Isolation And Expression Of CML21 Promoter And CML Genes From Camellia Sinensis

Tea plant[Camellia sinensis(L.)O.Kuntze]is a perennial,evergreen,woody crop,belonging to theaceae Camellia.C sinensis is thermophilic and afraids of low temperature."Tea" is made of tea leaves,which is known as one of the world's three major non-alcoholic drinks.Environmental stresses not only limit the distribution of tea planting area,but also cause economic losses.Among the environmental stresses,cold is the common stress affecting tea growth,tea yield and quality.Therefore,improving the cold tolerance of C.sinensis deserves more attetions.However,cultivating cold-resistant tea varieties through physiological and ecological method has a long breeding season and is difficult to obtain ideal phenotype.So,it is significant to understand the molecular mechanisms of C.sinensis responding to abiotic stress and improve the resistance of C.sinensis by biotechnology means.In response to the above scientific questions,the cultivar of C.sinensis'Longjingchangye' was used to analyze the calmodulin like 21 promoter(pCsCML21)and five CML genes from C.sinensis were cloned.The sequences of pCsCML21 and CML geneswereanalyzed by bioinformatics softwares.The response mechanism of the CML genes on transcriptional level under cold,ABA,drought and salt stress was assessed by molecular biology technology.The main results and conclusions are as follows:1.The pBI101::CsCML21 and pBI101::CsCML21-M of expression vector were introduced into Arabidopsis thaliana by Agrobacterium tumefaciens(GV3101).Transformation of Arabidopsis plants(F and M mutant plants)was identified by RT-PCR.The GUS reporter gene of the M(lack the ABRE element pCsCML21 promoter)and F(pCsCML21 promoter)mutants was analyzed by GUS histochemical staining method.In the leaves,wild-type Arabidopsis thaliana(WT)did not have GUS activity,and the M and F mutants had GUS gene expression.Besides,the F mutant plant was more active than the M mutant plant,indicating that the absence of the ABRE element weakens the activity of the pCsCML21 promoter.2.In order to study the function of CML gene family,we searched for transcriptome data(SRR5075641),and 10 homologous sequences were obtained.Five EST sequences of CsCML were obtained after removing the repeat sequences.The 3' and 5' ends of the incomplete sequence were obtained by RT-PCR and RACE.After the splicing,the full length of the cDNA was analyzed by Bioxm software to obtain the largest open reading frame(ORF).The specific primers were designed at both ends of the sequence.Five genes containing complete ORF were obtained with the size of 480bp,474bp,501 bp,435bp and 609bp,respectively.These sequences were named CsCML16,CsCML18-1,CsCML18-2,CsCML38 and CsCML42.Phylogenetic tree analysis showed that CML genes family of C.sinensis has high homology with the CML gene sequence corresponding to other species,which indicates that the CML gene of C.sinensis are orthlogs of CML genes from other species.Structure analysis showed that five CsCML proteins had no transmembrane motifs,and all of proteins located in inner of membrane with exception of CsCML 18-1 in outer of membrane.3.The expression level of CML gene in tea leaves under various abiotic stress was analyzed by quantitative real time PCR.The results showed that CML genes were up-regulated under low temperature stress except CsCML18-1.Under the drought stress,these genes were down-regulated,except CsCML38.The expression of CsCML16,CsCML18-1 and CsCML38 was down-regulated by ABA,but the expression of CsCML18-2 and CsCML42 was up-regulated.Under high salt treatment,the expression levels of CsCML16,CsCML18-2 and CsCML42 were upregulated,while the expression patterns of CsCML18-1 and CsCML38 were the opposite.The results of issue-specific expression showed that the mRNA accumulation of CsCML was detected in roots,stems,leaves and flowers of C.sinensis.