Cloning and expression analysis of calmodulin-like protein gene VdCML8 in Vitis davidii and determination of its promoter transcriptional activity

【Objective】Calmodulin-like protein gene（VdCML8）in Vitis davidii and its promoter sequence were cloned，and the expression of VdCML8 gene was analyzed，and the transcriptional activity of its promoter was determined to provide theoretical reference for further exploring the anti-anthracnose biological function of this gene in grapes. 【Method】V. davidii Ziqiu was as mate-rials. The VdCML8 gene and its promoter sequence were cloned by RT-PCR，and the physicochemical properties and se-condary structure of VdCML8 protein were analyzed by bioinformatics. Real-time fluorescence quantitative PCR was used to detect the expression characteristics of CML8 gene in V. davidii Ziqiu and V. vinifera cv. Red Globe after inoculation with Colletotrichum gloeosporioides and external salicylic acid（SA）and jasmonic acid（JA）treatments. β-glucosidase（GUS）fusion vector was constructed and transformed tobacco for transcriptional activity detection.【Result】The open reading frame（ORF）of VdCML8 gene was 450 bp，encoded 149 amino acids residues， and had an EF-hand domain. In the secondary structure，the proportion of α-helix was 65.10%，that of extension chain was 4.70%，that of random coil was 20.13%，and that of β-turn was 10.07%. The protein was localized in the cell membrane. According to phylogenetic tree，VdCML8 protein in V. davidii was closely related to V. vinifera VvCML8 and V. vinifera VrCML8. The VdCML8 gene promoter sequence（pVdCML8）was 1050 bp. It contained a large number of CAATbox and TATA-box，and also contained some light response elements（L-box，chs-CMALa and CTT-motif），episisic acid （ABA）response element ABRE，anaerobic induction response element（ARE），defense and stress element（TC-rich repeats），wounding response element（WUN-motif）. pVdCML8::GUS，a transient expression vector of pVdCML8，was constructed. It was found that pVdCML8 had transcriptional activity and could drive the expression of VdCML8 gene after transient transformation of tobacco. The expression of V. davidii Ziqiu VdCML8 gene and V. vinifera VdCML8 gene were up-regulated and reached the peak at 12 h after inoculation. The relative expression levels of both were as 22.08 times and 9.30 times as that of control group（water treatment）. After SA treatment for 3 h，the relative expression of VdCML8 gene was as 7.68 times as that of control and as 2.76 times as that of VdCML8 gene. After JA treatment for 6 h，VdCML8 gene reached its peak and was as 22.25 times as that of control group and as 9.04 times as that of VdCML8 gene.【Conclusion】 VdCML8 gene is the downstream regulatory gene of SA and JA signaling pathways. SA and JA can induce its efficient expression，participate in the response process of grape anthracnose，and have certain effects in improving plant disease resistance.