| Atorpa belladonna is the only medicinal plant species and the main commercial source of hyoscyamine and scopolamine in China(authorized by pharmacopoeia of The People’s Republic of China2011). Compared to hyoscyamine, scopolamine is the most potent reagent with relatively low side effects, so its demand is large. The content of scopolamine is very low in wild A. belladonna, and about the commercial cultivation of A. belladonna, content of scopolamine is only0.01-0.08%of dry weight. In recent years, with the development of molecular biology research of tropine alkaloids (TAs) biosynthetic pathway, great progress of metabolic regulation of TAs biosynthesis including hyoscyamin and scopolamine, has been made at molecular level. Several important functional genes involved in the biosynthetic pathway of scopolamine have been identified that can be used to genetically modify the TAs pathway.Among the TAs biosynthetic genes, pmt、trI and h6h are important ones in TAs metabolic pathways, separately encoding putrescine N-methyltransferase (PMT; EC2.1.1.53), tropinone reductase-I (TRI; EC1.1.1.206) and hyoscyamine6β-hyroxylase (H6H; EC1.14.11.11), and located in the upstream, middle branch point and downstream of the pathway respectively. Many researches have shown that in A. belladona hair root and plant, the high expression levels of pmt, trI and h6h are related to changes of hyoscyamine and scopolamine contents. Overexpression of tobacoo pmt in Datura metel increased hyoscyamine content2times more than control, and3times for scopolamine content. Strong expression of D.stramonium trI in A.belladona is accompanied with3-fold more hyoscyamine and5-fold more scopolamine compared with control roots. Introduce h6h of Hyoscyamus niger into Hyoscyamus muticus hair root. The best clone produces17mg/L DW of scopolamine, which is over100times more than the control clones. But the effects of endogenous genes are not clear. Based on research before, this paper study the influence of two-gene strategy of A. belladonna (pmt、trl、h6h)for the tropine alkaloids content of A. belladonna hairy root. Constructing two-gene efficient expression vectors of p1304+-pmt-trl, p1304+-pmt-h6h and p]304+-trI-h6h, respectively. Transform the constructed vectors into "disarmed" C58C1to obtain C58C1-p1304+-pmt-trI, C58C1-p1304+-pmt-h6h and C58C1-pl304+-trI-fh6h, for genetic transformation of A. belladonna.In transgenic C58C1-pl304+-pmt-trI hair root, PCR studies confirmed their presence in6hairy root clones. Continue training in liquid medium, then detect content of TAs and genes expression level. TAs contents in4hair root clones have been improved, PT11> PT17>PT13>PT3in turn. Total TAs content in PT11is2.5times than control hair root. There are2hair root transgenic lines contents remain unchanged or reduce significantly, but scopolamine content has been improved. The best line(PT11) increases2times in hyoscyamine content and5.7times in scopolamine content. Except PT2pmt gene, the pmt and trI genes expression levels of the other hair root are higher than NC. In transgenic C58C1-pl304+-pmt-h6h hair root, obtain5transgenic hair root clones. Total TAs content in2hair root clones enhanced, PH2>PH14. PH2TAs content increased2.7times than NC. There are3hair root transgenic clones hyoscyamine contents remain unchanged or reduce significantly, but scopolamine content have been improved. The best line(PH2) increases1.8times of hyoscyamine content and8.2times of scopolamine content. Except PH32pmt gene, the pmt and h6h genes expression levels of the other hair root are higher than NC. In transgenic C58Cl-p1304+-trI-h6h hair root, In transgenic C58C1-p1304+trI-h6h hair root, it has succeed to get4hairy root clones. TAs content in3hair root clones have been improved, HT2>HTA1>HTA3in turn. Total TAs content in HT2is2.6times than NC. TAs contents of One transgenic line reduced, but scopolamine content enhanced. The best line(HT2) increases2.1times of hyoscyamine content and5.6times of scopolamine content. Expression of HT2h6h genes are far higher than NC. The expression of HTA1、HTA3and HT16h6h gene are closed to NC, but trI enhanced significantly. The genes expression is not completely consistent with the level of TAs content. This may be an expression suppression due to the two transgenic genes. Under high expression of genes, the transgenic hair root content remain the same or reduced. Above all, two genes in the tropine alkaloids biosynthetic pathway may exist synergies. Despite of existing difference among the individual in transgenic hair root, the trend of improving scopolamine content doesn’t change. It proves that two-gene strategy can break the rate-limited step in A.belladonna TAs biosynthetic pathway, and produce more TAs content including hyoscyamine and scopolamine.pmt is the first important enzyme gene in TAs biosynthetic pathway. Its overexpression can provide more precursor. trI gene is a branch point of TAs biosynthetic pathway. It makes more flux to scopolamine. The last important enzyme gene is h6h. The overexpression of h6h can deliver more scopolamine. This study proves that A. belladonna endogenous genes Abpmt, Abtrl and Abh6h, can enhance TAs content. |
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