Molecular Cloning Of3-hydroxy-3-methylglutaryl Coenzyme A Reductase And Its Regulation On The Biosynthesis20-Hydroxyecdysone In Cyanotis Arachnoidea

20-hydroxyecdysterone (20E), one of the most important secondary metabolites inCyanotis arachnoidea C. B. Clarke, is not only widely used in sericulture, but also hasmany pharmacological activities. It can stimulate the synthesis of protein and nucleic acidof vertebrates, promote the metabolism of carbohydrates and lipids, and regulate theimmunological function and the central nervous system. So it attracts much attention ofscientific researchers all over the world. However, due to its complex structure, thechemical synthesis cannot be used in its commercial production. Since the supply of20Efrom the plants is in great shortage, its application is always restricted.20E, one of themost prevalent and abundant phytoecdysteroid produced by plants, is mainly synthesizedby mevalonate (MVA) pathway. In the MVA pathway,3-hydroxy-3-methylglutaryl-CoA(HMG-CoA) is catalyzed into mevalonate by3-hydroxy-3-methylglutaryl-CoA reductase(HMGR) which is a rate-controlling step. Therefore, HMGR is key enzyme of the MVApathway and is very important for regulation of isoprenoid biosynthesis. As a result, it ismeaningful to study the regulation of HMGR for terpenoids and phytoecdysteroidsbiosynthesis in the MVA pathway.Firstly, a full length cDNA encoding3-hydroxy-3-methylglutaryl coenzyme Areductase (designated as CaHMGR) was cloned and characterized from C. arachnoidea forthe first time. The full length cDNA of CaHMGR was2037bp containing91bp5，untranslated region,146bp3，untranslated region and a1800bp open reading frameencoding a599amino acid polypeptide, with a putative isoeletric point7.25and calculatedmolecular weight about64.0kDa. The results of sequence alignment and BLASTP search in Genbank database showed CaHMGR had high homology with many other plantHMGRs. In the putative polypeptide, two HMG-CoA-binding motifs (ELPIGFVQLP andTTEGCLVA) and two NADPH-binding motifs (DAMGMNM and GTVGGGT) werefound. The3-D structure of CaHMGR was predicted by Swiss-Model using sequencehomology-based structural modeling and the catalytic domain of CaHMGR consists ofthree domains including L-domainm, N-domain and S-domain, which is similar to otherplant HMGRs reported previously which suggested that CaHMGR is a functionalreductase, with similar catalysis between other HMGRs. A phylogenetic tree of differentHMGRs from different organisms including plants, insects, fungi and animals wasconstructed. The result revealed that HMGRs originated from a common ancestor andevolved into different groups including plants, fungi, insects and animals. According to thephylogenetic tree, CaHMGR belongs to the plant HMGRs, as expected, and has the closestrelationship with Amomun villosum HMGR.We established real time quantitative PCR method for quantification of CaHMGRexpression with specific primers CaHF and CaHR when treated by methyl jasmonate(MeJA). The absence of nonspecific products or primer dimmers was confirmed bymelting curve analysis. The melting curve analysis showed a single clear melting peak forCaHMGR in real time PCR assays and no formation of nonspecific products. The DNAsegments of amplication showed a constant peak Tm of82.50℃for CaHMGR. Typicalstandard curve of CaHMGR was generated with a10-fold dilution series of plasmidcontaining targets insert. There was a strong liner negative relationship between Ct andlogarithm of the concentration of CaHMGR with correlation coefficient of0.9974. Theregression equation appear to be the best fit the standard curve data was Ct=-3.1116×lg(Concentration of CaHMGR)+37.05and the amplification efficiency was102.6%. Thevalues of Ct were linearly correlated with logarithm of the concentration of the target gene,indicating that the reference standard was suitable as a quantitative assay. We performedquantitative RT-PCR to investigated expression of CaHMGR in leaves, stems, roots andwhole plants of C. arachnoids. As shown in the results, the RNA transcript levels varied considerablely from tissue to tissue, with the highest expression in stems, followed byroots and the lowest in leaves. The expression of CaHMGR in different tissues was inducedby MeJA treatment for3days except stems. In C. arachnoids leaves and roots, CaHMGRexpression levels were about two fold of those in the controls. The results revealed thatCaHMGR was elicitor-responsive and could be effectively elicited at least at transcriptionlevel.In our study, when9-days-old C. arachnoidea cell suspension cultures were treated byMeJA, YE and AgNO3for3days,20E contents in C. arachnoidea cell suspension cultureswere stimulated. The maximum content was obtained when treating cell suspensioncultures by0.2mM MeJA and was about8-fold higher than that of control, followed by25μM AgNO3(about6-fold higher than that of control) and100mg/L YE (about2-foldhigher). Our results suggested that MeJA, YE and AgNO3treatment might be an effectivemethod to induce higher20E production in C. arachnoidea cell suspension cultures bylager scale bioreactors. To investigate the effect of MeJA, YE and AgNO3on the transcriptlevels of CaHMGR in cell suspension cultures, C. arachnoidea cell cultures were subjectedMeJA, YE and AgNO3treatment and the expression levels of CaHMGR were analyzed byquantitative RT-PCR. The expressions of CaHMGR in C. arachnoidea cell cultures weresignificantly induced by MeJA, YE and AgNO3treatment for3days. Our results revealedthat CaHMGR was elicitor-responsive and CaHMGR expression in cell suspensioncultures was strongly induced by elictors which implied that more MVAs can besynthesized which are necessary for high production of isoprenoids or.phytoecdysteroid.In this study, we have successfully cloned and chanracterized a new gene encodingHMGR involved in the biosynthesis of20E from C. arachnoidea. In addition, weinvestigate the effect of MeJA on the transcript levels of CaHMGR in various tissues. Theregulatory effects of MeJA, YE and AgNO3on20E biosynthesis and CaHMGR expressionlevels in C. arachnoidea cells were studied. The cloning, characterization and expressionanalysis of CaHMGR will be helpful to understand more about its role in the20Ebiosynthesis pathway, which provide the basis for improving20E production by lager scale bioreactors in the near future.