茶树CLH基因家族的鉴定与转录调控研究及其在白化茶树中的表达分析

Identification and Transcriptional Regulation of CLH Gene Family and Expression Analysis in Albino Tea Plants(Camellia sinensis)

叶绿素酶(Chlorophyllase,CLH)是叶绿素降解过程中的关键酶,将叶绿素a脱去植醇,形成脱植基叶绿素a。以白化茶树白鸡冠新梢叶片为材料,克隆获得3条CsCLHs基因cDNA全长序列,并进行生物信息学分析。结果表明,3条CsCLHs基因分布于2个亚家族,其蛋白质编码区(Coding sequence,CDs)长度为894~975 bp,编码氨基酸个数为297~324,蛋白质分子量为31.99~34.91 kDa,等电点为4.89~7.61,不稳定系数为38.94~48.24,其中CsCLH1.1和CsCLH1.2为不稳定蛋白,CsCLH2为稳定蛋白。Cell-PLoc亚细胞定位预测结果表明,3个CsCLHs蛋白均定位于叶绿体;而WolfPsort亚细胞定位预测结果显示,CsCLH1.1和CsCLH1.2定位于细胞质,CsCLH2定位于叶绿体。遮阴和恢复光照处理下的qRT-PCR结果显示,遮阴抑制白鸡冠叶片CsCLHs的表达,光照诱导白鸡冠叶片CsCLHs的表达。不同品种中CsCLHs表达模式分析表明,CsCLH1s在白化叶中高表达。另外,酵母单杂交结果表明,CsCDF5可以与CsCLH1.1和CsCLH2启动子结合。综上所述,CsCLHs在白化茶树叶片中可能参与叶绿素降解,在叶片白化过程中发挥重要作用,结果可为进一步探究茶树CLH基因家族的功能及茶树叶片白化机理提供参考。

Chlorophyllase(CLH)is the key enzyme in the degradation of chlorophyll,stripping its phytol to form dephytolithochlorophyll a.The full-length cDNA sequences of three CsCLHs genes were obtained from the second leaves of albino tea cultivar‘Baijiguan’,and bioinformatics analysis was performed.The results show that the three CsCLH genes could be divided into two subfamilies.The full length of CsCLHs was 894-975 bp,encoding 297-324 amino acids.The protein molecular weights were 31.99-34.91 kDa.The isoelectric points were 4.89-7.61,and the instability coefficients were 38.94-48.24.CsCLH1.1 and CsCLH1.2 were unstable proteins,while CsCLH2 was a stable protein.The subcellular localization prediction results of Cell Ploc show that three CsCLH proteins were located in chloroplast,while the results of Wolf Psort show that CsCLH1.1 and CsCLH1.2 were located in cytoplasm and CsCLH2 was located in chloroplast.The qRT-PCR results on the‘Baijiguan’leaves indicated that expressions of CsCLHs were inhibited by shading treatment and light induced CsCLHs’expressions.Expression pattern analysis of CsCLHs shows that CsCLH1s were highly expressed in the albino cultivars.In addition,it was identified that CsCDF5 could bind to the CsCLH1.1 and CsCLH2 promoters according to the yeast one hybrid system.In conclusion,CsCLHs in albino tea leaves might be involved in chlorophyll degradation and play an important role in the process of albino leaf,which provided a reference for further investigation in the function of the CLH gene family and the albinism of leaves in tea plants.