Functional Characterization Of The Structural Genes Involved In Rotenoid Biosynthesis And Metabolic Engineering Rotenoid Biosynthetic Pathway In Mirabilis Himalaica

Mirabilis himalaica, namely Ba Zhu in Tibetan native language, is a Tibetan traditional herb and an endangered plant species localized in Tibet Platue, of which roots are used as the medicinal part. Rotenoid is the typical natural product isolated from M. himalaica with a range of anti-tumor bioactivities. In the presenty, hairy root cultures of M. himalaica were established to investigate the influences of several factors on rooting rate, root growth and rotenoid accumulation; and three rotenoid-biosynthetic genes including phenylalanine ammonia-lyase (MhPAL), chalcone synthase (MhCHS) and chalcone isomerase (MhCHI) were isolated and analyzed from M. himalaica; finally, substrate feeding was used to identify the function of the purified recombinant MhPAL; transgenic technology was applied to study the roles of MhCHS/MhCHI involved in rotenoid biosynthesis in M. himalaica.Agrobacterium strain C58C1 (pRiA4) induced hairy roots from tender leaves of M. himalaica. The different factors, such as explants, preculturing time, bacterial infection time, had different influences on rooting rate. Tender leaves were the best explants for hairy root initiation, and the rooting rate reached 32%; 2-3 d preculturing time was suitable to induce hairy roots at the rooting rate of 26-32%; bacterial infection on tender leaves for 20 min led to the highest rooting rate,34%. The hairy root lines were confirmed by genomic PCR:a 423-bp specific fragment of rolB and a 626-bp specific fragment of rolC could be simultaneously amplified from the positive control Ri plasmid and hairy root cultures of M. himalaica, while these fragments could not be amplified from wild roots. Further, when respectively cultured in MS,1/2MS, B5 and 1/2B5 liquid medium, hairy root cultures of M. himalaica produced more biomass. Hairy root cultures produced much higher level of rotenoid when cultured in 1/2MS or MS liquid medium, while hairy root cultures did not produce rotenoid when cultured in 1/2B5 liquid medium.Rotenoid biosynthesis started from the biochemical steps catalyzed by phenylalanine ammonia-lyase, chalcone synthase and chalcone isomerase. In this study, three rotenoid-biosynthetic genes including MhPAL, MhCHS and MhCHI were cloned and functionally analyzed in M. himalaica.The 2299-bp full-length cDNA of MhPAL encoded a 713-amino-acid polypeptide with the theoretical molecular weight of 77.55 kDa and the calculated pI of 5.9, both of which were similar with those of plant PAL proteins. The multiple alignments showed that MhPAL was very similari with PAl of Dianthus monspessulanus at the sequence level (>87%identity); structural modeling demonstrated that the catalytic core of plant PALs, composed of the three conserved amino acid sites Y102-N254-Q342, was found in MhPAL.The 1468-bp full-length cDNA of MhCHS encoded a 392-amino-acid polypeptide with the theoretical molecular weight of 43.2 kDa and the calculated pI of 6.02, both of which were similar with those of plant CHS proteins. The multiple alignments showed that MhCHS was very similari with CHSs of Dianthus monspessulanus and Polygonum cuspidatum at the sequence level (>80%similarity); structural modeling demonstrated that the catalytic core of plant CHS, composed of four conserved amino acid sites C165-F216-H304-N337, was found in MhCHS. The 819-bp full-length cDNA of MhCHI encoded a 221-aa polypeptide with the oretical molecular weight of 24.0 kDa and pI 5.18, both of which were almost the same with those from Leguminosae family. The catalytic core composed of the four conserved amino acid sites T50-Y108-Q115-T192 was found in MhCHI based on sequence comparison and structural modeling; and the phylogenetic analysis revealed that MhCHI might be closer to CHI II.The gene relative expression levels of MhPAL, MhCHS and MhCHI were detected by real time quantitative PCR (qPCR) in roots, stems and leaves of M. himalaica. The expression level of MhPAL was much higher in roots that in stems and leaves; MhCHS was expressed in roots, stems and leaves but at different levels; the highest expression level of MhCHS was found in roots, lower levels in stems and leaves; there was no significant different of expression levels of MhCHS between stems and leaves. The tissue profile of MhCHI was similar with that of MhCHS and MhPAL. MhCHI had higher expression levels in roots than that in other organs. The similarity of MhPAL, MhCHS and MhCHI tissue profiles suggested the gene expression coordination between them, and as a result, the coordinate expression of the three genes facilitated metabolite biosynthesis. As expected, the higher rotenoid content was detected in roots by HPLC, and this was consistent with the tissue profile of MhPAL, MhCHS and MhCHI. This suggested that roots were the main organ in which rotenoid was biosynthesized. Additionally, the results above provided the scientific evidence supporting the facts M. himalaica roots were used as medicinal parts.Then, the recombinant protein of MhPAL was obtained and purified from E. coli. The molecular weight of recombinant MhPAL was 77kDa. When feeding by phenylalanine as substrate, MhPAL catalyzed phenylalanine to for trans-cinnamic acid as product, which was detected by TLC and HPLC, finally confirmed by LC-MS. The further enzymatic kinetics research demonstrated that the Km of MhPAL was 0.1001mM and Vmax,2.212 mM/min. The functional identification of MhPAL will provide a candidate gene for genetically engineering the rotenoid biosynthetic pathway in M. himalaica.Finally, MhCHS and MhCHI were respectively used to engineering rotenoid biosynthesis in transgenic hairy root cultures of M. himalaica. The transgenic hairy root lines with MhCHS or MhCHI overexpression were confirmed by genomic PCR. A 1405-bp specific fragment of both 35S promoter and MhCHS, the 423-bp rolB fragment and the 626-bp rolC fragment were simultaneously amplified from MhCHS-overexpressed hairy root lines; a 782-bp specific fragment of both 35S promoter and MhCHI, the 423-bp rolB fragment and the 626-bp rolC fragment were simultaneously amplified from MhCHI-overexpressed hairy root lines. The qPCR analysis showed that the transgenes including MhCHS and MhCHI had higher expression levels in their corresponding transgenic hairy root lines. Finally, HPLC analysis demonstrated that the rotenoid contents in MhCHS/MhCHI-overexpressed hairy root cultures were higher than the nontransgenic lines. This suggested that rotenoid biosynthesis was fortified by overexpressing MhCHS or MhCHI. In summary, this study provided a promising way to develop transgenic M. himalaica with high content of rotenoid based on metabolic engineering strategy of breaking through committed steps involved in biosynthetic pathways.