*4.2. Cloning of CsWRKY7 Gene and Sequence Analysis*

The expressed sequence tags (EST) were obtained from different transcriptome databases [44–46]. The full length of CsWRKY7 cDNA sequence was identified from different tissues of 'Longjing 430 by KOD DNA Polymerase (Toyobo, Tokyo, Japan) and RT-PCR. Gene-specific primers were as follows; 50 -ATGGCCGTCGAGCTCGTGAT-30 (forward) and 50 -TCAAGAAGACTCTAAGATAAG-30 (reverse). The purified RT-PCR products were inserted into pEASY-blunt simple cloning vector (TransGen Biotech, Beijing, China) and subsequently sequenced. The predicted molecular weight and theoretical isoelectric point were analyzed by ProtParam (http://web.expasy.org/protparam/, accessed on: 22 May 2017) [47]. The homology of CsWRKY7 protein with other species was analyzed by NCBI BLAST website. The amino acid sequences of WRKY II d subfamily members were analyzed by ClustalX 2.0 and DNAMAN 6.0 software. The MEGA 5.0 software was used to analyze their evolutionary relationships. CsWRKY7 genomic DNA (gDNA) sequence was searched in 'Shuchazao' tea plant genome database, and the promoter region was determined by aligning the open reading frames of the gDNA sequence and those of corresponding gene. The promoter region of CsWRKY7 (1680 bp upstream of start codon) was amplified from 'Longjing 430 gDNA by PCR using KOD DNA polymerase (Toyobo). The regulatory elements in promoter region were predicted by PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on: 9 March 2019) [48].
