Functional Characterization of Key Genes CsANS and CsLAR Involved in Catechin Biosynthesis in Camellia sinensis
Two key enzyme genes CsANS and CsLAR involved in the catechin synthetic pathway from'Yinghong 9'were cloned,and successfully identified their functions by prokaryotic expression,subcellular localization and transgenic overexpression analyses.The results showed that the ORFs(open reading frame)of CsANS and CsLAR were 1 068 and 1 029 bp,respectively,which were 99％homologous to the corresponding genes from'Shuchazao'in TPIA database.Soluble proteins of CsANS and CsLAR can be obtained in prokaryotic induced expression system and can be successfully purified by GST tag.Transient expression of CsANS and CsLAR in Nicotiana benthamiana leaves showed that proteins they encoded were localized in the cytoplasm and nucleus.Furthermore,CsANS and CsLAR were overexpressed in tea calli by Agrobacterium-mediated genetic transformation.The results showed that both of them could significantly promote the biosynthesis of total catechins in tea calli,in which CsANS mainly increased content of EC and EGCG,while CsLAR mainly promoted the accumulation of trans-catechins C,GC,CG and GCG.It is speculated that the function of CsANS is mainly to promote the synthesis of cis-catechins,while CsLAR is more conducive to the synthesize trans-catechins in'Yinghong9'.
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