Study On The Regulation Of Plant Growth By Terpene Synthesis Gene SMpGPPS

Terpenoid is a large group of compounds derived from isoprene as the building unit in nature.It is the largest family of plant secondary metabolites discovered in nature.More than30000 terpenoid molecules have been identified from organisms.To date,it is appropriately determined that isoprene is biosynthesized from two metabolic pathways in plants.One is the mevonolate(MVA)pathway,which is localized in the cytosol.The other is the2-C-methyl-D-erythritol 4-phosphate /1-deoxy-D-xylulose 5-phosphate(MEP/DOXP)pathway,which is localized in plastids.Due to this subcellular compartmentization of the MVA and MEP/DOXP pathways,different subgroups of terpenoids are synthesized in either the cytosol or plastids.To date,it has been determined that sequiteprenoids,triterpenoids,phytols,and other plant steroids are mainly biosynthesized from isoprene formed via the MVA pathway in the cytosol,while hemiterpenes,monoterpenes,limonene(limonene,cymene,Eucalyptus brain),diterpenoids,abscisic acid and erythroid,gibberellic acid,Mycin,carotenoids,chlorophyll,and others,are mainly biosynthesized from isoprene formed via the MEP/DOXP pathway.Numerous terpenoids have important chemical and ecological functions to protect plants from herbivores,pathogens,and stresses.Moreover,GAs and ABA are essential plant hormones.GAs control plant development,growth,flowering,and fruting.ABA controls fruit riping and senensing.Therefore,terpenoids have been utilized for economic improvements in agriculture,food,and chemical industries.In comparision,the utilization of terpenoids for plant biomass improvement remains continuous investigations.In this study,we aim to investigatate to what extent a manipulation of a key step of terpenoids biosynthesis can affect plant growth and biomass in both tobacco model plants and economically important trees.One novel gene was designed from three heterologous gene sequences of three orgaanisms: At RBCSIA(Atlg6709)sequence fragment(PTP)from Arabidopsis thaliana,AAY33491.1 from Myzus persicae(Mp GPPS),and 27-bp human influenza hemagglutinin(HA)tag c DNA sequence c DNA fragement(Hemagglutinin).After codon optimization,three c DNA fragments were synthesized together to form a novel c DNA sequence,namely,PTP-Mp GPPS-HA,which could transport protein to plastids.Meanwhile,another short Mp GPPS-HA,namely Mp GPPS-HA,was synthesized to localize protein in the cytosol.Two enzymes ecoded by PTP-Mp GPPS-HA and Mp GPPS-HA were named to be Pl-s Mp GPPS and Cy-s Mp GPPS.Two novel genes were then cloned to the binary vector PMDC84.The resulting recombinant vectors were named PMDC84-PTP-s Mp GPPS and PMDC84-Cy-s Mp GPPS,which were introduced to Agrobacterium tumefaciens GV3101 for genetic transformation of tobacco(Nicotiana tobacco Xianthi)and poplar 84K(P.alba × P.glandulosa).Multple transgenic plants from tobacco and poplar were obtained by selection on agar-solidified medium containing antibiotics.Genotyping analysis with PCR and RT-PCR identified multiple positive transgenic plants,which were further grown in the pot soil in the greenhouse for phynotyping,including growth rate,leaf numbers,leaf sizes,nodal numbers and lenghs,plant heights,and biomass.In addition,flowering times were compared in transgenic versus wild-type tobacco and vector control transgenic plants.Subcellular localization of transgenic novel protein and terpenoid metabolism were analyzed for transgenic versus wild type and vector control plants.The main results are as follows:1.Two novel PTP-Mp GPPS-HA and Cy-Mp GPPS-HA c DNAs were synthesized with gene sequences from human influenza,aphids,and Arabidopsis thaliana.Two c DNAs were successfully introduced to tobacco and poplar plants.Multiple transgenic plants were obtained from both tobacco and poplar for genotyping and phenotyping.2.Two novel c DNAs were fused with GFP for subcellular localization.Hypocotyl sections of 5-day-old seedlings geminated on MS medium were used for confocal microscopic analysis.The results showed that Cy-s Mp GPPS-HA-GFP was localized in in the cytosol of Cy-Mp GPPS-HA-GFP transgenic plants.Pl-s Mp GPPS-HA-GFP was localized in plastids in plastidial localization of fused PTP-s Mp GPPS-HA transgenic plants.The positive control using GFP alone was localized in the cytosol.In addition,the localization of Pl-s Mp GPPS-HA-GFP was characterized by actively cytosolic streaming in cells of hypocotyls.These data demonstrate that two novel enzymes are localized in the subcellular locations as designed.3.The results of morphological and physiological phynotyping showed that the overexpression of two novel genes in tobacco significantly promoted growth and early flowering and fruiting.Compared with wild type,the average height,leaf area,number of leaves of transgenic plants were significantly increased by 40%,35%～50%,and by 20%,respectively.The flowering time of transgenic plants was 3～4 days earlier than those of wild type plants.The growth rate of transgenic plants were two-fold faster than that of wild type and vector control plants.The dry biomass and seed yield of transgenic plants were by 50-80% and35%,respectively.4.The results of metabolite analysis showed that the carotenoid content in tobacco overexpressing the PTP-Mp GPPS-HA gene was significantly higher than that that in wild type,while the carotenoid content in tobacco overexpressing the Cy-Mp GPPS-HA gene was significantly lower.The isoprene emission from transgenic plants was significantly lower than that from wild type and vector PMDC84 control plants.The daily production of isoprene was was significantly lower in PTP-Mp GPPS-HA and Cy-Mp GPPS-HA transgenic plants than in wild-type tobacco in one day.In addition,the emission of isoprene from tobacco plants was stronger during midday than during night time.All these features were similar between PMDC84 control transgenic and wild type plants.5.Non-polar metabolites in transgenic versus control tobacco plants were preliminarily analyzed with GC-MS.Total ion chromatogram profiles were recoded to characterize genome-wide peaks of metaoblites.The resuling data showed that the overexpression of two novel genes apparaently modified non-polar metabolite profiles in transgenic tobacco plants..6.Mutltiple PTP-Mp GPPS-HA and Cy-Mp GPPS-HA transgenic poplar plants were obtained.Phynotyping comparison showed the height,the ground diameter,and the leaf number of transgenic poplar plants were significantly increased by 17%～1%,by 10%～15%,and by 20%,respectively the average growth rate of transgenic popolar was increased by nearly 50%.In conclusion,the novel synthetic PTP-Mp GPPS-HA and Cy-Mp GPPS-HA genes are valuable for molecular breeding to promote growth and increase biomss of both herbaceous and woody plants.This study further demonstrates the significance of synthetic biology in the application of agriculture and forest improvement for high economic values.