马氏珠母贝Sox11基因的克隆及时序表达模式分析

为探究Sox(SRY-related HMG-box genes)基因家族在马氏珠母贝个体发育及性别分化中的作用, 研究首先利用兼并引物从马氏珠母贝基因组中克隆到一个HMG框(high mobility group box), 利用RACE-PCR技术从SMART cDNA文库中克隆到一个Sox基因的cDNA全长, 通过Clustal X和MEGA 4软件对该序列进行比对分析并构建系统进化树; 通过荧光定量PCR技术对该基因在不同组织及发育不同时期性腺中的表达情况进行分析。结果显示, 马氏珠母贝该Sox基因的cDNA全长为1579 bp, 其中开放阅读框(ORF)为1008 bp, 编码336个氨基酸, 5'非编码区为126 bp, 3'非编码区为445 bp。同源性分析表明, 马氏珠母贝Sox基因与太平洋牡蛎Sox11基因的同源性(Identity)最高, 为80%, 故命名为pmSox11; 系统进化树分析也显示pmSox11与太平洋牡蛎Sox11基因的亲缘关系最近。荧光定量PCR分析组织表达特异性显示, pmSox11在马氏珠母贝神经节分布较多的组织如外套膜、鳃、足、消化盲囊等大量表达, 在神经节相对较少的闭壳肌和卵巢中表达量较少; 时序表达图谱显示, pmSox11在3月龄幼贝性腺和1年龄发育早期精巢中表达量最高, 在2年龄成熟精巢和2年龄性转换性腺中表达量降低, 而在2年龄卵巢中表达量最低。研究表明, pmSox11基因可能在马氏珠母贝早期神经系统发育和性别发育的调控方面起到重要作用。

MOLECULAR CLONING AND EXPRESSION PATTERNS OF SOX11 GENE IN PINCTADA MARTENSII

To evaluate the expression of Sox (SRY-related HMG-box) gene during development and sex differentiation of Pinctada martensii, we cloned and characterized the HMG box and the full-length cDNA of Sox gene in Pinctada martensii. DNA sequences and phylogenetic tree were analyzed. RT-PCR was used to measure the expression of Sox gene in different tissues and in gonads of different phases of Pinctada martensii. The results showed that the full-length cDNA of Sox was 1579 bp, including a 5'UTR of 126 bp, a 3'UTR of 445 bp and an open reading frame of 1008 bp, which encoded a deduced protein of 336 amino acids. Sequence comparison indicated that Sox in Pinctada martensii (pmSox11) and Sox11 in Crassostrea gigas (CgSox11) had 80% homology. Phylogenetic analysis demonstrated that pmSox11 was clustered with CgSox11. pmSox11 expressed highly in mantle, gill, foot and digestive diverticulum with more ganglia distribution and lowly in adductor muscle and female gonad with fewer ganglia distribution. Moreover, pmSox11 expressed highest in 3-month-old gonad and 1-year-old testis, modest in 1-year-old testis and 2-year-old testis, and lowest in 2-year-old ovary. The results indicated that pmSox11 might be involved in development of early nervous system and regulation of sex differentiation in Pinctada martensii.