Cloning And Studies Of Jasmonate Biosynthetic Pathway Key Genes From Catharanthus Roseus

Catharanthus roseus (L.) G. Don possesses a large number of terpenoidindole alkaloids in which over130compounds have been isolated andidentified, such as vindoline, catharanthine, ajmalicine, vinblasitne andvincristine. However, the contents of most alkaloids are low and the mostvaluable dimeric indole alkaloids in C. roseus, vinblastine and vincristine, areimpossible to be yielded in hairy root culture and suspend cell culturesystems. So the strategies to obtain TIAs from hairy root culture and suspendcell culture systems are limited.Jasmonate is a kind of significant plant growth regulator, it plays a key rolein plant growth, development, defense system and secondary metabolitessynthesis pathway.The transcription factor ORCA3of C. roseus can beinduced by methyl jasmonic acid, and ORCA3can further enhance severalgenes expression which are related in TIAs biosynthesis metabolic pathway,including DXS、ASΑ、TDC、STR、D4H. Some researches have shown that plant hormones can improve TIAs content. So it is expected to improve thecontent of secondary metabolites by gene engineering of the JA syntheticpathway instead of hormone treatment. Therefore, gene cloning and analysisof JA biosynthetic pathway are important.In jasmonate biosynthetic pathway of plants, allene oxide synthetase andallene oxide cyclase catalyze the most important steps.To study the genesrelated in jasmonate synthesis and find their functions in secondarymetabolism, we cloned and analyzed the two genes AOS and AOC from C.roseus. We found that both of AOS and AOC were expressed in differentorganizations and the expression profile under stresses were also different.We have some basic recognition about the two genes and their proteinscharacteristics which can be useful in further genetic engineering study.Research results obtained are as follows:1) Cloning and expression of allene oxide synthase gene from C. roseusIn the study, a full-length cDNA of AOS gene (named as CrAOS,JQ364955) was cloned from C. roseus. The gene was2118bp and itcontained an open reading frame (1638bp) encoding545amino acids.Comparative and bioinformatic analysis revealed that the deduced protein ofCrAOS was highly homologous to AOSs from other plant species. Southernblot analysis revealed that it was a low-copy gene. Real-time Quantitative PCR （RT-qPCR） analysis showed that CrAOS mRNA accumulatedabundantly in old leaves and least in young alabastrums. The RT-qPCRanalysis revealed that wound, low temperature, methyl jasmonic acid,ethylene treatments significantly enhanced CrAOS expression, and salicylicacid had no influence.2) Cloning and expression of allene oxide cyclase gene from C. roseusIn the study, a full-length cDNA of AOC gene (named as CrAOS,JQ364956) was cloned from C. roseus. The gene was1049bp and itcontained an open reading frame756bp encoding251amino acids,5′terminal UTR122bp and3′terminal UTR171bp. There are two introns inthe DNA sequence located in the354~355bp and478~479bp。Comparativeand bioinformatic analysis revealed that the deduced protein of CrAOC washighly homologous to AOCs of other plant species. Southern blot analysisrevealed that it was a single-copy gene. Rt-qPCR analysis showed thatCrAOC mRNA accumulated most abundantly in alabastrums, followed bystem, and the expression in leaf was a little higher than that in the root. TheRT-qPCR analysis revealed that wound, methyl jasmonic acid, ethylene andsalicylic acid treatments significantly enhanced CrAOC transcript expression,while low temperature had no significant influence. We also found thatvindoline, catharanthine and vinblastine content were enhanced after MeJA treatment, but the content level of anhydrovinblastine was lowerthan controls. Anhydrovinblastine is the precursor, so we predict that more ofanhydrovinblastine flowed downstream to synthetize vinblastine.3) Overexpression of CrAOC in transgenic tobaccos enhanced nicotineproductionThe allene oxide cyclase catalyzed step is the most important one injasmonate biosynthetic pathway of plants. Here the heterogenous CrAOCgene from C. roseus was transformed into Nicotiana tabacum to investigatethe influence of CrAOC overexpression on the nicotine content as a testingmode. PCR analysis showed CrAOC was transformed into tobaccos. HPLCshowed that the content of nicotine in transgenic tobacco was enhanced.Therefore, this study proved that transgenic engineering of JA biosyntheticpathway genes was expected to replace the tedious exogenous hormonetreatment to improve the content of secondary metabolites product, andprovided an effective gene engineering strategy.