家蚕C8表皮形态突变体相关基因的鉴定与分析

发布时间：2018-03-12 13:22

本文选题：近等基因系　切入点：表皮形态突变　出处：《安徽农业大学》2016年硕士论文　论文类型：学位论文

【摘要】：家蚕(Bombyx mori)属于鳞翅目、蚕蛾科,是鳞翅目昆虫的典型代表。随着家蚕基因组精细图的完成,人们对家蚕的研究正式进入后基因组时代。家蚕有400多种突变品系,其中表皮形态的突变体有枝蚕、竹蚕、石蚕等。对于引起表皮形态突变的基因及调控机制的研究尚处于起步阶段,许多表皮形态突变体的形成机制还有待研究。本研究以一种家蚕表皮形态突变体构建的近等基因系C8与其回交亲本C108为材料(家蚕C8与其回交亲本C108相比具有几个明显特征:体型细短、表皮硬实等),通过扫描电镜观察C8与C108表皮之间的形态结构差异;再以SDS-PAGE结合质谱分析鉴定在C8与C108之间差异表达的表皮蛋白,再通过对其cDNA序列的相似性分析,鉴定可能的突变基因,并用RT-qPCR分析其差异表达模式;进一步对候选基因进行连锁遗传分析,研究结果如下:1.C8表皮外表面上的外突比C108的更为明显,但C8的表皮比较致密,这种致密的表皮结构可能是引起C8表皮硬实的原因。2.利用SDS-PAGE筛选出8条在C8和C108表皮中差异表达的蛋白条带,并通过MALDI-TOF-MS质谱分析获得9个蛋白,其中含有3个表达差异的表皮蛋白:Bm CPH34、BmCPG4和BmCPR41,BmCPH34在C8中的表达量高于C108,而Bm CPG4和BmCPR41在C8中的表达量低于C108。3.对3个表皮蛋白基因:BmCPH34、BmCPG4和BmCPR41以及前期获得的在C8与C108中差异表达的表皮蛋白基因BmCPR2的cDNA序列进行克隆测序,结果表明:BmCPR2与BmCPR41基因所编码的氨基酸序列在C8与C108以及SilkDB数据库中的P50之间并没有差异;BmCPG4的基因的cDNA序列在C108和P50中存在较大差异,而在C8与P50之间只有1个碱基发生替换,并引起1个氨基酸残基发生突变。推测这3个基因并不是引起C8突变表型的直接因素。与C108和P50相比,BmCPH34基因的cDNA序列在C8中存在15 bp的碱基重复,并有9个碱基的替换,在其编码的氨基酸序列中存在5个氨基酸残基的重复,并引起2个位点的氨基酸的突变。我们推测,BmCPH34基因可能参与C8表皮形态突变的形成。4.利用RT-qPCR技术分别检测这4个表皮蛋白基因在C8与C108之间的表达差异,结果显示:4个表皮蛋白基因在C8中的表达量均高于C108,并且达到差异显著水平,表明这4个表皮蛋白基因在一定程度上参与C8表皮形态突变的形成。进一步对BmCPH34基因的发育时相分析表明该基因主要在家蚕的头、表皮、脂肪体和气管丛中表达,在家蚕幼虫四龄时表达量最高。因此,推测BmCPH34基因与C8表皮形态的形成存在着一定的联系。综上所述,在本研究中,我们共鉴定出4个可能与C8表皮形态突变相关的基因:BmCPH34、BmCPG4、BmCPR2和BmCPR41。但C8表皮形态形成的具体原因还需进一步研究。
[Abstract]:Bombyx morii (Bombyx mori) belongs to Lepidoptera, Silkworm Moth, which is a typical representative of Lepidoptera. With the completion of genome fine map of Bombyx mori, the study of Bombyx mori has entered the post-genome era. There are more than 400 mutant lines in Bombyx mori. Among them, the mutants of epidermis morphology include branch silkworm, bamboo silkworm, stone silkworm, etc. The study on the genes and regulation mechanism of epidermal morphology mutation is still in its infancy. The formation mechanism of many epidermal mutants remains to be studied. In this study, C8, a near-isogenic line constructed from a mutant of Bombyx mori epidermis, was used as a material with its backcross parent C108. One obvious feature: thin and short, The morphological and structural differences between C8 and C108 epidermis were observed by scanning electron microscope, and the differential expression of epidermal protein between C8 and C108 was identified by SDS-PAGE and mass spectrometry, and the similarity of cDNA sequence was analyzed. The possible mutant genes were identified and their differential expression patterns were analyzed by RT-qPCR. Further linkage genetic analysis of candidate genes was carried out. The results are as follows: 1. The exophthalmos on the outer surface of C8 epidermis is more obvious than that of C108, but the epidermis of C8 is denser than that of C108. This dense epidermal structure may be the cause of C8 epidermis. 2. Eight differentially expressed protein bands in C8 and C108 epidermis were screened by SDS-PAGE, and 9 proteins were obtained by MALDI-TOF-MS mass spectrometry. Three epidermal proteins: BmCPH34-BmCPG4 and BmCPR41BmCPH34 were expressed higher in C8 than C108, while BmCPG4 and BmCPR41 were lower in C8 than C108.The three epidermal protein genes: BmCPH34BmCPG4 and BmCPR41 were compared in C8 and C108. the three epidermal protein genes: BmCPH34BmCPG4 and BmCPR4BmCPG4 and BmCPR41 were obtained in C8 and C108 respectively. The cDNA sequence of the expressed epidermal protein gene BmCPR2 was cloned and sequenced. The results showed that there was no difference between the amino acid sequence of the BmCPR41 gene and that of the BmCPR41 gene between C8 and C108 and P50 in the SilkDB database. The cDNA sequence of the BmCPG4 gene was significantly different between C108 and P50, but only one base was substituted between C8 and P50. The cDNA sequence of BmCPH34 gene was 15 BP repeats in C8 gene, and 9 base pairs were replaced, compared with C108 and P50, the cDNA sequence of BmCPH34 gene was 15 BP in C8. In the amino acid sequence it encodes, there are repeats of five amino acid residues. We speculated that BmCPH34 gene may be involved in the formation of C8 epidermal morphologic mutation. The expression of the four epidermal protein genes in C8 and C108 was detected by RT-qPCR technique. The results showed that the expression of four epidermal protein genes in C8 was higher than that in C108, and the difference was significant. These four epidermal protein genes were involved in the formation of C8 epidermal morphologic mutation to some extent. The developmental phase analysis of the BmCPH34 gene showed that the gene was mainly expressed in the head, epidermis, fat body and trachea of Bombyx mori. Therefore, it is speculated that BmCPH34 gene is related to the formation of C8 epidermis. We identified four genes: BmCPH34, BmCPG4, BmCPG4BmCPR2 and BmCPR41, which may be related to C8 epidermal morphologic mutation, but the specific causes of C8 epidermal morphology need to be further studied.
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q78

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