龙须菜细胞周期检查点相关基因的分离鉴定及在不同品系四分孢子体中的表达分析  

Isolation and Characterization of Cell Cycle Checkpoint Related Genes and Their Expression Analysis in Different Tetrasporophyte Strains of Gracilaria lemaneiformis

作　　者：王璐[1] 吴琼[1] 银静茹 隋正红[1] Wang Lu;Wu Qiong;Yin Jingru;Sui Zhenghong(Key Laboratory of Marine Genetics and Breeding(Ocean University of China),Ministry of Education,Qingdao 266003,China)

机构地区：[1]中国海洋大学海洋生物遗传学与育种教育部重点实验室,山东青岛266003

出　　处：《中国海洋大学学报（自然科学版）》2024年第11期65-82,共18页Periodical of Ocean University of China

基　　金：国家自然科学基金项目(32072953);中国农业农村部现代化产业技术体系项目(CARS-50)资助。

摘　　要：为了探究细胞周期检查点的调控对龙须菜(Gracilariopsis lemaneiformis)四分孢子体发育的影响,本研究中对龙须菜细胞周期检查点部分基因进行分离鉴定及基因表达分析。从不同品系不同发育阶段的数字表达谱数据中,筛选得到与细胞周期检查点通路相关的差异表达基因ATR、ATM、RAD17和RAD9。通过基因克隆、序列比对和结构域预测,确认了其存在于龙须菜中,对4个基因上游2000 bp进行启动子预测发现,4个基因均含有与光照和植物激素相关的启动子元件;通过荧光定量PCR检测4个基因在良种981、WLP-1品系和野生型龙须菜的四分孢子体发育Ⅰ、Ⅱ、Ⅲ期的表达水平变化趋势发现,在WLP-1品系的Ⅰ期,4个基因表达呈现上升趋势;在该品系Ⅱ期,ATR、ATM和RAD9表达呈上升趋势,WLP-1在四分孢子体发育的Ⅰ期,细胞周期检查点相关基因表达相对活跃,表明此时其DNA复制和细胞分裂旺盛,这可能是WLP-1品系高育的原因。良种981在Ⅰ期ATR、ATM和RAD17相对表达量与野生型相比无显著变化,而RAD9有显著降低的趋势。进入Ⅱ和Ⅲ期后,与野生型相比,ATR、ATM和RAD17的相对表达量呈现显著上升的趋势,结合对其形成四分孢子性状的观察,推测在良种981四分孢子体放散的Ⅱ期,DNA损伤可能没有完全修复,因而最终导致低育。In order to explore the influence of the regulation of cell cycle checkpoint on the development of tetrasporophyte of Gracilaria lemaneiformis,some genes of cell cycle checkpoint of G.lemaneiformis were isolated,characterized and analyzed in this study.From the Digital Gene Expression Profiling at different stages of development of different strains,the differential expression genes ATR,ATM,RAD17 and RAD9 related to the pathway of the cell cycle check point were screened.The cloning,sequence alignment and domain prediction of the four genes were carried out,and their existence in G.lemaneiformi s were confirmed.The promoter prediction of the 2000 bp upstream of four genes showed that all four genes contained promoter elements related to light and plant hormone response.Through qPCR technology,the expression trends of four genes in tetrasporophyte development stagesⅠ,ⅡandⅢof cultivar 981,WLP-1 as well as the wild type strain were detected.The results showed that the expression of four genes displayed an upward trend in stageⅠof WLP-1 strain.The gene expression of ATR,ATM and RAD9 showed an upward trend in PhaseⅡof this strain.The relative promoted of cell cycle checkpoint related genes of WLP-1 strain in phaseⅠof tetrasporophyte development suggested active status of DNA replication and cell division,which may be the reason for the high fercility of WLP-1 strain.The relative expression of ATR and ATM,RAD17 genes of the cultivar 981 in PhaseⅠshowed no significant change compared with that of the wild type,and RAD9 showed a significant downward trend,while the relative expression of ATR,ATM,RAD17 in PhaseⅡandⅢshowed a significant upward trend compared with that in PhaseⅡandⅢof the wild type Gracilaria lemaneiformis respectively.Combined with the observation of its tetraspore forming character,it is speculated that the DNA damage of the cultivar 981 in PhaseⅡof the tetraspore release process was not completely repaired,finally,it leads to low fertility.

关 键 词：龙须菜 细胞周期检查点 荧光定量PCR 育性 四分孢子体 

分 类 号：Q751[生物学—分子生物学]