斑节对虾性腺差异表达基因的筛选

Screening of differentially expressed genes of gonads from the pond-reared black tiger shrimp(Penaeus monodon)

以池塘养殖250日龄的斑节对虾性腺组织为材料,利用mRNA差异显示(differential display,DD)筛选性腺差异表达基因,获得173条雌、雄性腺差异表达片段。随机选取44条差异片段进行回收、克隆、测序及Real-timeRT-PCR验证,最终获得10条阳性差异表达片段,其中OA7-1、OA7-2、OG8-1、OG8-2、OC1-3、OC4-2、OC8在卵巢中表达量极显著(P<0.01)高于精巢,TG6-3、TG8在精巢中表达量极显著(P<0.01)高于卵巢,TC1为精巢特异表达片段。序列分析表明OA7-2为脱氧尿嘧啶三磷酸核苷酸水解酶(dUTPase)(E值6e-54);OG8-1为真核细胞翻译起始因子3亚单位4(eIF-3delta)(E值9e-58);OC1-3为未命名蛋白产物(unnamed protein product)(E值2e-17);OC4-2为60S核糖体蛋白L7(RPL7)(E值6e-40);OC8为60S核糖体蛋白L3(RPL3)(E值5e-36);OA7-1、OG8-2和TC1与BLASTx比对同源性较低(10-4<E值<10);TG6-3和TG8未检索到同源序列(E值>10),为新的EST序列。对TC1转录水平分析表明其在精巢中表达,在卵巢及雌、雄性个体的眼柄神经节、大脑组织、肝胰腺、肌肉中均不表达,雌、雄个体基因组DNA均能扩增出TC1片段,推测TC1可能为一条由转录水平调控参与精子发生及繁殖相关的重要基因。对精巢表达量远高于卵巢的差异片段TG6-3进行雌、雄个体不同组织转录水平的对比分析发现,其在眼柄神经节组织中不表达,在雌性脑组织中不表达而在雄性脑组织中表达且在性腺组织中表达水平最高,表明TG6-3有可能为一条参与斑节对虾性腺发育调控的基因,为进一步阐明对虾性腺发育的分子调控机制提供了有价值的信息。

In order to clone and analyze the differentially expressed genes of gonads of domesticated broodstock Penaeus monodon Fabricius, total RNA was extracted from the ovary and testis, and differential display technique was performed to screen the differentially expressed genes by using a set of 3 anchored primers for cDNA synthesis and 8 arbitrary primers in the PCR. The differential display DNA bands were separated by denaturing urea-polyacrylamide gel electrophoresis （PAGE） and displayed by silver staining. Finally 173 cDNA fragments were isolated, and 44 cDNA fragments were excised. After cloning and sequencing ,11 differences in expression of the screened cDNA fragments were confirmed by real-time RT- PCR, and 10 cDNA fragments proved to be real difference. Results show that these 10 fragments were expressed differently between ovary and testis, 7 fragments were expressed significantly higher level in ovary, two fragments were expressed significantly higher level in testis, one fragment was particularly expressed in testis. The nucleotide sequence analysis revealed that two fragments were not homologous to of the known genes or ESTs, may be novel genes. TC1 was only expressed in testis, and there was no any difference between female and male DNA level. TG6-3 revealed a preferential expression level in testes than ovaries, and it was only expressed in male brain but no expression in female brain. This research provided a foundation for further studies of the molecular mechanism of sexual differentiation and gonadal development of Penaeus monodon.