金花茶多酚氧化酶基因的克隆及其体胚发生过程中的表达分析  

Cloning and expression of PPO gene during somatic embryogenesis in Camellia nitidissima Chi.

作　　者：高宇琼[1,2] 林玉玲[1] 赖钟雄[1] 

机构地区：[1]福建农林大学园艺植物生物工程研究所,福建福州350002 [2]铜仁学院生物科学与化学系,贵州铜仁554300

出　　处：《福建农林大学学报（自然科学版）》2014年第6期602-608,共7页Journal of Fujian Agriculture and Forestry University:Natural Science Edition

基　　金：福建省科技平台建设项目(2008N2001);国家科技支持计划项目(2007BAD07B03)

摘　　要：以金花茶胚性培养物为材料,对体胚PPO基因克隆以及在体胚发生过程中的转录水平进行q PCR分析和酶活性变化检测.结果表明:克隆获得的金花茶PPO基因c DNA序列1788 bp,含有完整的开放阅读框(ORF),编码595个氨基酸;与登录Gen Bank的山茶属其他植物PPO基因序列进行核苷酸和氨基酸比对发现,同源性分别为74%和72%左右.同时,以18S rRNA作为内参基因进行的q PCR分析表明:金花茶体胚发生过程中PPO基因表达量呈下降趋势,球形胚PPO基因表达量最大,子叶胚和心形胚次之,成熟胚最小;而正常子叶胚和花状多子叶胚的表达量存在明显差异.此外,对金花茶体胚发生过程中PPO活性变化检测结果表明:在此过程中PPO活性明显比较低,变化趋势与PPO基因定量表达结果相似,推测PPO基因可能在金花茶体胚发生早期发挥重要作用.The embryogenic cultures were used as the material for gene cloning of PPO gene from somatic embryos,and analysis of relative expression of PPO gene by using real-time quantitative PCR technology and detection of PPO activities during somatic embryogenesis in Camellia nitidissima Chi..The results showed that the sequence of cDNA of PPO gene was 1788 bp,with entire open reading frame （ORF）,and encoded 595aa.The homologous of the PPO gene sequence in C.nitidissima and those of the other plants of Camellia reported in GenBank was 74％,and the homologous of the deduced amino acid sequence was 72％.In the meantime,18S rRNA gene was used as the control for PPO gene expression analysis by real-time quantitative PCR technology during somatic embryogenesis in C.nitidissima,and the results showed that the expression of PPO gene showed a tendency to decline,the relative expression levels at the stage of globular embryos was the highest and then a small drop appeared at the stage of cotyledon embryos and heart-shape embryos,the relative expression levels at the stage of mature embryos was the lowest,the obvious differences of the relative expression levels were found between normal cotyledon embryos and MCFS embryo （embryo with multiple cotyledons and flower-like shape）.Besides,the trend of PPO activity changes was detected during somatic embryogenesis in C.nitidissima,and the results showed that the PPO activities were lower,its changeable tendency was similar with the expression of PPO gene,which suggested that the PPO gene may play an important role in the early somatic embryogenesis of C.nitidissima.

关 键 词：金花茶 体胚发生 多酚氧化酶(PPO) 基因克隆 实时荧光定量PCR PPO活性 

分 类 号：S682.31[农业科学—观赏园艺]