【作者】 郑济芳；

【导师】 朱睦元；

【作者基本信息】 浙江大学 ， 遗传学， 2003， 博士

【摘要】 Sox基因均含有一个编码高速泳动蛋白(High-Mobility-Group，HMG)的盒区，其对应的氨基酸组成与位于哺乳动物Y染色体上的SRY基因的HMG盒区相比，至少有50％的同源性。据不完全统计，目前已从包括哺乳类、鸟类、爬行类、两栖类、鱼类以及果蝇等不同物种中克隆了150多个Sox基因。Sox基因家族分成7个亚族(A，SRY；B，Sox-1，-2，-3，-14，-21；C，Sox-4，-11，-12，-22，-24；D，Sox-5，-6，-13，-23；E，Sox-8，-9，-10；F，Sox-7，-17，-18；G，Sox-15，-16，-20)，其中，除了Sox3和SRY基因分别位于哺乳动物的X和Y染色体上，其他Sox基因均位于常染色体上。哺乳动物Sox3基因被认为是睾丸决定基因SRY的起源基因。Sox3、Sox9等其它Sox基因也可能与脊椎动物的性别决定和分化有关。然而，对于温度决定型性别决定的分子机理至今仍不清楚。 本研究以性别决定方式为温度决定型(Temperature-dependent sex determination，TSD)的扬子鳄(Alligator sinensis)和中华鳖(Trionyx sinensis)为实验对象，采用兼并引物PCR，从扬子鳄和中华鳖的基因组DNA中分别克隆并测序8个和7个HMG盒区，每个盒区长度均为216bp。扬子鳄的8个HMG盒区命名为AS41～48基因，中华鳖的7个HMG盒区命名为TS41～47基因。8个AS基因组成3组(Class)，7个TS基因也组成3组，但AS与TS之间没有互相重叠。同源序列比较表明：AS43～46和TS43～46与鼠Sox12的HMG盒区有95％的氨基酸同源性，表明是鼠Sox12的同源基因(homologues)：AS47～AS48与鼠Sox4的HMG盒区分别有100％和97％的氨基酸同源性，可以认为AS47～48是鼠Sox4的同源基因；与鼠、鸟Sox21的HMG盒区相比，TS47分别有98％和100％的氨基酸同源性。分析结果表明：AS47和TS47是直向同源基因(orthologues)。 与鼠的Sox1、Sox2或Sox3的HMG盒区相比，AS41、AS42、TS41、TS42分别有81％、79％、80％、72％的氨基酸同源性，表明AS41～42和TS41～42是鼠Sox-1、-2、-3的同源基因。进一步序列分析表明：AS41～42、TS41～42与非爬行类脊椎动物的Sox-1、-2、-3的氨基酸同源性低于非爬行类脊椎动物包括哺乳类、鸟类、两栖类和鱼类的肠工一1、一2、一3相互之间的氨基酸同源性，。分子系统发生分析表明AS41~42和Z弘1碑2的成簇(Cluster)偏离非爬行类脊椎动物肋工一1、一2、一3同源基因之间的成簇，显示了AS41~42和了万碑1一2在物种内的特异性进化，并且序列变化一定发生在羊膜动物多样化之后。本研究首次在爬行动物中克隆了哺乳动物辜丸决定基因(SR力的起源基因(加力)的同源序列，有助于进一步研究5’Ox基因与温度决定型性别决定的关系。 为了克隆到与胚胎发育有关的新基因，以孵化一星期的中华鳖(Trto尸砂优sinensis)胚胎的头部、生殖晴混合组织为原始材料，采用SMART但witching丛eehanism丛5’end of旦NA Iranseript)和长距离PCR伍D一PCR)技术，构建T一个中华鳖cDNA表达文库。分析结果表明，该未扩增的cDNA文库大约含有4.134xlo，个克隆。任意挑取了192个克隆，提取质粒后用助I酶切鉴定表明:没有插入片段的克隆为9个，插入了CDNA片段的克隆为183个，插入的。DNA片段大小范围为0.4一3.skb。其中，插入片段在0.4一1.0 kb之间的克隆为19个，在1.1一2.okb之间的克隆为53个，在2.1一3.okb之间的克隆为92个，大于3.Ikb的克隆为19个。 从插入片段在2.1一3.Okb之间的克隆中，任意挑选12个克隆，提取质粒后，从5’端进行测序，序列分析表明:12个序列均有开放阅读框(旦pen一:eading丘ame，oRF)的第一个密码子(ATG)，没有内含子。Poly(A)信号序列伍AUAAA)也在招月晒因D中被证实。Blast分析表明，12个序列代表5种类型的基因:核糖体蛋白基因(ts RpL4，tsRp丈6，招天尸乙26)、组织特异性表达的基因(钻尸乃从招月叨火D)、线粒体基因(tsC“叨、结构蛋白基因伽DC从括人从石，，ts刀一ctin)、编码转录因子的基因(招月沏吟1，tsDA尸5)。其中，招月沏吟1、招刀CN、仃尺尸乙26、招材从护和招刃只PS基因在爬行动物中为首次克隆。该发育阶段特异性cDNA文库可以进一步用于中华鳖胚胎发育过程中相关基因的鉴定分离、结构功能分析、以及表达调控机制研究。采用”GW一pCR区andom助med少ne里alki飞pCR)方法，从经及。天I消化过的中华鳖(Trio矛移优sinensis)基因组DNA中克隆了c眨治xZ基因(72ObP)，它与鼠、鸟的及戏2基因分别有85%和91%的核昔酸同源性，93%和97%的氨基酸同源性。进一步分析表明，已巴滩2编码的238个氨基酸多肤链含有两个保守的功能区域:位于N端的HMG盒区(1一78aa)和位于C端的转录激活区(131一236aa)，以及一个保守的特征性B亚族同源区域(79一90aa)与HMG盒区的C端相连。高度的序列同源性和结构相似性表明C泛治戈2是中华鳖的直向同源基因。肠工2基因在哺乳类、鸟类、爬行类以及两栖类中极端保守，说明sbxZ功能在进化中可能也是非常保守的。C心以2基因的HMG盒区外的序列信息可以进一步研究它的功育旨。更多还原

【Abstract】 Members of the Sox gene family are characterized by a HMG-box which shows sequence similarity with that of the mouse testis-determining gene SRY. Using degenerate primers PCR, seven and eight HMG-box motifs of SRY-related genes from respective genomic DNA of Trionyx sinensis and Alligator sinensis with TSD were cloned and sequenced, which were called TS41-TS47 in Trionyx sinensis and AS41-AS48 in Alligator sinensis respectively. TS41-TS47 form three major gene classes, which are not completely overlapped with those from AS41-AS48. TS43-TS46 and ,AS43-AS46 were demonstrated to be the mouse Sox12 homologues. ,AS48 was identified to be the mouse Sox4 homologue. TS47 may be a Sox21 orthologue of Trionyx sinensis, and ,AS47 may be a Sox4 orthologue of Alligator sinensis based on Blast search. Blast analyses showed that the percentage of identity at the amino acid level in the HMG-box region between TS-41, -42, AS-41, -42 and the non-reptilian Sox-1, -2, -3 was lower than that among the non-reptilian Sox-l, -2, -3. The molecular phylogenetic analysis indicated that the clustering of TS-41, -42 and AS-41, -42 was distant to the clustering of the non-reptilian Sox-l, -2, -3 homologues identified previously in species, including fish, amphibian, bird and mammalian. This suggests that TS-41, -42 and ,AS-41, -42 may represent the species-specific evolution and share a unique function. The unexpected sequence change in TS-41, -42 and ,AS-41, -42 may have occurred after divergence of amniotes. It is the first report that the sequence homologues of the progenitor of the mammalian testis-determining gene SRY were identified in reptile. These findings might provide molecular evidences that SRY-related genes may be involved into TSD.In order to clone new genes expressed during early embryonic development of Trionyx sinensis, we constructed and characterized a cDNA expression library from poly (A+) mRNA isolated from 250 mg of cranial/kidney/gonad complex tissues of one-week-old embryos of Trionyx sinesis using the SMART (Switching MechanismAt 5’ end of RNA Transcript) cDNA synthesis and LD-PCR amplification strategy. The measured complexity of the cDNA library is 4.134X 10s directional clones. The insert lengths of 192 randomly selected clones ranged from 400 to 3800 bp. In all, 12 clones were randomly picked and sequenced, all showing typical cDNA characteristics. All sequences had the first codon of the ORFs. These sequences represent five groups: including ribosomal protein gene(tsRPL4, tsRPL6, tsRPL26), tissue-specific gene (tsFTH, tsHBAD), mitochondria! gene (tsCOXI), structure protein gene (tsDCN, tsMMIP, tsB-Actiri), transcriptional factor gene (tsHMG1, tsDAP5). The cDNA library can be used to provide expressed sequence tags (ESTs). The stage-specific cDNA library will be a useful resource for the study of gene structure, expression and regulatory during the early process of embroygensis of Trionyx sinensis.Using Random Primed Gene Walking PCR (RPGW-PCR), CtSox2 gene was cloned and sequenced from the digested genomic DNA of Trionyx sinensis. Compared with the mouse and chicken Sox2, CtSox2 shared 86% and 91% nucleotide homology respectively, and 93% and 97% amino acid identity respectively. The predicated amino acid sequences contain two conserved functional domains: a DNA-binding domain (HMG-box) and a transactivation domain. The conserved motif of group B homology lies immediate adjacent to C-proximal region of the HMG-box. Similar structure and identity of Sox2 gene among mammals, birds, amphibians and reptiles suggest that Sox2 gene have evolutionary conserved roles. Cloning of CtSox2 gene shows the validation of RPGW-PCR used for isolating genes from the digested genomic DNA.更多还原