Molecular Cloning And Expression Analysis Of ACS, ACO And Cyclophilin Genes Of Tea Plant (Camellia Sinensis)

Ethylene is one of the important plant hormones, it has significant physiological role in the life of plant. ACC synthase (1-aminocyclopropane-l-carboxylic acid synthase, ACS) and ACC oxidase (1-aminocyclopropane-1-carboxylicacid oxidase, ACO) are very important enzymes in the ethylene biosynthesis of the plant. They play important roles in the regulation of ethylene biosynthesis. So the research on the two genes has both theory and practicality significance.Based on the ESTs from the cDNA library sequencing program of tea plant, ACO and ACS genes encoding the two enzymes were cloned using the RACE and RT-PCR methods. The GenBank Accessions are EF205149 and DQ904328, respectively. The ACS is 1579 bp in length, encoding 478 amino acids, the putative molecular weight is 53.7 KD and the pI is 8.039. The ACO is 1237 bp in length, encoding 320 amino acids, the putative molecular weight is 36.2 KD and the pI is 5.41. The phylogenetic trees constructed by the software Mega showed that both ACS and ACO genes are closely related to those of Diospyros kaki. Cloned of the two genes will do some spadework for the research of its effect on the resist of adversity in tea plant later.Primers were designed based on the ACS and ACO sequences, the gene expressions of 7 cultivars, Anji Baicha, 6/8, Hangzhou Daye, Longjing Changye, Yabukita, Longjing 43 and Fuding Dabaicha after stress of high temperature in the summer and low tempeture in the winter, as well as Longjing 43 during different periods in the winter were assayed using RT-PCR methods and theβ-actin as a positive control. The results showed the expressions of ACS and ACO genes had some extent relationship with the resistance of the cultivars. Similarly results were also found in the different time of winter for Longjing 43.Cyclophilin exists widely in organisms. It has very important roles in plant growth and metabolism. A cDNA clone, encoding cyclophilin gene, was obtained from the expressed sequence tag sequencing of tea plant. The full-length cDNA of the cyclophilin gene was 953bp, encoding 164 amino acids. The predicted MW and pI were 17.47 kD and 8.54, respectively. A "CAAT" signal in 5' untranslated region and a polyadenylation signal of "AATAA" poly-A in 3' untranslated regions of cyclophilin mRNAs were found. The presumed protein sequences of tea plant were aligned with those of other 26 organisms through software CLUSTAL W. The phylogenetic analysis based on the Neighbor-Joining method showed the similarity was greater than 85% between cyclophilin genes of tea plant and Oryza sativa (japonica cultivar-group), Triticum aestivum etc. Primers were designed on the open reading frame of the cyclephilin gene of tea plant to construct the expressive vector pET/Csin-Cyp. A recombinant protein about 23kD in the Escherichia coli BL21 (DE3) was induced. This will do beneficial to research its effects on the physiology and regulation in the tea plant from the level of gene and protein.