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Alternative TitleEST analysis of Chinese sturgeon pituitary and the studies on cloning and expression characterization of GTH and GH genes
Thesis Advisor桂建芳
Degree Grantor中国科学院水生生物研究所
Place of Conferral水生生物研究所
Keyword中华鲟 垂体 Est Asgh Asgth-α i Asgth-α Ii Asfshβ Aslhβ
Abstract中华鲟 (Acipenser sinensis Gray) 是我国的一级保护动物,隶属于硬骨鱼纲,软骨硬鳞下纲。目前,有关其生长,繁殖和性别分化的分子机制还知之甚少。因此,我们构建了中华鲟垂体SMART cDNA的质粒文库,选取的分别是人工养殖的4-龄雄性中华鲟和野外捕获的刚产完卵的24-龄雌性中华鲟。通过测序分别筛选到944个和2025个EST。将所得EST与GenBank数据库中的序列进行比对,发现在4-龄雄性中华鲟垂体库中,阿黑皮素原基因 (POMC) 表达量最高,占总表达量的10.17%。而且在该文库中,我们还重点研究了7个未能在公共数据库中找到同源序列信息的新基因,结果发现,其中的EG009337和EG009340,只在垂体和卵巢中特异性表达;而EG009334和EG009338,只在垂体中特异性表 达。这些基因有可能是与生殖和性腺分化有关的基因,值得进一步的研究。 在24龄雌性中华鲟垂体的SMART cDNA质粒文库中,我们总共得到了2025个EST克隆,881个基因,包括407个未能在公共数据库中找到同源序列信息的新基因,其中有75个含有开放阅读框,这些基因有可能是一些从未被人报道过的新基因或中华鲟特有的基因。我们还发现了8种生殖与内分泌调控相关基因。其中,促性腺激素I型和II型的α亚基 (AsGTH-α I, AsGTH-α II) 与阿黑皮素原I型和II型 (AsPOMC-I, AsPOMC-II) 是整个EST库中表达量最高的两组基因,分别占总表达量的16.64%和7.26%。通过氨基酸序列比对我们发现,和AsGTH-α I 相比较,AsGTH-α II与进化上更加原始的鱼类GTH-α同源性更高,可能是更为原始的一种AsGTH-α基因。我们还发现了促性腺激素释放激素受体基因,这也是该基因第1次在鲟形目鱼类中的报道。 从垂体SMART cDNA质粒文库中筛选到的AsGH基因的全长cDNA序列,全长为954bp,编码214个氨基酸残基的成熟多肽。AsGH与其它鱼类GH基因有很高的同源性。RT-PCR和Western blot分析表明,在中华鲟胚胎发育的14个特定时期,GH一直处于高效表达。 从垂体SMART cDNA质粒文库中筛选到的AsGTH-α I, AsGTH-α II,AsFSHβ和AsLHβ基因的全长cDNA分别为659、642、1101和584bp,分别编码115、128、99和137个氨基酸残基的成熟多肽。和其它鱼类相比,在这些基因中, GTH-α基因的保守性最强,LHβ基因的保守性次之,而FSHβ基因的保守性最弱。Western blot分析发现,GTH的3个亚基均为垂体特异表达,并且在刚产完卵的野生雌性性成熟 (17-龄) 中华鲟垂体中,GTH 的3个亚基的表达量均很高。而在人工养殖的1-,2-,3-,4-龄中华鲟垂体中,无法检测到GTH-α和LHβ蛋白,另外FSHβ蛋白也只能在4-龄中华鲟垂体中检测到而在其它年龄段的中华鲟垂体中无法检测到。中华鲟垂体切片免疫荧光定位研究发现,在刚产完卵的野生雌性性成熟 (24-龄) 中华鲟垂体中,GTH-α的表达集中在腺垂体的PPD,RPD和PI等部位,FSHβ的表达集中在腺垂体PPD的中间一小部分区域,LHβ的表达范围主要集中在腺垂体PPD,PI和靠近RPD的外围部分。在4-龄中华鲟垂体中,FSHβ主要在腺垂体RPD和PPD的外围部分表达,与24-龄中华鲟相比,FSHβ在4-龄中华鲟垂体中的分布范围要更广泛一些。
Other AbstractChinese sturgeon (Acipenser sinensis Gray) is an endangered anadromous fish, and was listed as a Grade I protected animal in China. It mainly reproduces in Yangtze River. Its reproduction migratory route was blocked in 1981 by the Gezhouba Dam, which caused a drastic decline of the natural population. However, its molecular information is very limited at this time. In order to isolate specific functional genes involved in reproduction and endocrine regulation and to reveal their evolutionary mechanisms in Chinese sturgeon, we constructed its pituitary cDNA library. In teleost, pituitary plays significant roles in many physiological functions, such as growth and reproduction. In this study, the SMART cDNA plasmid libraries of the pituitaries from 4-years old male Chinese sturgeon and 24-years old female Chinese sturgeon were constructed respectively. 944 and 2025 ESTs were obtained respectively from the two pituitary plasmid libraries. Among all the 944 EST clones of the 4-years old male Chinese sturgeon, 461 (48.83%) clones were identified as known genes and 483 (51.17%) as unknown genes. The 461 clones of known gene products represent transcripts of 132 genes. Functional categorization indicated that the most abundantly expressed functional gene, the proopiomelanocortin (POMC), accounted for almost 10.17% of the overall expression, indicating its important function in the pituitary. Interestingly, the expression patterns of 7 unknown genes were analyzed in various tissues. Several unknown ESTs, such as EG009334, EG009337, EG009338 and EG009340, were detected to be pituitary-specific, or pituitary and ovary-specific genes. Clustering and assembly of the 2025 ESTs of the 24-years old female Chinese sturgeon revealed 1305 ESTs that match with some known genes and 720 ESTs that have no significant homology to any sequences. And, a total of 881 different genes were identified. The known genes were categorized into 10 groups according to the predicted and unknown functions. Additionally, 75 different genes, including 20 contigs and 55 singletons, were identified from the 720 unannotated ESTs. Moreover, we found 8 reproduction and endocrine-related genes, and the 8 genes accounted for 557 in the 2025 ESTs. Interestingly, we observed 2 subtypes of gonadotropin subunit common α (GTH-α) and 2 subtypes of proopiomelanocortin (POMC), and found that they were the most redundant expression genes. They accounted for 16.6% and 7.3% of all the sequenced ESTs, respectively. Furthermore, we analyzed their phylogenetic significance and suggested that AsGTH-α II might be an ancestral gene, and might play significant roles in reproduction regulation of ancestral fishes. Additionally, GnRH receptor was firstly identified from sturgeons of Acipenseriformes. The nucleotide sequences of Chinese sturgeon growth hormone (AsGH) cDNA contains 954 nucleotides and encodes 214 amino acid residues (aa). High homology were observed by amino acid sequence alignment and identity comparison of the mature peptides of AsGH with that of other fishes. RT-PCR and Western blot analysis showed that a considerable high level of GH transcript has been detected in the 14 embryo stages. These findings suggest that the AsGH in eggs may play a key role in early development of Chinese sturgeon The nucleotide sequences of Chinese sturgeon common α I (AsGTH-α I), common α II (AsGTH-α II), FSHβ (AsFSHβ) and LHβ (AsLHβ) subunit cDNAs are 659, 642, 1101 and 584 bp in length, and encode for mature peptides of 115, 121, 129 and 138 aa, respectively. Three polyclonal antibodies were prepared from the in vitro expressed AsGTH-α I, AsFSHβ and AsLHβ mature proteins, respectively. A western blot analysis showed that the expression of the three gonadotropin subunits (AsGTH-α I, AsFSHβ and AsLHβ) can be detected in the individuals with 17-years-old (sex matured). While in other individuals, only AsFSHβ expressed in the pituitary of 4-years-old sturgeon. Immunofluoresence localization were performed and indicated that in the pituitary of 24-years-old individual (sex matured), the AsFSHβ cells mainly distributed in the middle area of proximal pars distalis (PPD), while the AsLHβ cells distributed in the area of PPD, pars intermedia (PI) and the external periphery of pituitary near to the side of rostral pars distalis (RPD). The AsGTH-α I might be expressed in both AsFSHβ and AsLHβ cells. In the pituitary of 4-years-old individual, the AsFSHβ cells mainly distributed in the external periphery of pituitary near to the side of RPD and PPD. Compared with the 24-years-old sturgeon’s pituitary, AsFSHβ cells distributed more widely in the pituitary of 4-years-old sturgeon. Our results provide the basis for future investigations on reproduction biology of Chinese sturgeon.
Document Type学位论文
Recommended Citation
GB/T 7714
曹宏. 中华鲟垂体EST分析以及促性腺激素和生长激素的克隆和表达特征研究[D]. 水生生物研究所. 中国科学院水生生物研究所,2008.
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