Genetic Transformation Of Nicotiana Tabacum L. And Hypericum Perforatum L. By Agrobacterium Tumefaciens Carrying Melatonin Synthetase Genes And The Enhancement Of Antioxidative Capability In Transgenic Plants

In most vertebrates,melatonin is synthesized primarily in the pineal gland as a kind of nerve-neuroendocrine hormone.As a hormone melatonin may play a key role in circadian rhythmicity and seasonal photoperiod regulation,also in sleep regulation and immunological enhancement.This molecular may also act as an antioxidant and involves in scavenging different types of reactive oxygen species.At present, melatonin role in plants is little known,only in conclusive attempts to determine the content of melatonin in some plants.Transgenic researches of tobacco(Nicotiana tabacum L.)and Hypericum perforatum L.(St John'swort),using the key genes encoding the enzymes involving in the biosynthesis process of melatonin,have not yet been reported so far.Arylalkylamine N-acetyltransferase(AANAT)and Hydroxyindole O-methyltransferase(HIOMT)were the key regulation enzymes in the melatonin biosynthesis pathway.The genes were cloned and constructed into binary plant expression vector YXu55,provided by Dr.Xu Yao,Vanderbilt University,USA.Then the genes were transferred into the explants of tobacco and H.perforatum L.by Agrobacterium mediated method.The results of PCR and Southern blot showed that the genes were integrated into plant genome.The contents of melatonin and the characteristics of antioxidation in transgenic plants were analyzed.The obtained results were as follows. The characterization of plant expression vector YXu55 showed that the sequences of both AANAT and HIOMT were 100%homologous with that of AANAT and HIOMT genes in GeneBank.The sequence of HIOMT of YXu55 plasmid was HIOMT(A)-6+7(6 and 7 were splicing alternative exons).The expression cassettes of AANAT,HIOMT and gentamycine resistant genes were tandem cloned into plant expression vector YXu55.An efficient protocol for high-frequency regeneration system of H.perforatum L. was established by optimizing the combination of different phytohormones contained in medium.The calluses were induced from leaf segments after 10～15d of culture on MS medium added 1.0mg/L 2,4 -D and 0.1mg/L NAA.These calluses showed high differentiation ability.Cluster buds were regenerated on MS medium supplemented with 2.0 mg/L 6BA and 0.2mg/L NAA,and the frequency of callus differentiation even reached to 100%.Roots would be induced in 10 days after the regenerated shoots were cultured on MS medium with 0.2mg/L NAA.Agrobacterium strains,pZP122(only containing the gentamycin-resistance gene, acting as positive control)and YXu55(containing the gentamycin-resistance gene,AANAT gene and HIOMT gene)were adapted for the genetic transformation of two plant species,tobacco and H.perforatum L.Factors affecting the efficiency of Agrobacterium mediated transformation were optimized,such as the pre-culture time of the explants,the suitable infection duration and the working concentration of the Agrobacterium.The proper conditions were,Being pre-cultured for 2-3d firstly,the explants were infected by Agrobacterium solution at the concentration about OD₆₀₀= 0.5 for 20 min,being selected by 60mg/L(for tobacco)and 50mg/L(for H.perforatum L.)gentamycin respectively.Some positive transgenic clones of tobacco and H.perforatum L. were obtained.The PCR,Southern blot and RT-PCR identification of the transgenic plants showed that the AANAT-HIOMT genes have already integrated into the genome of the two transgenic plants and the target genes could express at the level of RNA transcription. A Reversed phase high performance liquid chromatographic procedure,coupled with ultraviolet detection(RP-HPLC-UV),was established for the determination of melatonin in these two plants.It employed a Waters S ymmetryC₁₈column(150 mm×4.6mm,5.0μm)and a mobile phase of 100mmol/L ammonium acetate-water (80:20,v/v)at a flow rate of 0.8mL·min^-1.The transgenic plant samples were extracted and separated with methanol solution,then centrifuged to remove protein residuals and finally assayed by HPLC with UV detection at 265.8nm for 8.8 min.The method was proved to be linear in the range of 20～500ng·mL^-1with a regression coefficient of 0.9992,The minimum detection limit was 1.0 ng·mL^-1 The recoveries were 98.09%(n=6)and the RSD were 2.21%.The results suggest that this procedure was simple,fast,accurate and convenient.By use of RP-HPLC method,the analyses of contents of melatonin analyses in some transgenic plants of tobacco and H.perforatum L.showed that the contents of melatonin in YXu55 transgenic plants were higher than that in pZP122 transgenic plants and non-transgenic plants,but the contents of melatonin in pZP122 transgenic plants were the same with that in non-transgenic plants.Physiological and biochemical analyses of melatonin over-accumulated transgenic plants were carried out.Compared to the controls(positive control transgenic plants with pZP122 and non-transgenic plants),the AANAT-HIOMT transgenic plants expressed improvement of the capacity of total antioxidantion, enhancement of the activities of superoxide dismutase(SOD),peroxidase(POD) and catalase(CAT),and the content of glutathione reductase(GSH)was higher than that in the controls.At the same time,the malonaldehyde(MDA)content did not appear remarkable difference between transgenic plants lines and the controls.The above mentioned facts indicated that the accumulation of melatonin in transgenic plants might play a role in reducing the damage of oxidative stress.