Application of virus-induced gene silencing approach in Camptotheca acuminata

Abstract

Camptotheca acuminata is the major plant for the production of camptothecin (CPT), an important anticancer drug used for the treatment of various cancers throughout the world. The low accumulation of CPT in plants limited its supply in the market and led to urgent need of promoting its accumulation by means of plant metabolic engineering, which relied on deep understanding of CPT biosynthesis pathway. However, missing of most of pathway genes restricted the attempts for regulating CPT biosynthesis. To unveil the CPT biosynthesis pathway, many efforts have been done for pathway gene identification and the application of large-scale RNA-sequencing technique accelerated screening of candidate genes which could be involved in CPT biosynthesis. To identify the function of these candidate genes in planta, it needs to develop an effective approach, such as virus-induced gene silencing (VIGS) in C. acuminata. In this work, a Tobacco Rattle Virus-based VIGS method was developed and the application of this method successfully silenced the expression of four known genes involved in the early steps of CPT biosynthesis and resulted in clear decrease of CPT and 10-hydroxycamptothecin (10-HCPT) accumulation. This VIGS approach could be further applied to functional identification of candidate genes to elucidate CPT biosynthesis in C. acuminata.

Microwave-Assisted Extraction of Multiple Trace Levels of Intermediate Metabolites for Camptothecin Biosynthesis in Camptotheca acuminata and Their Simultaneous Determination by HPLC-LTQ-Orbitrap-MS/MS and HPLC-TSQ-MS

“…As shown in Figure 1 , in the MAE method, when aqueous acetonitrile of 70% was used as the extraction solvent, the recoveries of the three target compounds ranged between 88% and 103%, and acetonitrile had the highest extraction efficiency for two of the key intermediates (loganic acid, strictosamide). The most commonly employed solvent for the extraction of CPT and 10-HCPT in plant samples is methanol [ 8 , 13 , 17 ]. However, fewer systematic studies on intermediates extraction were reported.…”

“…Seed germination and plant growth were conducted as described by Jin et al [ 8 ]. Briefly, seeds were germinated on wet filter paper in a growth chamber with a 12 h photoperiod at 25 °C until cotyledons appeared.…”

“…Like many other MIAs, CPT is derived from the common central alkaloid precursor strictosidine, which is synthesized by coupling indole-containing tryptamine and the monoterpenoid glycoside secologanin under the catalysis of strictosidine synthase [ 6 , 7 ]. In the upstream MIA pathway, tryptamine is produced through the shikimate pathway by decarboxylation of tryptophan, while secologanin is from the mevalonate (MVA) pathway or 2-methyl-( d )-erythritol-4-phosphate (MEP) pathway [ 8 , 9 , 10 , 11 ]. However, at present, the downstream pathway of CPT biosynthesis, namely the pathway following strictosidine (isovincoside), which is an important and specific metabolic pathway, is yet unclear [ 5 , 8 , 9 ].…”

“…In the upstream MIA pathway, tryptamine is produced through the shikimate pathway by decarboxylation of tryptophan, while secologanin is from the mevalonate (MVA) pathway or 2-methyl-( d )-erythritol-4-phosphate (MEP) pathway [ 8 , 9 , 10 , 11 ]. However, at present, the downstream pathway of CPT biosynthesis, namely the pathway following strictosidine (isovincoside), which is an important and specific metabolic pathway, is yet unclear [ 5 , 8 , 9 ]. Therefore, the extraction and detection of intermediate metabolites in the downstream pathway are urgently needed.…”