本文選題：近等基因系　切入點(diǎn)：表皮形態(tài)突變　出處：《安徽農(nóng)業(yè)大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：家蠶(Bombyx mori)屬于鱗翅目、蠶蛾科,是鱗翅目昆蟲的典型代表。隨著家蠶基因組精細(xì)圖的完成,人們對家蠶的研究正式進(jìn)入后基因組時代。家蠶有400多種突變品系,其中表皮形態(tài)的突變體有枝蠶、竹蠶、石蠶等。對于引起表皮形態(tài)突變的基因及調(diào)控機(jī)制的研究尚處于起步階段,許多表皮形態(tài)突變體的形成機(jī)制還有待研究。本研究以一種家蠶表皮形態(tài)突變體構(gòu)建的近等基因系C8與其回交親本C108為材料(家蠶C8與其回交親本C108相比具有幾個明顯特征:體型細(xì)短、表皮硬實等),通過掃描電鏡觀察C8與C108表皮之間的形態(tài)結(jié)構(gòu)差異;再以SDS-PAGE結(jié)合質(zhì)譜分析鑒定在C8與C108之間差異表達(dá)的表皮蛋白,再通過對其cDNA序列的相似性分析,鑒定可能的突變基因,并用RT-qPCR分析其差異表達(dá)模式;進(jìn)一步對候選基因進(jìn)行連鎖遺傳分析,研究結(jié)果如下:1.C8表皮外表面上的外突比C108的更為明顯,但C8的表皮比較致密,這種致密的表皮結(jié)構(gòu)可能是引起C8表皮硬實的原因。2.利用SDS-PAGE篩選出8條在C8和C108表皮中差異表達(dá)的蛋白條帶,并通過MALDI-TOF-MS質(zhì)譜分析獲得9個蛋白,其中含有3個表達(dá)差異的表皮蛋白:Bm CPH34、BmCPG4和BmCPR41,BmCPH34在C8中的表達(dá)量高于C108,而Bm CPG4和BmCPR41在C8中的表達(dá)量低于C108。3.對3個表皮蛋白基因:BmCPH34、BmCPG4和BmCPR41以及前期獲得的在C8與C108中差異表達(dá)的表皮蛋白基因BmCPR2的cDNA序列進(jìn)行克隆測序,結(jié)果表明:BmCPR2與BmCPR41基因所編碼的氨基酸序列在C8與C108以及SilkDB數(shù)據(jù)庫中的P50之間并沒有差異;BmCPG4的基因的cDNA序列在C108和P50中存在較大差異,而在C8與P50之間只有1個堿基發(fā)生替換,并引起1個氨基酸殘基發(fā)生突變。推測這3個基因并不是引起C8突變表型的直接因素。與C108和P50相比,BmCPH34基因的cDNA序列在C8中存在15 bp的堿基重復(fù),并有9個堿基的替換,在其編碼的氨基酸序列中存在5個氨基酸殘基的重復(fù),并引起2個位點(diǎn)的氨基酸的突變。我們推測,BmCPH34基因可能參與C8表皮形態(tài)突變的形成。4.利用RT-qPCR技術(shù)分別檢測這4個表皮蛋白基因在C8與C108之間的表達(dá)差異,結(jié)果顯示:4個表皮蛋白基因在C8中的表達(dá)量均高于C108,并且達(dá)到差異顯著水平,表明這4個表皮蛋白基因在一定程度上參與C8表皮形態(tài)突變的形成。進(jìn)一步對BmCPH34基因的發(fā)育時相分析表明該基因主要在家蠶的頭、表皮、脂肪體和氣管叢中表達(dá),在家蠶幼蟲四齡時表達(dá)量最高。因此,推測BmCPH34基因與C8表皮形態(tài)的形成存在著一定的聯(lián)系。綜上所述,在本研究中,我們共鑒定出4個可能與C8表皮形態(tài)突變相關(guān)的基因:BmCPH34、BmCPG4、BmCPR2和BmCPR41。但C8表皮形態(tài)形成的具體原因還需進(jìn)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.
【學(xué)位授予單位】：安徽農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：Q78

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