Cloning And Expression Level Analysis Of Transcription Factor CsEIN3/EILs In Tea Plant

Ethylene plays an important role in plant growth and development and stress response.The physiological role of ethylene is achieved by regulating the expression of genes involved in signal transduction pathways.Ethylene-insenstives3(EIN3)and EIN3-like(EIL)are key transcription factors in the ethylene signal transduction pathway.However,the transcriptional factors of EIN3/EILs in tea plant and their expression in response to various stress conditions are still unclear.It is further important to explore the expression characteristics of CsEIN3/EILs transcription factor family under different abiotic stresses for breeding new varieties of tea plant with strong resistance.In this paper,the cDNA cloning,identification and expression analysis of four CsEIN3/EILs genes,verification of transcriptional activation activity and expression analysis of transgenic Arabidopsis thaliana were carried out.The main findings are as follows:1.Based on the tea plant genome and the salt stress transcriptome database,four CsEIN3/EILs genes were cloned and named CsEIL1,CsEIL2,CsEIL3 and CsEIL4.The open reading frame of CsEIL1,CsEIL2,CsEIL3 and CsEIL4 are 1827 bp,1872 bp,1885 bp and 1917 bp respevtively,encodeing 609,624,595 and 639 amino acids.The physicochemical properties showed that the molecular weights of CsEIL1,CsEIL2,CsEIL3 and CsEIL4 are 68.7,70.3,67.2 and 71.6 kD,respectively,and the theoretical isoelectric points are 5.14,5.53,6.31,5.60.Online TARGETP1.1 predicts that CsEIL1,CsEIL2,CsEIL3 and CsEIL4 are all localized to the nucleus.Multiple sequence alignments revealed that the four CsEIN3/EILs proteins are highly conserved at the N-terminus which contain multiple highly conserved EIN3 functional domains,including acidic amino acid region,basic amino acid cluster and proline-rich region,while C-terminal conservation is lower.2.Based on qRT-PCR data analysis,the expression levels of four CsEILs genes vary from different tissues.The expression levels of CsEIL1,CsEIL3 and CsEIL4 are lower in the stems and higher in the leaves.There was no significant difference in the expression levels of CsEIL2 in roots,stems and flowers,which are lower than their expression levels in leaves.In addition,CsEIN3/EILs were induced by ethylene,ABA,NaCl,PEG and temperature,but their expression levels and expression characteristics were different.Maining in the following repects: under the treatment of 200 mM NaCl,the maximum expression levels of the four CsEILs appeared at 6 h,but the peaks were different,which were 5,11,2 and 4.5 times of the control,respectively.It indicates that there is a difference in the degree of response of CsEILs to salt stress.When treated with 0.5% ethylene solution,the expression level of CsEILs increased and then decreased,while CsEIL1,CsEIL2 and CsEIL4 increased significantly to the maximum expression level at 3 h after treatment,while CsEIL3 rised to the maximum at 6 h after treatment,indicating that ethylene can induce expressions of CsEILs at the transcriptional level and different members have different sensitivity to ethylene.After PEG simulated drought treatment for 6 h,the expression of CsEILs increased sharply,indicating that it was involved in response to drought stress.Under high temperature(40 °C)stress,the expression levels of CsEILs decreased slightly,and then gradually induced.Compared with other stress treatments,the variation of expression levels is not much different.CsEILs showed a rise and then decrease under low temperature(4 °C)stress,followed by a small increase and finally decreased to the control level,indicating that CsEILs play an important role in regulation of temperature stress in tea plant.3.Based on qRT-PCR data analysis,the gene expression levels of CsEIL1 and CsEIL2 transgenic Arabidopsis are significantly increased,and the transgenic Arabidopsis ERF1 expression is also significantly higher than wild type.ABA treatment induced a gradual increase in the expression level of CsEILs,which reached the maximum at 48 h after treatment,indicating that it was not sensitive to ABA response.