Cloning And Studies Of Jasmonate Biosynthetic Pathway Key Genes From Medicinal Plants

Plant secondary metabolites are a wide variety of low-molecular-weight compounds whose productions are often enhanced in response to both biotic and abiotic stresses. Nevertheless,most of the important secondary metabolites are of low content,or can only be produced with elicitation.Recently,metabolic engineering has been widely applied in order to achieve higher yields of specific metabolites.With the increasing number of cloned genes involved in biosynthesis and the expanding investigation of biosynthesis pathway,some effective approaches have showed encouraging progresses.Under biotic or abiotic stress,plants produce increased amounts of hormones such as abscisic acid(Sasaki et al.),SA(salicylic acid),ethylene and jasmonate(JA).These hormones may interact with one another in regulating stress signaling,plant stress tolerance and secondary metabolism.Many of the responses are mediated by a class of hormones named as JAs.JAs play significant roles in the biosynthesis of many plant secondary metabolites as well as in distinct developmental stages.In jasmonate biosynthetic pathway of plants,allene oxide synthase(AOS,EC 4.2.1.9)and allene oxide cyclase(AOC,EC 5.3.99.6)catalyzes the most important steps.Hyoscyamus niger is a medicinal plant which produces a class of jasmonate-responsive pharmaceutical secondary compounds named as tropane alkaloids.Honeysuckle(Lonicera spp.,named "jin-yin-hua" in Chinese,also known as Japanese Honeysuckle,whose flowers enjoy name of Flos Lonicerae as medicinal part),is an important crude herb frequently used in Chinese medicines,containing various metabolites with chlorogenic acid(CA,the major important active ingredient) as the indicator compound to characterize the quality of this herb.1)Allene oxide cyclase gene from H.nigerA novel cDNA encoding for allene oxide cyclase from H.niger,named HnAOC (GenBank accession;AY708383.)was cloned by RACE,showing highly homologous to the AOCs of other species.The full-length cDNA of HnAOC was 1044 bp with 5' and 3' UTR,polyA tail and a 747-bp ORE The comparison between the flall-length cDNA and the genomic DNA of HnAOC revealed that the genomic DNA contained two introns,one was 94 bp and the other one was 402 bp.The putative protein was 248 AA(amino acid)with a molecular weight of 27.0 kDa and a pI of 8.74.Southern blot analysis showed that it was a multi-copy gene.RT-PCR analysis revealed that the expression of HnAOC could be regulated by various stresses and elicitors,indicating the alternative ways to regulate AOC expression and jasmonate biosynthesis.2)Allene oxide synthase gene from H.nigerA full-length cDNA encoding allene oxide synthase,the first commit step enzyme in jasmonate biosynthesis pathway,which was named as HnAOS(GenBank accession; EF532599)was cloned by RACE.The full-length HnAOS was 1652 bp with 5'-UTR, a 1485-bp ORF,3'-UTR(un-translated regions)and poly-A tail.The putative protein was 494 AA(amino acid)with a molecular weight of 55.8 kDa and a pI(Isoelectric Point)of 8.82.HnAOS was a novel member of the cytochrome P450(CYP74A) subfamily.There was no intron present in the genome DNA among HnAOS.Real-time quantitative PCR analysis showed that HnAOS mRNA mainly accumulated in stems. The expression level of HnAOS significantly increased with wounding or MeJA treatment,and MeJA showed the induction-effect earlier than wounding.Evidently,it was consist with that wounding acted its role by inducing JA biosynthesis.3)Overexpression of allene oxide cyclase from H.niger in transgenic tobaccos with the effects of both improving stress-tolerance and enhancing nicotine production.It was speculated that genetic engineering of jasmonate biosynthetic pathway might enhance the endogenous jasmonates concentrations thus stimulate the production of jasmonate-responsive secondary metabolites.The allene oxide cyclase catalyzed step is the most important one in jasmonate biosynthetic pathway of plants.Here the heterogenous AOC gene from H.niger was transformed into Nicotiana tabacum cv. Petit Havana to investigate the influence of HnAOC overexpression on the nicotine content as a testing mode.The results revealed that the overexpression of HnAOC in tobacco not only enhanced stress-tolerance but also stimulated nicotine biosynthesis. Therefore,it was validated that without the cost of extraneous hormones,genetic manipulation of jasmonate biosynthetic pathway genes could be another alternative approach in secondary metabolic engineering for the production of valuable jasmonate-responsive secondary metabolites. 4)Allene oxide synthase gene from Lonicera japonicaHoneysuckle(Lonicera sp.)is an important Chinese herbal medicinal plant whose flowers(named "Flos Lonicerae" as a medicinal material)are widely used as anti-inflammatory agent in oriental area since ancient times.The content of chlorogenic acids,which represents the quality of this medicinal material,varies according to different tissues and distinct stages of development.So it is significantly important for farmers and medicinal manufactures to control the quality of this material.Jasmonates(JAs)are a class of phytohormones which play a central role in plant defense responses as well as in distinct stages of plant development.In order to investigate whether genetic manipulation of jasmonates biosynthesis pathway could regulate the development of honeysuckle coupled with the corresponding content of chlorogenic acids,we firstly cloned a novel cDNA from Lonicera japonica Thunb., named LjAOS(GenBank accession;DQ303120),which was homologous to other AOSs.The full-length cDNA of LjAOS was 1942 bp with 5' and 3' UTR,poly-A tail and contained a 1584-bp ORF.The putative protein was 527 AA with a molecular weight of 59.2 kDa and a pI of 8.41.No intron was present in the genomic DNA. Southern blot analysis revealed that it was a multi-copy gene.Real-time quantitative PCR analysis showed that LjAOS mRNA accumulated the most abundantly in alabastrums,in which the content of chlorogenic acids was also the highest.It indicated the positive relationship between JAs biosynthesis and the levels of chlorogenic acids.The study of JAs biosynthesis pathway in Lonicera japonica would help to facilitate the production of this medicinal material.In conclusion,the study of JA biosynthetic pathway in H.niger and L.japonica presented a knowledge of JA biosynthetic pathway and its regulation in these two medicinal plants.The results also helped to further elucidate the mechanisms and evolution of JA biosynthetic pathway in planta.Simultaneously,the examination of stress-tolerance and nicotine production in transgenic tobaccos as a testing mode would greatly shed light on the application of JAs in plant secondary metabolic engineering.