The present study was carried out to silence the transcription factor genes ZCT1, ZCT2 and ZCT3 via lipofectamine based antisense LNA GapmeRs transfection into the protoplasts of established photomixotrophic cell suspensions. The photomixotrophic cell suspensions with a threshold of 0.5% sucrose were raised and established using two-tiered CO₂ providing flasks kept under high light intensity. The photomixotrophic cell suspensions showed morphologically different thick-walled cells under scanning electron microscopic analysis in comparison to the simple thin-walled parenchymatous control cell suspensions. The LC–MS analysis registered the vindoline production (0.0004 ± 0.0001 mg/g dry wt.) in photomixotrophic cell suspensions which was found to be absent in control cell suspensions. The protoplasts were isolated from the photomixotrophic cell suspensions and subjected to antisense LNA GapmeRs silencing. Three lines, viz. Z1A, Z2C and Z3G were obtained where complete silencing of ZCT1, ZCT2 and ZCT3 genes, respectively, was observed. The Z3G line was found to show maximum production of vindoline (0.038 ± 0.001 mg/g dry wt.), catharanthine (0.165 ± 0.008 mg/g dry wt.) and vinblastine (0.0036 ± 0.0003 mg/g dry wt.). This was supported by the multifold increment in the gene expression of TDC, SLS, STR, SGD, d4h, dat, CrT16H and Crprx. The present work indicates the master regulation of ZCT3 knockdown among all three ZCTs transcription factors in C. roseus to enhance the terpenoid indole alkaloids production. The successful silencing of transcription repressor genes has been achieved in C. roseus plant system by using photomixotrophic cell cultures through GapmeR based silencing. The present study is a step towards metabolic engineering of the TIAs pathway using protoplast transformation in C. roseus.

进行本研究以通过基于脂质体的反义LNA GapmeRs转染到已建立的光合营养细胞悬液的原生质体中沉默转录因子基因ZCT1、ZCT2和ZCT3 。使用两层 CO _{2提高和建立具有 0.5% 蔗糖阈值的光合营养细胞悬浮液}提供保持在高光强度下的烧瓶。与简单的薄壁薄壁对照细胞悬浮液相比，光混合营养细胞悬浮液在扫描电镜分析下显示出形态不同的厚壁细胞。LC-MS 分析记录了光混合营养细胞悬浮液中的文多林产量（0.0004 ± 0.0001 mg/g 干重），而对照细胞悬浮液中不存在这种情况。从光混合营养细胞悬浮液中分离原生质体并进行反义LNA GapmeRs沉默。三行，即。Z1A、Z2C 和 Z3G 在ZCT1、ZCT2和ZCT3完全沉默的情况下获得分别观察到基因。发现 Z3G 系显示出最大的长春花碱（0.038 ± 0.001 mg/g 干重）、长春花碱（0.165 ± 0.008 mg/g 干重）和长春碱（0.0036 ± 0.0003 mg/g 干重）产量。这得到了TDC、SLS、STR、SGD、d4h、dat、CrT16H和Crprx基因表达的多倍增加的支持。目前的工作表明在C.roseus中所有三种 ZCTs 转录因子中ZCT3敲低的主要调节以提高萜类吲哚生物碱的产生。通过基于 GapmeR 的沉默使用光混合营养细胞培养物，已在 C.roseus 植物系统中成功沉默了转录抑制基因。本研究是在C.roseus中使用原生质体转化对 TIAs 途径进行代谢工程化的一步。