陆地棉HB红花近等基因系差异表达基因分析

发布时间：2018-04-14 04:14

本文选题：棉花 + HB红花性状　；参考：《山东师范大学》2016年硕士论文

【摘要】：HB红花性状来源于野生二倍体比克氏棉,陆地棉HB红花基因被初步定位于7号染色体上。在本研究中,利用SSR分子标记技术对HB红花基因进行精细定位,基因芯片和蛋白质组学技术也被应用于评估陆地棉HB红花近等基因系的相关功能。1、利用SSR分子标记技术,对HB红花基因进行精细定位。根据近等基因系与轮回亲本基因表达模式的差异,最终得到了5对差异引物,分别为A07-0646、A07-0592、A07-0594、CH07-44、CH07-53,并构建了一个全长81.8cM的连锁群,成功将HB红花基因定位于A07-0592与A07-0594之间,与它们的遗传距离分别是7.9cM和37.6cM。对比目前已有的陆地棉基因组测序信息,A07-0592和A07-0594之间物理距离为33Kb,包含21个基因,其中大于400bp的基因有4个,分别为MT-A70家族蛋白甲基转移酶、谷胱甘肽转移酶、类谷氨酰胺转酰胺酶类家族蛋白、八氢番茄红素合成酶,为下一步克隆重要性状相关基因并进行功能分析与验证奠定基础,为陆地棉种质创新和遗传改良提供依据。2、利用棉花全基因组cDNA芯片进行表达差异分析,获得陆地棉HB红花近等基因系与轮回亲本间明显差异表达基因80个(表达差异10倍以上),其中上调基因56个、下调基因24个,主要是与代谢和次生代谢调节相关基因、生物合成、细胞凋亡、信号传导等有关联的cDNAs及某些未知确切功能的基因。利用实时定量PCR技术,对通过基因芯片筛选出的4个明显差异表达基因进行分析,分析结果进一步明确了相关基因的表达调控规律,探讨红花性状形成的因素。3、应用差异蛋白组学方法,对陆地棉HB118与其轮回亲本118之间不同器官、不同生育时期的差异表达蛋白进行了研究,检测出表达丰度上在2倍以上的差异蛋白157个,其中花期叶片差异蛋白57个、絮期叶片差异蛋白56个、花差异蛋白44个,依据功能分为以下几类:(1)与碳水化合物代谢相关蛋白;(2)光合作用相关蛋白;(3)细胞骨架结构相关蛋白;(4)细胞防御相关蛋白;(5)转运相关蛋白;(6)转录与翻译调控蛋白;(7)分子伴侣类蛋白;(8)假定蛋白;(9)其他蛋白。明确了部分相关蛋白质结构、功能及互作网络集群调控规律。利用实时定量PCR技术,对通过蛋白质双向电泳中筛选出的10个差异表达蛋白进行验证,其主要参与了能量代谢、光合作用、转录翻译等过程,为今后开展蛋白功能方面的进一步研究提供基础。
[Abstract]:The trait of HB safflower originated from wild diploid Bickler cotton, and the HB gene of Upland cotton was located on chromosome 7.In this study, the SSR molecular marker technique was used to locate the HB gene, the gene chip and proteomics were also used to evaluate the function of the near isogenic line of HB safflower in Upland cotton, and the SSR molecular marker technique was used.The HB gene of safflower was carefully mapped.According to the difference of gene expression patterns between near-isogenic lines and recurrent parents, five pairs of differentially expressed primers were obtained, namely A07-0646, A07-0592, A07-0594, CH07-0594, CH07-4CH07-53, and a linkage group of full-length 81.8cM was constructed. The HB gene was located between A07-0592 and A07-0594.Their genetic distances were 7.9cM and 37.6 cm, respectively.The physical distance between A07-0592 and A07-0594 is 33kb, and contains 21 genes, of which 4 genes are larger than 400bp, which are MT-A70 family protein methyltransferase and glutathione transferase, respectively.Glutamine-like transaminase family proteins, octahydrolycopene synthase, lay the foundation for cloning, functional analysis and verification of genes related to the importance of lycopene.To provide basis for innovation and genetic improvement of upland cotton germplasm, the difference of expression was analyzed by using cotton genome cDNA microarray.A total of 80 differentially expressed genes (more than 10 times of the difference in expression) were obtained, including 56 up-regulated genes and 24 down-regulated genes, which were mainly related to metabolism and secondary metabolism regulation.Biosynthesis, apoptosis, signal transduction and other related cDNAs and some unknown functional genes.The four differentially expressed genes screened by gene chip were analyzed by real-time quantitative PCR. The results further clarified the regulation and regulation of the related genes.The factors of formation of safflower traits. The differential expression proteins between HB118 and their recurrent parents 118 in different organs and different growth stages were studied by differential proteomics.157 differentially expressed proteins were detected, including 57 in florescence leaf, 56 in floc, 44 in flower.According to their functions, they can be divided into the following categories: 1) and carbohydrate metabolism-associated protein 2) photosynthesis-associated protein (P3)) cytoskeleton structure related protein (PIA4)) cell defense related protein (T5) transporter associated protein (FY6)) transcription and translation regulatory protein (F7).Molecular chaperone protein (MCP 8) hypothetical protein 9) other proteins.The structure, function and regulation of some related proteins were clarified.Ten differentially expressed proteins screened by two-dimensional electrophoresis were verified by real-time quantitative PCR, which were mainly involved in energy metabolism, photosynthesis, transcriptional translation and so on.It provides a basis for further research on protein function in the future.
【学位授予单位】：山东师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S562

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