【作者】 周鹏；

【导师】 王艺磊；

【作者基本信息】 集美大学 ， 水产养殖， 2009， 硕士

【摘要】 以大黄鱼(Larimichthys crocea)性腺为材料,采用SMART(switching mechanism at 5’end of RNA transcript)技术和DSN (duplex-specific nuclease)处理构建了大黄鱼性腺线性化cDNA文库,并从文库中随机挑取了3961个克隆进行测序,共获得3535条高质量的表达序列标签(Expressed Sequence Tag, EST)。序列经聚类分析后共得到2916条Unigene,包括415条contigs和2501条singletons。初步分析结果表明：该线性化文库的库容为4.3×106pfu/mL,文库重组率达95.8%,EST的非冗余率为82.49%, Unigene的平均长度为355 bp。经生物信息学分析,1785个为已知基因占总ESTs的61.21%；其中与性腺发育相关的基因66个,占已知基因的3.70%。微卫星分析发现共有129条EST序列含有149个微卫星重复序列,包括6条与性腺发育相关的ESTs。利用实时荧光定量PCR (qRT-PCR)技术分析了11个与性腺发育相关基因在大黄鱼性腺的表达情况。结果显示精巢特异性LRR基因(testis-specific LRR)只在大黄鱼精巢中表达(P<0.05)；雄性特异性致死3样1基因(MSL3L1),雄激素相互作用受体核蛋白激酶基因(ARINPK),精子发生凋亡相关蛋白基因(SARP),热休克蛋白70(HSP70)和细胞周期蛋白A1基因(cyclin A1)在精巢中的表达量高于卵巢(P<0.05)；相反,组织蛋白酶C基因(Cathepsin C),核自身抗原精蛋白基因(NASP)和细胞周期蛋白B1 (cycli B1)在卵巢中的表达量高于精巢(P<0.05)；促分裂原激活蛋白激酶1(MAPK1)和泛素C末端水解酶L5 (UCH-L5)在精巢和卵巢中表达没有显著差异(P>0.05)。在上述结果的基础上,利用SMART-RACE技术或RT-PCR技术克隆了大黄鱼性腺发育相关基因cyp19a、cyp19b、cyclin B1、CDC2、UBE2D和UBC9全长cDNA序列,其长度分别为1805 bp、2268 bp、1882 bp、1151 bp、798 bp和846 bp,可分别编码518、500、397、303、147和148个氨基酸的前体蛋白。利用qRT-PCR技术分析这些基因在大黄鱼各器官的表达情况,结果显示：cyp19a在卵巢中的表达量显著高于精巢(P<0.01),且cyp19a在精巢和卵巢中的表达均极显著高于其它各器官(P<0.01); cyp19b在端脑、中脑、下丘脑、脾和肝有较高表达量,在雄性大黄鱼的血中表达量最高。在性腺表达量极低。cyclin B1和CDC2基因在性腺中的表达量极显著高于其它各器官(P<0.01),且在卵巢中的表达量较精巢高(P<0.05)。UBE2D在脾脏和性腺中表达量较高,其它器官表达量较低,在性腺中的表达极显著高于其它各器官(除脾脏外)(P<0.01)。UBC9在性腺中表达量较高,且极显著高于其它各器官(P<0.01)。更多还原

【Abstract】 To clone the gonad-specific or gonad-related expressed genes of large yellow croaker Larimichthys crocea, a normalized gonad cDNA library was constructed using the combination of SMART and TRIMMER techniques. A total of 3961 clones randomly picked from the library were single-pass sequenced and 2916 unigenes were clustered from the 3535 high quality ESTs (expressed sequence tags). The content of cDNA library was 4.3×106pfu/mL, the recombinant percentage of the normalized cDNA library was 95.8%, the percentage of unique sequence of the library was 82.49% and the average length of unigenes was 355 bp. Searching Genbank by using these nucleotide sequences indicated that 1785 ESTs showed significant homology with known genes in the NCBI database. Among the 1785 known genes, about 66 significant gonad-related genes were found, accounting for 3.70% of the total unigenes. Six gonad-related microsatellite-containing ESTs were identified from the 129 ESTs containing a total of 149 microsatellites. Expression patterns of 11 gonad-related homologues in testes and ovaries were selected for quantitative relative-time PCR (qRT-PCR). The results showed that gene encoded testis-specific LRR protein was expressed in testes but not ovaries (P<0.05), and MSL3L1, ARINPK, SARP, HSP70 and cyclin A1 were expressed more in testes than ovaries (P<0.05). Conversely, Cathepsin C, NASP and cyclin B1 were expressed more in ovaries than testis (P<0.05), and expression levels of MAPK1 and UCH-L5 in ovaries and testes were not significantly different (P>0.05).Based on the above results, full length cDNAs of cypl9a, cyp19b, cyclin B1, CDC2, UBE2D and UBC9 from the large yellow croaker were obtained by using SMART-RACE PCR or RT-PCR. The full length cDNAs are 1805 bp,2268 bp,1882 bp,1151 bp,798 bp and 846 bp respectively, and the lengths of deduced protein sequence of these genes are 518,500,397,303, 147 and 148 amino acids respectively. The results of qRT-PCR showed that the expression level of cyp 19a in ovary was significantly higher than in testis (P<0.01), and the expression level of cypl9a in both ovary and testis was also much significantly higher than in other organs (P<0.01). However, cypl9b has lower expression levels in gonads, and has higher expression levels in midbrain, forebrain, hypothalamus, spleen and liver, and the highest expression level in blood. Cyclin B1 and CDC2 were expressed the most in gonads, and the expression level in ovaries is significantly higher than in testes (P<0.05). UBE2D was most expressed in spleen and gonads, and the expression level of UBE2D in testis is significantly higher than in ovary, and the expression levels in gonads were also significantly higher than in other organs (excluding spleen) (P<0.01). The expression levels of UBC9 in gonads are significantly higher than in other organs (P<0.01).更多还原