Cloning, Expression And Analysis Of Some Related Genes On The Pathway Of GA Synthesis In Camellia Sinensis

In this paper, using buds and leaves of strain’1005’(Camellia sinensis), we detected the content of esterified catechins in different leaf positions, cloned the full-length cDNAs of aroB and aroDE, analized their biological information and determined their expressions in different leaf positions of Camellia sinensis 1005.1 Content of esterified catechins in different leaf positions on Camellia sinensis 1005The content of esterified catechins of strain’1005’ranged from 6.71% to 22.49% refering to different leaf positons as the following order:1st leaf>bud>2nd leaf>3rd leaf>4th leaf>5th leaf>6th leaf>old leaf. The content of epigallocatechin-3-gallate (EGCG) is extremely higher than the other 3 esterified catechins, range from 2.26% to 13.09%, showing a similar variation with the total catehins content. As a whole, the content of cis-esterified catechins is significantly higher than the trans-form ones.2 Full-length cDNA cloing and bio-informatic analysis of aroB in Camellia sinensisThe full length cDNA sequence of aroB (named as Cs1005-aroB, GenBank ID: KM009084) was cloned from Camellia sinesis strain’1005’, which is 1509bp, including 70bp 5’UTR,89bp 3’UTR and 1350bp ORF, coding 449 amino acids. The bio-informatic prediction results showed:Cs1005 aroB was a highly conserved hydrophilic, non-secretory proteinwith high stability; its three-dimensional structure is extremely similar to that of Actinidia chinensis; it is closed to Fagus sylvatica in genetic relationship; it has a characteristic structure domain of DHQS. Comparing all results with other previous studies, it was stuggested that Cs1005-aroB protein was synthesized in chloroplast, and the Fe2+was required to bind and activate its function.3 Full-length cDNA cloing and bio-informatic analysis of aroDE in Camellia sinensisThree cDNA sequences of aroDE genes, namely Cs1005-aroDEl, Cs1005-aroDE2 and Cs1005-aroDE3, were cloned using RACE clone technique.The full-length ofCs1005-aroDEl, Cs1005-aroDE2 and Cs1005-aroDE3 cDNA were 2118bp,1990bp and 1957bp, respectively, coding 519,532 and 525 amino acids. The homology of sequence of the three cDNAs was 64.32%, and that of the three amino acides sequence was 65.36%. The distribution of highly homology sequence was discontinuously.The bio-informatic prediction results showed that proteins encoded by Cs1005-aroDEl, Cs1005-aroDE2 and Cs1005-aroDE3 were hydrophilic, non-secretory proteins with high stability; although the homology of sequences were low, their three-dimensional structures were extremely similar, and also they were all semblable to that of Arabidopsis thaliana; There were amount of phosphorylation sites in the structure of these three proteins, in where had one DHQase_I domain, one Shikimate_dh_N domain and one NAD_bind_Shikimate_DH domain, although Cs1005-aroDE2 protein had one extra shikimate binding site; the most probable location for the synthesis and reaction of Cs1005-aroDE2 and Cs1005-aroDE3 was chondriosome, and that for Cs1005-aroDEl was cytoplasm; they were all closed to Vitis vinifera in genetic relationship, but a little far relatives from each other.4 Relative quantitative expressions of aroB and aroDE in different leaf positions on Camellia sinensis 1005The expression of four genes, Cs1005-aroB, Cs1005-aroDEl, Cs1005-aroDE2 and Cs1005-aroDE3, were detected using SYBR Green QPCR method and the method of 2-ΔΔCt was applied for the calculation. The expression of the four showed a normal distribution trend. All the genes expressed most in the third leaf except Cs1005-aroDE3, which expressed most in the second leaf. The expression of aroB was least in bud, which was significant different with three aroDEs genes.