Abstract
The biosynthesis of catechins begins with phenylalanine and is catalyzed by over a dozen key enzymes. Among them, anthocyanidin synthase (ANS), anthocyanidin reductase (ANR), and leucoanthocyanidin reductase (LAR) are downstream genes in the biosynthesis pathway of catechins. ANR catalyzes anthocyanins to produce epicatechins (EC, EGC), and LAR converts the leucoanthocyanidins to corresponding catechins (C, GC). Number of successful experiments have been conducted in plant multi-gene genetic engineering studies. To fortify catechin content through genetic engineering, we selected three key genes involved in the biosynthesis pathway of catechins, CsANS, CsLAR, and CsANR, as the metabolic genes for catechin biofortification.
Firstly, we performed the codons optimization of three genes (CsANS, CsLAR, and CsANR) and carried out the chemical synthesis. Each gene expression unit is connected between the 35S promoter and NOS terminator by a PAGE-mediated overlapping extension PCR method. The three genes (CsANS, CsLAR, and CsANR) were arranged in series for co-expression, employing the same-end enzyme technique. Subsequently, we transformed the recombinant vector pYC1301 into tobacco. We measured the catechin content of the wild-type and two transgenic lines by ultra-performance liquid chromatography (UPLC). Several catechin monomers (C, GCG, EC, ECG, EGC and EGCG) were detected in transgenic tobacco.
Our study confirmed that the heterologous tandem co-expression of CsANS, CsLAR, and CsANR genes in tobacco clearly promotes catechin biosynthesis.This work provided a new perspective for understanding the synthesis and regulation mechanism of catechin in tea plant, as well as the accumulation of catechin.
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