR. The results showed that the early and late eggshell genes were expressed in the early stage (i~iii) and late stage (ix~xi) of the eggshell period, while the medium-term eggshell was expressed in the whole eggshell period. DNA library search has identified the transcriptional transcripts of bmcho-1 and bmcho-96 in the spermary. In the follicle epithelial cells and the sperms, the bmcho-1 transcript has the same exon sequence. The first exon is different. The bmcho-1 transcriptional first exon is located in the intron of the follicle epithelial cell transcript. The bmcho-96 transcript in the sperm nest is transcribed. In the cDNA library of the home egg embryo, the bmcho-11 transcript is identified in the cDNA library of the eggs of the family silkworm eggs. The sequence of the transcriptional coding region in the embryo is 27 bases in the 5 'end than in the follicle epithelial cells, and 9 multiple coding sequences are predicted. In addition to the mature transcriptional transcript, the amino acid analysis found that in addition to the mature transcriptional transcript, there is a splice intermediate form (splicingintermediate), which contains 5 'UTR, exons, introns, 3' UTR and polyadenine sequence.3. egg white matter analysis by lc-ms/ms mass spectrometry, in P50 eggs 260 proteins are identified in the shell, including 88 egg shell proteins, 28 ovarian specific proteins (non egg shell proteins) and 144 other types of proteins. The silkworm egg shell gene is mainly distributed on the genome in the form of gene pair (genepair), each gene contains a Class A and one B gene, and two genes share the 5 'flanking promoter region. Domain, a and B genes are transcribed in the opposite direction. In the identified egg shell protein, the gene encoding B in the gene is more easily identified, suggesting that the bi-directional promoter may have stronger transcriptional activity to the B gene. A specific type of ovarian specific protein is also identified in the egg shell. The genes encoding this kind of protein are mainly distributed in number 2, No. 10, and 15. On chromosome number and chromosome 16, most of them are specific genes of the silkworm, which are unknown in function. 12 ovarian specific genes are distributed within the range of the 100kb upstream of the egg shell gene site of chromosome 2. 10 of them are encoded in the egg shell. Although their functions are not known, they are presumed to be expressed in the egg shell gene or in the egg shell. Structural form construction plays an important role in the mutation mechanism of the.4. silkworm egg (Grk). The Grk is a gray egg mutant caused by the mutation of the egg shell gene of the silkworm. The mutant gene is located in the egg shell gene locus (2-7.2). The phenotype is: the homogenous (grk/grk) egg is spherical and the fertilization rate is low; the heterotype (grk/+) ovum is normal and the egg color is The fertilization rate is normal, and the normal type (+ / +) egg color to the dark brown.Sem observation shows that there is no obvious difference in the surface of the three genotypes of Grk, but the egg shell and the oval holes are different. On the egg shell, the normal type of Grk is made up of the lamellar parallel to the egg cell, while the lamellar layer of the heterozygous egg shell is perpendicular to the layer. The phenotype of the surface of the egg cell; the middle layer of the homozygous egg shell has a greater degree of destruction and almost disorganized. On the oval structure, the Grk normal oval holes are surrounded by petal markings, the stripes of the inner layer protrude the oval surface, and the ovum has 4 oval orifice. The petal pattern around the heterozygote is obviously more than the normal type. The size of the markings in the inner layer is smaller and the number of the oval holes is normal; the markings at the oval holes are also smaller, the markings in the inner layer are almost no protruding phenotypes, the oval orifice is only 2.Grk normal, and the number of petals in the heterozygous and homozygous oval inner layer decreases in turn. By observing the Grk egg shell, we can infer the Grk process. The large change in the shell shell structure of the egg shell is the basis of the appearance of the grey egg phenotype. The decrease of the orifice of the ovum is the reason why the fertilization rate of the egg is greatly reduced. When the egg shell structure protein is extracted, the Grk normal type is found, the heterozygous type and the homozygous egg shell structure in the 8m urea / 35mmdtt lysate decreases in turn. Increasing the concentration of DTT on the basis of liquid, it will increase the solubility of Grk mutant, but can not completely dissolve. If increase of 1% SDS, it can completely dissolve the Grk mutant egg shell. This indicates that the changes in the eggshell structure of the Grk mutant may affect the dissolving of the eggshell protein of the three genotypes of the egg shell of the egg shell protein, the bmcho-20, bmcho-41, BM. The contents of cho-57, bmcho-62, bmcho-73 and bmcho-115 in normal Grk, heterozygous and homozygous egg shells are increased in turn, of which BmCho-20 and BmCho-115 are early egg shell proteins, BmCho-73 is medium egg shell protein, BmCho-41, BmCho-57 and BmCho-62 are later egg shell proteins. The late egg shell protein rich in cysteine is mainly distributed outside the egg shell. The rich two sulphur health causes the egg shell to form a hard outer structure. Therefore, the up-regulation of the 3 later egg shell proteins increases the hardness of the Grk mutant egg shell, making it more difficult to dissolve. At the same time, the egg shell gene has a strict period specificity. If Grk up-regulated the egg shell protein by prolonging the expression time, the eggshell gene will disrupt the formation of the egg shell morphology. On the other hand, BmCho-17 is expressed in the normal and heterozygous egg shells, but is lost in the homozygous type, and the BmCho-17 is the medium-term egg shell protein, which should play a role in the construction of the middle layer of the egg shell. Therefore, its loss in the Grk homozygous egg shell is bound to affect the form of the middle egg shell to construct the.5.miRNA to the egg shell. The gene expression regulation analysis of gene expression has strict tissue and period specificity, and its expression regulation is one of the earliest model genes. However, the regulatory factors that have been reported can not fully explain the regulation mechanism of a large number of egg shell genes. Therefore, this study analyzed the regulation of the expression of miRNA in the egg shell gene. Role, through high throughput sequencing, 847 miRNA were identified in the follicle epithelial cells of the 8 day egg shell generation period of the family silkworm chrysalis, including 399 known miRNA and 448 newly identified mi RNA.qPCR results, and 33 mi RNA were expressed in the follicle epithelial cells. The target gene predicted that miRNA potentially regulates all types of eggshell genes and Its transcriptional factor and analysis also found that in the regulation of the egg shell gene in miRNA, there is a miRNA integration analysis between the pattern.MiRNA and the egg shell protein group based on the same target site sequence, and the miRNA also participates in the regulation of the gene of other structural proteins in the encoding egg shell. The double luciferase experiment shows that Banta M-3P regulates the expression of BmC/EBP, a transcription factor of Bombyx mori eggshell. The results of this study show that miRNA is involved in the regulation of eggshell gene expression.
【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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