【作者】 刘静；

【导师】 王勤；

【作者基本信息】 四川农业大学 ， 动物学， 2015， 硕士

【摘要】 Sox (SRY-box containing)基因在动物界广泛存在,目前已在鱼类、两栖类、爬行类、鸟类和哺乳类动物中发现超过100个成员,根据HMG盒的相似性,将其划分为A-K 11个亚族。Sox4基因是SoxC亚族成员之一,在胚胎发育中起着至关重要的作用,主要参与心脏、淋巴细胞、中枢神经系统、内分泌胰岛、造血细胞、性腺等多种器官的发育。Sox7基因属于Sox家族F亚族,主要参与胚胎发育和细胞命运的决定。Sox7基因不仅能够诱导Nodal信号分子、中胚层相关基因以及内胚层分化基因的表达,而且是内胚层Veg-T信号途径所需的标志物。本研究以中国特有的大型珍稀二级保护物种,唯一尚存的隐鳃鲵科两栖动物一中国大鲵(Andrias davidianus)为研究对象,采用逆转录聚合酶链反应(]RT-PCR)及cDNA末端快速扩增(RACE)技术克隆获得中国大鲵Sox4基因的3个同系物：Sox4a、Sox4b、Sox4c及Sox7基因的cDNA序列,对其进行生物信息学分析,并通过荧光定量PCR技术进行各组织间表达情况的分析,主要取得如下结果：1. Sox4a基因的cDNA序列为1153 bp,编码224个氨基酸,位于59-130之间的HMG-box是其主要功能区,同源性分析表明中国大鲵Sox4a编码蛋白与其他物种Sox4a蛋白同源性在63.8%-97.2%之间；对不同物种的Sox4a蛋白构建系统发育树可知：鲤鱼(Cyprinus carpio)与施氏鲟(Acipenser schrenckii)从进化树最先分出,随后两栖类的中国大鲵与饰纹姬蛙(Microhyla ornata)聚成一支,鳙鱼(Hypophthalmichthys nobilis)与斑马鱼(Danio rerio)最后分出,说明Sox4a基因是一个古老的基因,且从中国大鲵到鱼类的进化速度比较快,Sox4a基因的基因复制时间可能在鳙鱼与斑马鱼分化之前。Sox4b基因的cDNA序列为1058 bp,编码193个氨基酸,位于59-130之间的HMG-box是其主要功能区,同源性分析表明中国大鲵Sox4b编码蛋白与其他物种Sox4b蛋白同源性在60.3%-97.1%之间；对不同物种的Sox4b蛋白构建系统发育树可知饰纹姬蛙最早从进化树分出,随后是鲤鱼、瓣结鱼(Folifer brevifilis)等几种鱼类,中国大鲵与金线蛙(Pelophylax plancyi)聚成一支最后从进化树分出。Sox4c基因的cDNA序列为996bp,编码149个氨基酸,位于59-130之间的HMG-box是其主要功能区, Sox4c基因目前仅在饰纹姬蛙获得部分核苷酸片段,将其推导蛋白与中国大鲵Sox4c序列进行比对,两者同源性高达81.7%。2.Sox7基因的cDNA序列长为847bp,编码188个氨基酸,位于44-115之间的HMG-box是其主要功能区,中国大鲵Sox7编码蛋白与其他物种Sox7蛋白同源性在64.1-76.8%之间；对不同物种的Sox7蛋白构建系统发育树可知Sox7基因是一个比较古老的基因,在鱼类、两栖、爬行、哺乳均有存在,中国大鲵与非洲爪蟾、热带爪蟾两栖类聚在一起基本符合物种进化的历程。3.从NCBI下载、查阅文献及本实验室先前所得的中国大鲵Sox基因家族：Sox2、Sox3、Sox4a、Sox4b、Sox4c、Sox7、Sox9、Sox11、Soxl4a、Soxl4b、Sox19共11个成员。利用MEGA 5.0软件,采用邻接法(Neighbor-joining method)重复1000次对这11个Sox基因成员构建系统发育树,结果显示：Sox4a、Sox4b、Sox4c及Soxll汇聚在一起分化出来,显示其较原始的进化地位、Sox9及Sox7聚在一起随后分出,Sox14的2个同系物、Sox2、Sox19及Sox3最后分出。4.利用荧光定量PCR方法分析中国大鲵Sox4和Sox7基因的组织表达情况,结果显示：Sox4基因在性成熟的中国大鲵各组织表达量均比较低,相对而言在肠道表达量最高,心脏、卵巢、垂体的表达量次之,肝脏中的表达量极低,脑及肾脏中几乎不表达。Sox7基因在所有检测的组织中均有表达,且在肾脏中的表达量最高,肠道、心脏、脑以及卵巢的表达量次之,垂体中的表达量较低,肝脏表达量极低。更多还原

【Abstract】 The Sox (SRY-related HMG box) family of transcription factor is a large gene family that is widely distributed across the animal kingdom and has been subdivided into groups A to K based on their sequence similarity and gene structure. Sox4 belongs to the subgroup C, has key roles in heart outflow tract and valve development, lymphocyte differentiation, neural and glial cell development, endocrine islet formation, osteoblast development. Sox7 belongs to the subgroup F, involved in the regulation of embryonic development and the determination of cell fate. It was shown that Sox7 can induce expression of multiple nodal genes, as well as the expression of genes involved in mesodermal and endodermal differentiation, and is required for Veg-T-induced expression of endodermal markers. The Chinese giant salamander (Andrias davidianus) belongs to one of the most primitive orders of urodele amphibians, the Cryptobranchidae. It is the largest extant amphibian species, and found only in China. In this study, we have successfully cloned the Sox4a, Sox4b, Sox4c and Sox7 cDNA sequence from Andrias davidianus based on the techniques of homology cloning and RACE-RCR, and analyzed the amount of gene expression in different tissues by using fluorescence quantitative PCR technique, the main topics of this paper are as followed:1. Sox4a cDNA seqence was 1153 bp in length, which encoded 224 amino acids and the N-region 59-130aa as highly conservative HMG-box. The putative protein of Sox4a in Andrias davidianus had 63.8%-97.2% identity with other species. We constructed the phylogenetic tree by the amino acids sequences of Sox4a, the result showed that Cyprinus carpio and Acipenser schrenckii split off earlier, Microhyla ornata and Andrias davidianus which belongs to the amphibian in the same branch, while Hypophthalmichthys nobilis and Danio rerio split off at last. This reveals that the evolutionary rate from fish to Andrias davidianus is very fast, and the duplication of these three Sox genes occurred before the divergence of Hypophthalmichthys nobilis and Danio rerio. Sox4b cDNA seqence was 1058 bp in length, which encoded 193 amino acids and the N-region 59-130aa as highly conservative HMG-box. The putative protein of Sox4b in Andrias davidianus had 60.3%-97.1% identity with other species. We constructed the phylogenetic tree by the amino acids sequences of Sox4b, This reveals that Microhyla ornata split off earlier, various fishes then separate, Pelophylax plancyi with Andrias davidianus in the same branch split off at last. Sox4c gene with partial nucleotide fragment was only found in Rana plancyi at present. The putative protein of Sox4c in Andrias davidianus had 81.7% identity with that of Rana plancyi.2. Sox7 cDNA seqence was 847 bp in length, which encoded 188 amino acids and the N-region 44-115aa as highly conservative HMG-box. The putative protein of Sox7 in Andrias davidianus had 64.1%-76.8% identity with other species. We constructed the phylogenetic tree by the amino acids sequences of Sox7, the results showed that Sox7 is an ancient gene which exists in fish, amphibian, reptules and mammal, and Andrias davidianus, Xenopus tropicalis, Xenopus laevis which belong to the amphibian in the same branch, this conform to the course of long-term evolution.3. We obtained 11 members of the Sox gene family in Andrias davidianus which contain Sox2, Sox3, Sox4a, Sox4b, Sox4c, Sox7, Sox9, Soxll, Soxl4a, Soxl4b, Soxl9. Based on the amino acids sequences of these genes, we constructed the phylogenetic tree by Neighbor-joining method to repeat 1000 times in MEGA 5.0, the results showed that Sox4a, Sox4b, Sox4c with Sox11 gathered in the same branch, while Sox9 and Sox7 then separated, the two homologues of Soxl4, Sox2, Sox19 and Sox3 separated finally.4. By fluorescence quantitative PCR analysised the expression of Sox4 and Sox7 in differences tissues of Andrias davidianus. The results indicated that its slight maternal mRNA could be detecte in all tissues, while intestinal maintained a high leve relatively, moderate expression in heart, ovarian, pituitary, and the lowest expression in hepar, brain and kidneys. Sox7 gene was expressed in all tissues, and kidneys maintained a considerable high level, moderate expression in intestinal, heart, brain, ovarian, while the lowest expression in pituitary.更多还原