Exploring The Function Of Calmodulin-like Gene CsCML16 In Response To Cold Stress In Tea Plant

Tea plant(Camellia sinensis)originated in southwest region of China,mainly grows in the tropical and subtropical regions,the characteristics of sensitive to low temperature makes it vulnerable to cold stress.Therefore,it is essential to study molecular mechanism under cold hardiness between different cultivation physiological and molecular breeding way from the cold resistance of tea plant.Ca2+signaling pathways,one of the most major ways of plant for response to low temperature stress.The Calmodulin-like(CML)proteins are a kind of important calcium sensors,which play an important role in the Ca2+signal channel.One-year-old tea cutting seedlings of ’Longjing 43’,’Fuding Dabai’ and ’HuangJinya’ were used as materials to study the change of physiological indicators under low temperature stress between different varieties.Full length of CsCML16 gene was cloned from ’Longjing 43’ for biological information analysis and subcellular localization assay.The Arabidopsis strains over-expression CsCML16 were constructed for analyzing the functional validation of CsCML16.The main results are listed below:(1)The results of physiological characteristics of tea leaves under low temperature stress showed that there were significant differences to response to cold resistance between ’Longjing43’,’Fuding Dabai’ and’HuangJinya’,the size of cold resistance was ’Longjing43’>’Fuding Dabai’>’HuangJinya’.(2)The CsCML16 sequence was cloned from ’Longjing43’.Bioinformatics analysis results showed that CsCML16 protein belonged to EF-hand superfamily,containing two EF-hand domains,which possessed high similarity with other species.The promoter sequence of CsCML16 contained several cis-elements associated with abiotic stress,including binding sites(MYC:CATGTG/CATTTG;CCAAT)of low temperature responsive element.(3)qRT-PCR analysis showed different expression of CsCML16 among different tea varieties,which responded to low temperature stress.Under low temperature stress,CsCML16 was expressed in’Longjing43’,’Fuding Dabai’ and ’HuangJinya’,the expression level of CsCML16 in ’Longjing 43’was the highest.(4)The signal of CsCML16 was observed in both cell membrane and nucleus.(5)Over-expression vector was constructed of CsCML16,and the over-expression Arabidopsis were obtained by Agrobacterium-mediated method.CsCML16 overexpressed Arabidopsis showed higher tolerance to cold stress,and improved ROS scavenging capacity,cell membrane with less damage under cold condition compared to wild type plants.Furthermore,it was observed that the CsCML16 gene regulated the expressions of downstream genes of cold responsive pathway,such as CBF1,RD29A,COR47 and KIN1.To sum up,CsCML16 plays an important role in response to low temperature response in the tea plant.This study would provide insights for elucidating the molecular mechanism of CsCML16 under cold condition.