Identification And Functional Analysis Of CsSnRK2s And CsADC Genes Based On RNA-Seq Of Postharvest Dehydrate Leaves From Tea Plant

Tea tree is one of the important economic crops in our country.Persistent drought and low temperature and other abiotic stresses all affect the yield and quality of tea.In order to screen important genes related to drought resistance and to analyze the molecular network of dehydration stress signal transduction,In this study,the important protein kinase SnRK2 s in the ABA signal transduction pathway and the arginine decarboxylase(ADC),a key enzyme for the metabolism of the polyamine,were identified by sequencing analysis of the leaves of fresh tea leaves.The relationship between the two genes was detected by qRT-PCR,and the expression of the above genes in different tea cultivars was determined by qRT-PCR.The main results were as follows:1.The results showed that the quality of the data was high,and the total number of clean data was 104.12 G and unigene were 233,131.It is worth noting that the difference gene KEGG enrichment analysis showed that ABA signal transduction pathway related genes were significantly up-regulated at 3 h,12 h and 24 h after inoculation.2.The cDNA sequences of CsICE1,CsSnRK2.1,CsSnRK2.6,CsSnRK2.8 and CsADC genes were cloned in the E-tea No.1 tea cultivars.Bioinformatic analysis of the cloning CsICE1,CsSnRK2.1,CsSnRK2.6,CsSnRK2.8 and CsADC genes showed that the open reading frame were 1587 bp,1077 bp,1027 bp,1035 bp,2163 bp.The number of the encoded amino acids were 528,358,342,344,720.Hydrophobicity and cross-modeling analysis elaborated that the proteins encoded by the five amino acids were both hydrophilic and non-transmembrane.On the basis of phylogenetic tree,CsSnRK2.1,CsSnRK2.6 and CsSnRK2.8 was found in three group and Cs ADC was similar to CpADC.Multi-sequence alignment and protein super-secondary structure prediction analysis showed that CsICE1 had the bHLH domain;CsSnRK2s had Ser/Thr protein kinase domains;Similar to PLPDE,the type III member of pyridoxal phosphate dependence enzyme gene superfamily,CsADC also possessed both Orn-DAP-Arg-deC domain and Orn-DAP-Arg-2 pyridoxal-proline binding sites in C-terminus.In line with the analysis of protein secondary structure prediction,CsICE1 protein,CsSnRK2 s protein as well as CsADC protein,were all composed of ? helix,extended strand,?-turn and random coil.Additionally,there were multiple phosphorylation sites for Serine,Threonine and Tyrosine in three proteins mentioned above.It is predicted thatCsSnRK2 s was located in both cytoplasm and nucleus,CsICE1 was mainly located in nucleus,CsADC was found mostly in cytoplasm.3.Results of protein interaction exhibited that the full length of CsICE1 was active,but its short segments CsICE1-73 and Cs ICE1-146 were inactive.The short segments CsICE1-73 and CsICE1-146 did not interact with CsSnRK2.6 and CsADC.4.When the tea plant suffered from dehydration stress,relative expression analysis showed the expression levels of CsSnRK2.1,CsSnRK2.8,CsICE1 and CsADC in four cultivars were not significantly different.Whereas,the extent of up-regulation of relative expression of CsSnRK2.6 in Qian Fu No.4 was the highest when compared to E-Cha No.10,Zi Juan and E-Cha No.1: the expression level of CsSnRK2.6 was up-regulated by more than 5 times under 3 h dehydration stress treatment and more than 16 times under 9h dehydration stress,while after 12 h dehydration stress,the expression level of CsSnRK2.6,became slightly down-regulated.For E-Cha No.10,Zi Juan and E-Cha No.1,there was no significant difference in the expression level of CsSnRK2.6.