中华蜜蜂卵黄原蛋白基因的分子特征及表达研究

发布时间：2018-02-08 11:43

本文关键词： 中华蜜蜂卵黄原蛋白 DNA序列生物信息转录蛋白质合成　出处：《东北师范大学》2016年博士论文　论文类型：学位论文

【摘要】：中华蜜蜂(Apis cerana cerana,简称中蜂)是东方蜜蜂(Apis cerana)的指名亚种,也是我国本土蜂种资源,具有优良的蜂种特性和不可替代的经济、社会和生态作用,在我国蜂业经济发展中占有重要地位。未来中华蜜蜂资源的保护和开发利用迫切需要对中华蜜蜂展开系统深入的研究。目前,对中华蜜蜂的研究,多集中于地理形态学、分子系统学等方面,对于特定功能基因的研究尚处于起步阶段。卵黄原蛋白是当前蜜蜂生理生化研究中最活跃的领域之一。本文主要利用DNA克隆、测序和生物信息学分析方法,研究了中华蜜蜂卵黄原蛋白基因的全DNA序列及其蕴含的生物信息,应用SSCP-PCR技术分析了中华蜜蜂卵黄原蛋白基因外显子基因型特征,利用qRT-PCR技术研究了中华蜜蜂蜂王、工蜂和雄蜂在不同发育阶段卵黄原蛋白基因的mRNA转录表达特性,利用SDS-PAGE和Western blot技术测试分析了中华蜜蜂蜂王、工蜂和雄蜂在不同发育阶段卵黄原蛋白的合成特点。主要研究成果如下:1、中华蜜蜂卵黄原蛋白基因一级结构中华蜜蜂卵黄原蛋白基因序列由6496bp碱基组成,其中C+G含量为46.14%,与意大利蜜蜂(A.mellifera ligustica)的同源性为92%。中华蜜蜂卵黄原蛋白基因序列由7个外显子组成,总计编码1689个氨基酸。2、中华蜜蜂卵黄原蛋白预测结构合成中华蜜蜂卵黄原蛋白的氨基酸序列N-末端具有一个识别因子位点,此位点为丝氨酸富集区,是典型的真核生物基因结构特征;在846~856位点上具有一个锌指结构,即HSFPTETgLPF;具有翻译后修饰位点89个,其中有75个是磷酸化位点,磷酸化是蜜蜂卵黄原蛋白翻译后最主要的修饰方式。3、中华蜜蜂卵黄原蛋白基因外显子基因型在海南中蜂第五外显子中发现A、B、L、M、N五个等位基因,AA、AB、LM和LN四个基因型,其中AB型和LN型是海南中蜂特异的优势基因型,其它地区中华蜜蜂样本未见此基因型检出。海南中蜂种群处于Hardy-Weinberg平衡状态,具有独立的遗传结构特点。推断海南中蜂最小的外部形态长度指标(吻长、翅长、翅宽和3+4背板总长等)可能与其独特的基因型(AB型和LN型)相关联。4、中华蜜蜂工蜂卵黄原蛋白基因mRNA的转录模式基因转录模式与意大利蜜蜂不同,中华蜜蜂成年工蜂随着日龄,卵黄原蛋白基因的转录丰度呈现出间歇式的变化规律。在工蜂羽化出房第4天、第12天和第19天,卵黄原蛋白基因的mRNA转录出现峰值;在羽化出房第1天、第8天和第17天,卵黄原蛋白基因的mRNA转录活性最低。卵黄原蛋白基因可以在中华蜜蜂工蜂的头、胸和腹部表达,其中头部表达丰度最低,其次是胸部,腹部卵黄原蛋白基因mRNA水平最高(P0.05)。并且无论在头、胸、腹部还是血淋巴中,中华蜜蜂工蜂卵黄原蛋白表达丰度均显著低于意大利蜜蜂(P0.05)。5、中华蜜蜂蜂王卵黄原蛋白基因的表达特点中华蜜蜂蜂王羽化出房后一周内,卵黄原蛋白的表达丰度缓慢增加。出房一周后性成熟,卵黄原蛋白的表达水平迅速增加。蜂王在产卵期间卵黄原蛋白表达量最高。与工蜂相似,卵黄原蛋白基因可以在蜂王的头、胸和腹部表达,腹部卵黄原蛋白合成量最高(P0.05)。6、中华蜜蜂雄蜂卵黄原蛋白基因的转录与蛋白合成特点卵黄原蛋白基因的mRNA在中华蜜蜂雄蜂羽化出房1-3天内转录,但是表达丰度很低,出房第4天以后再未检测到mRNA信号;从各个日龄雄蜂血淋巴样本中均未检出卵黄原蛋白信号的存在。据此推测,卵黄原蛋白在雄蜂体内无法顺利合成或者合成微量,雄蜂体内的卵黄原蛋白功能表现可能是退化的。本研究结果将对蜜蜂主要功能基因的开发利用奠定理论基础,对保护和利用我国特色蜂种资源具有意义。
[Abstract]:The Chinese honeybee (Apis cerana cerana, referred to as the bee) is (Apis cerana) of Apis cerana subspecies, is our native bee species has bee species resources, excellent characteristics and irreplaceable economic, social and ecological effects, occupies an important position in China's economic development in the future. Bee Apis cerana resources protection the development and utilization of the urgent need to initiate a systematic study of Chinese bee. At present, the research on Chinese bee, focused on geographic morphology, molecular systematics, etc., for the study of specific genes is still in the initial stage. Vitellogenin is one of the most active areas of current research in honeybee physiological and biochemical. This paper mainly uses the DNA cloning, sequencing and bioinformatics analysis method, studies the DNA sequence of Chinese honeybee vitellogenin gene and its biological information contained in the application of SSCP-PCR technology in Chinese The bee vitellogenin gene exon genotypes, of Chinese bee queen using qRT-PCR technology, characteristics of workers and drones in the expression of mRNA transcription in different developmental stages of vitellogenin gene, analysis of the Chinese Bee King by SDS-PAGE and Western blot test, workers and drones in the synthesis of characteristics in different developmental stages of eggs vitellogenin. Main research results are as follows: 1, the honeybee vitellogenin gene level structure of Chinese honeybee vitellogenin gene sequence from the 6496bp base composition, the content of C+G was 46.14%, and the Italy bee (A.mellifera ligustica) 92%. homology of honeybee vitellogenin gene sequences from 7 exons encoding 1689 amino acids, total.2, the honeybee vitellogenin predicted amino acid sequence of N- terminal structure synthesis of honeybee vitellogenin has a recognition for Sub site, this site is the serine rich region, is a typical structure of eukaryotic gene; at 846~856 locus with a zinc finger structure, which has HSFPTETgLPF; post-translational modification of 89 loci, of which 75 are phosphorylation sites, phosphorylation of.3 is modified the main way of raw egg yolk bees white translation, the Chinese honeybee vitellogenin gene exon genotypes in Hainan bee exon fifth A, found in B, L, M, N five alleles, AA, AB, LM and LN four genotypes, including type AB and type LN is the predominant genotype in Hainan bee specific, other areas of Chinese bee samples but not in the genes detected in bee populations in Hainan. Hardy-Weinberg equilibrium, genetic structure has the characteristics of independence. Hainan bee inferred the smallest morphological length index (snout length, wing length, fin width and length of the 3+4 backplane etc.) may be related to its unique genotype ( AB type and LN type) associated.4 APIs ceranaceranafabricius vitellogenin gene transcription of mRNA gene transcription pattern model and Italy bee, Apis cerana worker bees with age, transcript abundance of vitellogenin gene showed a variation of intermittent. In fourth days out of real feather workers, and twelfth days the nineteenth day, mRNA transcription of vitellogenin genes in the emergence of real peak; first days, eighth days and seventeenth days, mRNA transcription activity of vitellogenin gene. The lowest vitellogenin gene in the bees on the head, chest and abdomen of the head, which expressed the lowest, followed by chest and abdomen of vitellogenin gene mRNA (P0.05) and the highest level. No matter in the head, chest, abdomen or blood Lin Bazhong, Apis ceranaceranafabricius vitellogenin expression abundance was significantly lower than the Italy bee (P0.05).5, the queen bee egg yolk The original gene expression characteristics of Chinese bee bee emergence room after a week, the expression of vitellogenin increased slowly out of the room. One week after sexual maturity, the expression level of vitellogenin increased rapidly during the spawning period. The queen Huang Yuan oval highest amount of protein expression. And the worker is similar to that of vitellogenin the Queen's head in gene expression, chest and abdomen, abdominal vitellogenin synthesis was the highest (P0.05).6, transcription and protein synthesis of vitellogenin gene of Chinese honeybee drone vitellogenin gene mRNA in the honeybee drone emerged housing within 1-3 days of transcription, but the expression is very low, after a room for fourth days and then did not detect the mRNA signal; from every day old male hemolymph samples were not detected in vitellogenin signal. We speculated that the vitellogenin in the body can not be synthetic or synthetic drone trace, drone body The function of egg yolk protein may be degraded. The results of this study will lay a theoretical foundation for the development and utilization of main functional genes of honeybees, and will be meaningful for the protection and utilization of bee resources in China.

【学位授予单位】：东北师范大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S891

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