【作者】 徐跑；

【导师】 夏德全； 吴婷婷；

【作者基本信息】 南京农业大学 ， 水产养殖， 2006， 博士

【摘要】 Sox9是与哺乳动物性别分化形成有关的重要基因之一。本文在脑、精巢和卵巢分离和克隆到了两个黄颡鱼Sox9基因，其中从精巢和脑中分离到的序列一致，通过序列比对分析，称Sox9a1；从卵巢中分离到的称Sox9a2。黄颡鱼Sox9a1 cDNA 1604 bp[不包括poly(A)]，包括5′非翻译区70 bp，3′非翻译区142 bp，阅读框1392 bp，编码464个氨基酸，计算的蛋白分子量为50.62 kDa，其中99-176个氨基酸为保守的sox-TCF HMG-box，305-351个氨基酸为富含P／Q的结构域，352-464个氨基酸为TA结构域(transcription activation domain)。黄颡鱼Sox9a2全长cDNA 1843 bp[不包括poly(A)]，包括5′非翻译区313 bp，3′非翻译区159 bp，阅读框1371 bp，编码457个氨基酸，计算的蛋白分子量为50.24 kDa。其中111-188个氨基酸为保守的sox-TCF HMG-box，321-358个氨基酸为富含P／Q结构域，359-457个氨基酸为TA结构域。黄颡鱼Sox9a1和Sox9a2核苷酸序列的相似性为64％，但阅读框序列的相似性为71％。黄颡鱼Sox9a1和Sox9a2的氨基酸的相似性为65％，黄颡鱼Sox9a1和其他动物Sox9的相似性平均为73％，其中只有与斑马鱼Sox9a相似性较低为61％；而黄颡鱼Sox9a2与这些动物Sox9的相似性平均为67％，由此可见，黄颡鱼Sox9a1和其他动物的相似性要高于Sox9a2和其他动物的相似性。由系统树可见Sox9明显可分成三支，一支包括两栖类、爬行类、鸟类和人Sox9；另外两支为鱼类Sox9，其中鲤鱼Sox9b、黄颡鱼Sox9a2和斑马鱼Sox9b三个为一支；另一支包括虹鳟、黄鳝、鲻鱼、鲀鱼的两个Sox9以及黄颡鱼Sox9a1和斑马鱼Sox9a，该支又明显地把同一鱼类中分离到的两个Sox9分成两个小支，如黄鳝Sox9a1和黄鳝Sox9a2，而斑马鱼的Sox9a和黄颡鱼的Sox9a1分别属于这两个分支。RT-PCR结果显示Sox9a1在雌雄黄颡鱼前脑、心脏、肝脏、脾脏、肾脏和性腺均能检测到其表达，而在肌肉没检测到其表达；Sox9a2只在卵巢检测到，在其他组织均没检测到。说明Sox9a1和Sox9a2的表达存在着组织表达特异性，它们的调控也肯定存在着一定的差异。本文分离了黄颡鱼Ftz-F1同源基因，包括Ad4BP／SF-1和LRH-1。在精巢分离和克隆的黄颡鱼Ad4BP／SF-1 cDNA，Ad4BP／SF-1全长cDNA 1986 bp[不包括poly(A)]，包括5′非翻译区79 bp，3′非翻译区461 bp，阅读框1446 bp，编码482个氨基酸，计算的蛋白分子量为53.9 kDa。其中22-88个氨基酸为DBD结构域，89-116个氨基酸为Ftz-F1结构域。在肝脏分离到的黄颡鱼LRH-1，LRH-1全长cDNA 1945 bp[不包括poly(A)]，包括5′非翻译区207 bp，3′非翻译区238 bp，阅读框1500 bp，编码500个氨基酸，计算的蛋白分子量为57.19 kDa。其中39-105个氨基酸为保守的DBD结构域，106-132个氨基酸为Ftz-F1结构域。另外，还分离到一个没有活性的不含锌指结构和没有AF-2结构域的黄颡鱼LRH-1′，其全长1663 bp，5′非翻译区283 bp，3′非翻译区330 bp，阅读框1050 bp，编码350个氨基酸，计算的蛋白分子量为39.7 kDa。黄颡鱼LRH-1和Ad4BP／SF-1的氨基酸相似性只有59％。黄颡鱼LRH-1和其他动物的LRH-1相似性为79％-85％，和其他动物的SF-1相似性为59％-66％；黄颡鱼Ad4BP／SF-1和其他动物的SF-1为57％-96％，其中和人SF-1只有57％，其相似性低于黄颡鱼LRH-1和人SF-1的相似性60％。Ftz-F1系统树显示可分成两大分支，其中，鱼类SF-1单独聚为一大支，另外一支又分成人等脊椎动物SF-1和LRH-1，提示鱼类SF-1和LRH-1很早就分化了，但人类SF-1和LRH-1则分化稍晚。RT-PCR结果显示黄颡鱼LRH-1除了在肌肉、心脏没检测到外，在脑、肝、脾、肾和性腺中均有表达；本实验使用RT-PCR法在所取组织均没检测到Ad4BP／SF-1的表达，其原因可能是在成鱼组织中表达量比较少，但也有可能是引物不合适。P-450芳香化酶(P450arom)是催化雄激素生物合成雌激素的关键酶。本文首次分离和克隆了黄颡鱼卵巢和脑P450芳香化酶基因P450arom A和P450aromB。黄颡鱼P450arom A cDNA全长1914bp[不包括poly(A)]，5′端非翻译区13 bp，3′端362 bp，阅读框(Open reading frame，ORF)1539 bp，翻译成513个氨基酸，计算的蛋白质分子量为58.7kDa。P450aromB cDNA全长2260 bp[不包括poly(A)]，5′端非翻译区有205bp，3′端555 bp，阅读框(open reading frame，ORF)1500 bp，编码500个氨基酸，计算的蛋白质分子量为56 kDa。黄颡鱼P450aroma和P450aromB cDNA序列相似性为49％，但两者阅读框的相似性为62％。黄颡鱼P450aromA和P450aromB的相似性只有60％；黄颡鱼P450aroma的氨基酸序列与鱼类卵型的同源性比较高，除了黄鳝60％外，其他均在70％以上，和脑型的则比较低为60％左右；相反，脑型P450aromB则和其他鱼类脑型的相似性较高达70％以上，和卵巢型的相似性也只有60％左右；它们与鸡和人P450arom的相似性稍低均为50％左右。从相似性比较可见黄颡鱼P450aromB和P450aromA均与鲶鱼的相似性最高，体现了两者亲缘关系较近。脑型和性腺型芳香化酶基因在芳香化酶高保守区包括Ⅰ-螺旋区、芳香化酶特异保守区Ⅱ和血红素结合区Ⅲ的相似性很高，以人芳香化酶序列为基准，Ⅰ区性腺型的相似性为70％-73％，脑型的为66％-70％；Ⅱ区则高达73％-86％和78％-82％；Ⅲ区则在73％左右。系统发育分析表明P450arom可分为两支，一支包括软骨鱼类、两栖类、爬行类、鸟类和哺乳类，另外一支为除了软骨鱼类外的其他鱼类，并且该支又明显地分为性腺型和脑型P450arom，黄颡鱼P450aromA与鱼类性腺型P450arom属于同一支，而P450aromB与鱼类脑P450arom同一分支，至于这两支芳香化酶是否与在鱼类进化的长河中早期出现的基因组复制有关，还没有直接证据，但现在普遍接受这一解释。荧光实时定量RT-PCR研究结果显示，P450aromA在性腺发育为Ⅱ期鱼的前脑、下丘脑、脑垂体、精巢、肝脏中没检测到其表达，只在卵巢中表达，这说明P450arom A的表达具有组织特异性，并由此可推测其对卵巢发育起着重要作用；而P450aromB在前脑、下丘脑、垂体、卵巢、精巢均有表达，但肝脏没检测到其表达，脑部表达量高于性腺，但性腺发育处于Ⅱ期的雌雄鱼脑部P450aromB的表达总量没显著差异，卵巢中P450aromA的表达量是P450aromB的18.7倍。使用荧光定量RT-PCR法测定了性腺发育处于不同阶段的鱼脑、肝、肾和性腺中Sox9a1、Sox9a2、LRH-1、P450aromB和P450aromA的表达。结果，Sox9a1在雌、雄鱼(性腺Ⅳ、Ⅴ期)的肝、脑以及精巢表达量相对较高，在肾和卵巢表达量明显低，其中，雌雄鱼之间脑、肝和肾的表达水平没显著差异，卵巢和精巢之间有显著差异。以雌鱼脑表达量为1，则肝的表达量平均为雌鱼脑的2倍左右，而肾脏的表达量只有30％左右。Sox9a1在精巢的表达量和性腺发育有关，Ⅳ、Ⅴ期精巢中的表达两大约为雌鱼脑的50％左右，但Ⅱ期精巢的表达量是其4倍左右。Sox9a1在发育早期的精巢和卵巢的表达没显著差异。Sox9a2在卵巢各阶段的表达呈现在Ⅲ和Ⅳ期出现高峰，Ⅳ期和Ⅵ期表达量相对较低。LRH-1在各组织表达量测定揭示该基因在卵巢、雌雄鱼的肝以及脑表达量比较高，在肾的表达量比较低。在不同发育阶段卵巢的表达显示在Ⅲ期和Ⅴ期卵巢表达量明显比其他各期高，和Sox9a2有相似的表达模式。P450aromB在雌鱼脑的表达量较雄鱼脑要高，但差异不显著。在卵巢不同发育期，该基因呈现出在Ⅱ期晚表达量开始上升，在Ⅳ期出现高峰，到Ⅴ期、Ⅵ期表达量则下降，从趋势上Ⅲ期的表达量应该比Ⅱ期晚高，但结果并非如此，有待继续验证。黄颡鱼P450aromB在卵巢不同发育期的表达模式和在金鱼和虹鳟的研究结果基本一致。P450aromA的表达量在Ⅱ期晚期、Ⅲ期表达量明显高于Ⅳ期、Ⅴ期和Ⅵ期，Ⅱ期早期和其他各期均没显著性差异，以Ⅱ期早期该基因的表达量为1，则Ⅱ晚期和Ⅲ期分别是其6倍和5倍左右，而Ⅳ期、Ⅴ期和Ⅵ期值有其表达量的10％以下。从Sox9a2、LRH-1和P450aromA的表达模式可见，Sox9a2和LRH-1具有类似的表达模式，说明了他们之间存在着调控关系，这和在哺乳类等的研究结果一致，在Ftz-F1的启动子区存在结合Sox9蛋白的序列。但芳香化酶基因的表达和Sox9、LRH-1没明显的相关性，和现有在芳香化酶基因启动子区域存在Sox9和Ftz-F1结合位点存在着不一致，很可能虽然有结合位点，但并不起主要调节作用，但需要进一步实验证实。更多还原

【Abstract】 Yellow catfish (Pelteobagrus fulvidraco) is a valuable carnivore.that has good flesh quality.There is a market potential for this fish in China, Japan, Korea and SE Asia. In naturalwater, yellow catfish grows slowly, with a small marketable size, seriously influencing itsmarket development. In term of production, the most effective way to increase the yield andefficiency of yellow catfish consists in utilizing the obvious growth difference betweenmale and female yellow catfish, and to maintain mono-sexual cultures, since males grow30％faster than females. However, studies on the molecular mechanism of yellow catfishsex determination still remain unavailable. Especially studies on the integrative regulationmodel. Here we isolated and cloned Sox9 Ftz-F1 and P450arom cDNA, which played keyfunction in vertebrate sex differentiation and gonad development, using RT-PCR and RACEfrom yellow catfish. And then we analyzed their tissue expression pattern, the expressionlevels response to the gonad development period, and their relationship using fluorescentreal time RT-PCR andβ-actin as internal control. Wish. to reveal their tissue expressionmodel and the function in gonad development through our study.Sox9 is one of the important genes related to mammal sex differentiation and determination.Two cDNAs encoding sox9 were derived from brain, testis and ovary of yellow catfishusing reverse transcriptase-polymerase chain reaction (RT-PCR) and RACE. Sequenceanalysis revealed the cDNA from testis is as same as from brain, using Blast analysis wecalled it Sox9a1. The cDNA from ovary called Sox9a2. Yellow catfish Sox9a1 cDNA was 1604 bp with 70 bp 5’UTR, 142 bp 3’UTR(excluding poly[A]), and 1 392 bp ORF, whichencodes 464 amino acids and has a Predicted mol wt of 50.62 kDa. Thereinto the stretchfrom 99 to 176 of residues is the highly conserved sox-TCF_HMG-box, from 305 to 351 isPro-and Gin-rich (P/Q) region, 352 to 464 is TA region. Moreover, the Sox9a2 cDNA was1843bp with 313bp 5’UTR, 159bp 3’UTR[excluding poly(A)], and 1371 bp ORF, whichencodes 457 amino acids and has a predicted mol wt of 50.24 kDa. Thereinto the stretchfrom 111 to 188 of residues is the highly conserved sox-TCF_HMG-box, from 321 to 358is the P/Q rich region which is shorter than in Soxal and without the characteristic Pro-andGin-rich, from 359 to 457 residues is TA region which is shorter than Sox9a1. The whole sequence identity is 64％between Sox9a1 and Sox9a2, but the ORF of Yellow catfishSox9a1 shares 71％sequence identity with Sox9a2. The amino, acids sequence identity is65％between Sox9a1 and Sox9a2. Sox9a1 shares average 73％sequence identity withother fish speciesand animals including rice field eel (M. albus),common carp (C. carpio),medaka (O.latipes),rainbow trout (O.mykiss),zebrafish (D. rerio),red fin east puffer fish(T.rubripes),and X. laevis, A.mississippiensis, chicken (G. gallus), and human (H. sapiens),with the most smallest similarity 61％with zebrafish Sox9a. And Sox9a2 shares 67％sequence identity with above mentioned animals. So the identities between Sox9a1 andother animals were higher than those of Sox9a2 and other animals.The phylogenetic tree of Sox9 consists three obvious branches, one of which includingSox9 of amphibian species, reptilian specie, birds and mammals; teleosts Sox9 weredivided into two other.branches, one including common carp Sox9b, yellow catfish Sox9a2and zebrafish Sox9b, the other branch has two smaller branches, which comprised otherfishes Sox9a or Sox9a1 and Sox9b or Sox9a2 including rainbow trout, rice field eel,medaka, pufferfish separated, yellow catfish Sox9a1 and zebrafish Sox9a belong to thesetwo smaller branches respectively. According to the unrooted tree, the farthest distance wasbetween zebrafish Sox9b and other animals Sox9, and thenext is yellow catfish Sox9a2.Yellow catfish Sox9a1 also. had farther distance with Sox9a1 of rainbow trout, medaka andrice field eel which were in the same branch. The yellow catfish Sox9a1 was detected inbrain, heart, liver, spleen and gonad of female and male fish using RT-PCR, but not inmuscle, otherwise Sox9a2 was only detected.in ovary. This indicated that there were tissuesspecific expression pattern of Sox9a1 and Sox9a2 and there were differences in theirregulation mechanism.The fushi tarazu factor-1 (FTZ-F1) is a member of the nuclear receptor Superfamily andwas originally found as a regulator of the Drosophila homeobox segmentation gene FTZ.These genes were nominated as a sub-superfamily by nuclear receptor committee in 1999,and this sub-family have be classified two sub-group, one is NR5A1 including SF-1/Ad4BP(steroidogenic factor-1/adrenal 4 binding protein), another is NR5A2 including LRH/FTF(liver receptor hormone/a-fetoprotein transcription factor), which regulates a-fetoproteinexpression and involved in cholesterol metabolism. The mammal SF-1/Ad4BP genes areimportant regulators of steroid biosynthesis by controlling transcription ofmany P450enzymes including P450scc、3β-HSD and P450arom. They are expressed in steroidogenic tissues and are involved in the embryonic development of adrenals and gonads. In adultmammals, LRH-1 expression is confined principally to tissues of endodermal origin, suchas the liver, pancreas and intestine-justifying its functional classification as anenterohepatic NR. Recently, in several species LRH-1 was highly expressed in ovary. Thissuggests LRH-1 and SF-1 are involved in the ovarian steroidogenesis. Here we isolatedyellow catfish Ad4BP/SF-1 cDNA, which is 1 986 bp with 79 bp 5’UTR, 461 bp3’UTR(excluding poly[A]), 1 446 bp ORF, which encodes 482 amino acids and has apredicted mol wt of 53.9 kDa. The conserved DBD domain was in residues from 22 to 88,and Ftz-F1 box was in 89 to 116 residues. Yellow catfish LRH-1 cDNA is 1945 bp with207 bp 5’UTR, 238 bp 3’UTR[excluding poly (A)], 1500 bp ORF, which encodes 500amino acids and has a predicted tool wt of 57.19 kDa. DBD domain was in residues from39 to 105, and Ftz-F1 box was in 106 to 132. At the same time of isolation LRH-1, we alsogot an inactive LRH-1’in which the zinc-finger domain and AF-2 motif is absent. TheLRH-1’is 1663 bp with 283 bp 5’UTR, 330 bp 3’UTR, 1050 bp ORF, which encodes 350amino acids and had a predicted tool wt of 39.7 kDa. Yellow catfish Ad4BP/SF-1shares48％sequence identity with P450aromB, the sequence similarities are 60％in ORE Theamino acid sequence identity is 59％between LRH-1 and Ad4BP/SF-1. The LRH-1 shares79％-85％sequence identity with LRH-1 of other animals, but only 59％-66％identity withthe SF-1. however, yellow catfish Ad4BP/SF-1 shares 57％-96％sequence identity withSF-1 of other animals, and only 57％with human SF-1 which is lower than 60％betweenyellow catfish LRH-1 and human SF-1. RT-PCR analysis indicated yellow catfish LRH-1was detected in almost all tissues except muscle and heart. Unfortunately yellow catfishAd4BP/SF-1 was not detected using RT-PCR in all tissues used in our experiment. Maybethe expression of Ad4BP/SF-1 in adult fish was hardly detected or the primers used inexperiment were not proper.Aromatase cytochrome p450 (P450arom; the product of the CYP19 gene) is therate-limiting enzyme which catalyzes the conversion of androgens to estrogens. Here wereport the cloning of two types of cDNA encoding P450arom derived.from yellow catfish(Pelteobagrus fulvidraco) brain, and ovary, using RT-PCR, and RACE. The brain-derivedcDNA Was 2260 bp with 205 bp 5’UTR, 555 bp 3’UTR[exclUding poly(A)] and 1500 bpORF, which encodes 500 amino acids and has a predicted mol wt of 56 kDa. Moreover, theovarian cDNA was 1914 bp with 13 bp 5’UTR, 362 bp 3’UTR[excluding poly(A)] and1539bp ORF, which encodes 513 amino acids and has a predicted mol wt of 58.7 kDa. Yellow catfish P450aromA shares 49％sequence identity with P450aromB, the sequencesimilarities are 62％and 3％in ORF and 3’UTR respectively. The amino acid sequenceidentity is 60％between P450aromA and P450aromB. The P450aromA shares 61％-90％sequence identity with ovarian aromatases of other fish species, but only 60％identity withthe homologous P450aromB, 51％and 52％with chicken ovarian and human beingplacenta aromatases. And P450aromB shares 59％-85％sequence identity withbrain-derived aromatases of other fish species, about 50％with human being placenta andchicken ovarian aromatases. But the percentage of identity/similarity was higher in theregions of high homology, including theⅠ-helix, an aromatase-specific conserved regionⅡ,and the heme-binding regionⅢ, vs. human P450arom, which were 67％～96％, 78％～86％and 78％～100％respectively. Phylogenetic tree of the P450arom shows two main branches,one of them clusters all teleosts (bonny fishes) leaving apart the elasmobranches(cartilaginous fishes) and the other branch includes the tetrapods and the elasmobranches.The teleosts group bifurcates into two clear branches, one of them containing the brainvariants of aromatase and the other one the ovarian variants. It is acceptable thatduplication of an ancestral gene early in the teleostean lineage results in the fishP450aromB and A paralogs though there is no directive evidence. The yellow catfishP450arom are the closest with channel catfish. This result is consistent with theresult ofcontradiction classification. The fluorescent real-time quantity RT-PCR was developed toanalyze tissue-specific expression of P450arom B and P450arom A in adult yellow catfish,and measure mRNA relative expression leveIs withβ-actin as internal standard. B-isoformwas preferentially expressed in brain of both males and females but also present at muchlower levels in ovary, testis, however, A-isoform expression was restricted to ovary, andthere was no expression either A-or B-isoform in liver. The high to low order of P450aromB relatively, expression level in tissues was female fish hypothermia, male fishhypothalamus, female fish pituitary, male fish pituitary, male fish fore-brain, female fishfore-brain, ovary, testis. However, there was no significant difference of the totalexpression level in brain between male fish and female fish. The comparison of P450aromA and P450arom B expression in ovary indicated the P450arom A was 18.7 times ofP450arom B.We detected the expression levels of Sox9a1、Sox9a2、LRH-1、P450aromB and P450aromAin brain, liver, kidney and gonad of yellow catfish which was at different developed phases of gonad with the approaches of fluorescent quantitative RT-PCR. Comparison the Sox9a1levels between female and male yellow catfish with the gonad development of phaseⅣandⅤshowed that Sox9a1 was high in the liver, brain and testis, but low in kidney andovaries. There is no significant difference between male and female yellow catfish brain.The Sox9a1 expression level of testis at phaseⅡwas higher than phaseⅣandⅤ. Thisindicated that Sox9a1 played important role in the early development of testis. Expressionlevel of Sox9a2 had two peaks in ovary with phaseⅢandⅤ, and low in phaseⅣandⅥ.The expression levels of LRH-1 in different organs revealed that LRH-1 expressed highquantity in ovary, liver and brain, but low in kidney. The levels were significantly higher inthe ovary with phaseⅢandⅤthan in ovary with other development phases. Thisexpression pattern was consistent with Sox9a2. The expression level of P450aromB in maleyellow catfish brain was higher than in female fish, however, the difference was no distinct.During the different developing phases of ovary, expression of this gene ascended in thelate phaseⅡand peak appeared in phaseⅣ, then the level decreased in phaseⅤand phaseⅥ. The trend is that expression in phaseⅢshould be higher than that in late phaseⅡ,however, the result didn’t accord to this trend, which needed to be identify in future. Thisexpression pattern of P450aromB is the same with the result of goldfish.and rainbow trout.The expression levels of P450aromA in ovary with lateⅡandⅢphase were higher thanthe ovary withⅣ,Ⅴ, andⅥphases. There was no significant difference between earlyⅡphase ovary and ovary with other development phases. The expression levels ofP450aromA in ovary with lateⅡandⅢphase were 6 and 5 times, of earlyⅡphase ovaryrespectively, however, the expression level was below 10％inⅣ,Ⅴ, andⅥphases.It was observed from the expression pattern of Sox9a2、LRH-1 and P450aromA, Sox9a2and LRH-1 had the similar expression pattern; it indicated that regulatory relationship waspresent between them. The similar results were found in mammals. There are bindingsequences of Sox9 protein in the region of Ftz-F1 promoter. However, there is no distinctcorrelation in the expression of aromatic enzyme, Sox9 and LRH-1.更多还原