The Structure and Single Nucleotide Polymorphism Analysis of Chalcone Synthase Genes in Tea Plant(Camellia sinenesis)

【Objective】 The objectives of this study were to determine the structures of CHS genes in Camellia sinensis(CsCHS) by obtaining gDNA sequences of these genes, and to analyze single nucleotide polymorphism. Besides, association analysis was also carried out in order to find potential SNP sites in CsCHS which would influence the polyphenol content in tea plant. 【Method】 Based on CsCHS sequences uploaded to NCBI, specific primers were designed using primer 3.0 software. Genomic DNA and cDNA of tea leaves were used as templates in polymerase chain reaction(PCR) to obtain gDNA and cDNA sequences of CsCHS1, CsCHS2 and CsCHS3, respectively. Gene structures were determined through blasting gDNA and cDNA sequences. The putative amino acid sequences were analyzed by bioinformatics softwares, such as Compute pI/Mw, SOPMA, and so on. Single nucleotide polymorphism of CsCHS were analyzed in 57 cultivars with great variation polyphenol contents. In order to obtain the coding region of CsCHS genes, PCR reactions with specific primers were carried out by using cDNA of individual tea cultivars as template. TASSEL software was introduced in association analysis.【Result】 cDNA sequences of CsCHS1, CsCHS2 and CsCHS3 were 1 277 bp, 1 320 bp and 1 242 bp, respectively. And an open reading frame(ORF) of 1 170 bp was found in each CsCHS gene. gDNA sequences of CsCHS1, CsCHS2, and CsCHS3 were 1 600 bp, 1 330 bp and 1 607 bp, respectively. By comparing gDNA and cDNA sequences of each CsCHS gene, combined with GT-AG rule, it was determined that CsCHS1 and CsCHS2 have two exons and one intron, respectively. And the intron in CsCHS1 is 323 bp, compared with 356 bp in CsCHS3. While no interruption region was found in CsCHS2, this might prove there is no intron in CsCHS2. Deduced amino acid sequences analysis suggests that identity of amino acid sequences are 92.6%-95.4%. All of the conserved amino acids found in CHS protein subfamily were also found in these deduced sequences. Furthermore, bioinformatics analysis showed highly similarities among CsCHS1, CsCHS2 and CsCHS3 protein structures. There are 71 SNP sites in CsCHS1's coding region, SNP frequency was 1SNP/16.48 bp, and no Indel was found. A total of 55 SNP sites were found in CsCHS2's coding region, suggesting one SNP in every 21.27 bp. And the nucleotide diversity(π) in CsCHS1(0.01088) was significantly higher than that of CsCHS2(0.00530). By correlation analysis, two SNP sites were positioned thought to be related to polyphenol content in tea plant in CsCHS1 and 4 in CsCHS2. No further analysis referring to CsCHS3 was carried out due to low success rate of PCR reaction.【Conclusion】 Both CsCHS1 and CsCHS3 were determined to be conserved CHS genes, and bioinformatics analysis of protein structures results show that CsCHS1, CsCHS2 and CsCHS3 are similar. CsCHS1 and CsCHS3 are active, there should be hot spots of mutation in their coding regions.