极大节旋藻生物钟基因的克隆和表达分析-细胞生物专业论文.docxVIP

KaiC蛋白中不仅发现N端酰基化位点、酪蛋白激酶Ⅱ磷酸化位点、蛋白激酶C KaiC蛋白中不仅发现N端酰基化位点、酪蛋白激酶Ⅱ磷酸化位点、蛋白激酶C 磷酸化位点、cAMP和eGMP依赖的蛋白激酶磷酸化位点、N端糖基化位点，而 且还发现了一些具有重要的功能的模体walkerA、DXXG等，二级结构分析发现 有18个Ⅱ螺旋、2Z个B折叠，与PDB数据库中聚球藻PCC 7942的KaiC蛋白 的折叠方式相近，结构一致性为81．6％。蛋白系统发生树分析发现，Kai蛋白都 比较保守，其中KaiC蛋白的保守性最高，KaiA蛋白的保守性最低。 本研究还对已知的kai基因簇设计探针，Southern杂交分别测定了kai基因 在节旋藻基因组内的拷贝数。杂交结果显示勋谢、勋培、勋酊基因均为单拷贝， 证明极大节旋藻FACI-IB 438只含有kai基因簇，而不含游离于基因簇外的kai 基因，间接证明kai基因是有功能的。 本研究还对92小时不同培养条件下的极大节旋藻的kai基因簇表达进行检 测，检测结果发现在持续光照条件下，节旋藻kai基因簇表达具有明显的12小 时的节律，而在对照组(12L／12D)中kai基因簇前36小时表达情况与光照组基 本一致，在36小时后生物钟节律相位发生变化。两组实验材料虽然基因表达节 律不同，但是kaiA、勋诏、妇酊3个基因的表达总是同步的，而且它们的相对表 达量基本保持一致。两组实验组kai基因表达节律的差异说明光信号对极大节旋 藻生物钟节律是有影响的。 关键词：节旋藻；实时定量P隙；生物钟节律；kai基因簇 Clone Clone and gene expression analysis of kai gene cluster Abstract Circadian rhythms help organisms keep the track of daily cycle．Circadian clock genes generate and control the running ofthe circadian rhythms．Cyanobacteda which are the simplest organism known to exhibit circadian rhythms have provided expriment model systems．More and more research fmd the cyanobacteria clock is known to control many physiological processes an other gene expression．Arthrospira is a kind of noticeable cyanobactefia not only in their nutritional potential but in their special biological characteristics．Although the molecular genetic studies of Arthrospira initiated earlier,it lagged behind in circadian clock gene research and it WaS unknow about circadian rhythms in Arthrospira． Four fosmid clones which contained a circadian clock gene k口iC was isolated from a fosmid DNA library of Ar历rospira maxima by PCR-pooling based on using partial known sequence．The full length kai gene cluster was cloned by sequence walking and the coding region ofkai gene cluster consists of3820bp nucleotide acids． The clock gene cluster consists of 3 genes that they arc肠“，勋培，and kaiC．The length of encode region of翰{A，妇iB，and妇iC genes are 858bp,315jbp，1554bp respectively．The average GC content of the coding region of勋谢，勋诏，and勋酊 gene arc close，respec