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The Production And Stress-Resistance Of Transgenic Nicotiana Sylvestris Plants Expressing Melatonin Synthetase Genes And The Influences Of Exogenous Melatonin On In Vitro Plant Growth

Posted on:2012-01-08Degree:DoctorType:Dissertation
Country:ChinaCandidate:L J ZhangFull Text:PDF
GTID:1103330332494096Subject:Genetics
Abstract/Summary:PDF Full Text Request
Melatonin (N-acetyl-5-methoxytryptamine) is a well-known animal hormone synthetized by the pineal gland and has many functions, including roles in circadian adjustments, nocturnal behavior, sleep modulation, seasonal reproduction, immunoresponsiveness, the antioxidant system, vascular regulation and cancer inhibition. Since 1995, melatonin has been detected in of a considerable variety of plant species, the levels and functions of melatonin in plants raises attention widely. Related reports focused mainly on the determination of content of melatonin in different plants and tissues. Melatonin has been found to exist at different levels in more than 100 plant species. In recent years, studies of plant melatonin focused on the function of melatonin on plant growth, morphogenesis, and on plant tolerance against the environmental stresses. However these studies are still preliminary for illustrating the function of melatonin in plants. By genetic transformation, the key enzyme genes regulating melatonin synthesis could be transferred into plant genome, and then the content of endogenous melatonin would be improved. Subsequently the trangenic plants and the wild type plants will be compared on the content and function of melatonin in plants. In addition, exogenous melatonin used in vitro culture system could explore the effects of melatonin on plant growth and the resistance to the environment stresses. The research in this respect will provide more scientific evidences to clarify the physiological function of melatonin in plants.Arylalkylamine N-acetyltransferase (AANAT) and Hydroxyindole O-methyltransferase (HIOMT) were the key regulation enzymes in the melatonin biosynthesis pathway. Two genes were transferred into Nicotiana sylvestris by Agrobacterium mediated method, and transgenic plants were obtained. The contents of melatonin were measured in transgenic plants and the impact of UV-B radiation and salt stress on transgenic plants was detected. Further more, the effects of exogenous melatonin on the growth of some medicinal plants UV-B radiation were investigated. The main results obtained are as follows:1. Transgenic plants of N. sylvestris expressing arylalkulamine N-acetyltransferase (AANAT) gene and hydroxyindole-O-methyltransferase (HIOMT) gene have been obtained using Agrobacterium tumefaciens-mediated transformation. The PCR, RT-PCR, real-time PCRand FISH identification of the transgenic plants showed that the AANAT-HIOMT genes have already integrated into the genome of transgenic plants and the target genes could express at the level of RNA transcription. 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 explants were pre-cultured for 2-3d firstly, infected by Agrobacterium solution at the concentration about OD600=0.5 for 15-20 min, and then selected by 120mg/L gentamycin respectively, some positive transgenic clones of N. sylvestris were obtained.2. Reversed phase high performance liquid chromatographic procedure, coupled with ultraviolet detection (RP-HPLC-UV), was employed for the determination of content of melatonin in transgenic plants. The concents of melatonin in transgenic plants transformed by pXU55 were higher than that in the wild-type or pZP122 (with gen gene, without AANAT and HIOMT gene) transformed plants. Solid Phase Extraction (SPE) cartridges were used to purify and enrich samples during the extraction of melatonin from plants, which effectively prevented from poor separation and irregular peak shape.The samples have been separated in good chromatographic peak.3. The method of comet assay was optimized. The conventional "sandwich" mathod was simplified; "spreader" was applied to spread out agarose-embedded cells on the slide. The modified procedure could rapidly prepare 5 or more comet assay samples on one slide, and made the manipulation and procession of multiple samples easier. The impvoved protocol was cost-and time-saving and provided substantial advantages over the conventinal comet assay.By the modified comet assay, DNA damage caused by different dose UV-B in protoplasts of transgenic plants and the wild type was evaluated. Under the intensity of 0.5 W·m-2 radiation, TailDNA% value in transgenic protoplasts was lower than that in the wild type protoplasts at the range of 0s-10s of UV-B radiation. The duration of UV-B radiation leading to the highest TailDNA% value in transgenic plants (30s) was also longer than that in non-transgenic ones (10s). It suggested that the expression of AANAT gene and HIOMT gene enhanced the resistance of transgenic plants to UV-B radiation.4. The apoptosis rate and mortality rates of suspension cells of the trangenic plants of N.sylvestris and the wild-type under the condition of NaCl stress (at 0,50,100,200mM) were preliminarily estimated. Although apoptosis rate of two type cells reached the highest value at 100μM NaCl, apoptosis rate of suspension cells of wild type (control) was 87.5%, while 63.3% in transgenic cells. In the whole range of concentrations, the death rate of transgenic cells was always lower than that in the wild-type.5. The effects of exogenous melatonin on the growth or resistance to UV-B radiation in three tissue cultured medicinal plants were evaluated.(1) Stem explants of Polygonum cuspidatum were treated by melatonin (at 0,3,6,12 and 24μM). Melatonin at lower concentrations (3μM,6μM) significantly promoted root and shoot differentiation of explants. The most suitable concentration of melatonin for shoot regeneration was 6μmM. The average height of shoots reached 7.63±1.45 cm, leaf area was 31.8±6.55cm2, the average root number was 2.44±1.42 and the length of roots was 2.79±1.20 cm. However the corresponding datum in control plants were 4.07±2.19cm,18.99±6.88 cm2,1.67±1.53,2.66±1.58cm respectively. The growths of seedlings were inhibited at higher concentration (24μM) of melatonin. The effect of melatonin on the growth of P.cuspidatum was similar to IAA.(2) The callus growth of Scutellaria amoena was affected remarkebly by exogenous melatonin added in the MS medium. After cultured for 3w the growth rate of callus in the control group (at 0μM of melatonin) was 84%;the bud differentiation rate was 5.63%. At the same time the growth rate of calli treated with 0.1μM melatonin reached the highest value (171%), the highest differentiation rate (30.4%) was obtained at 1.0μM melatonin treatment. It is obviously that melatonin at low concentration (0.1μM,1.0μM,10.0μM) promoted the calli proliferation and adventitious bud differentiation, but melatonin at the higher concentration (100.0μM) exhibited inhibition for calli proliferation and the differentiation of adventitious buds(3) The effects exogenous melatonin on DNA repairs of protoplasts of Gentiana macrophylla after irradiation with different doses of UV-B were evaluated by comet assay. Under the intensity of 0.5 W·m-2 radiation. TailDNA% value in UV-B+MEL (1μM or 10μM) treated group protoplasts was lower than that in the group irradiated by UV-B alone in the range of 0s-30s of UV-B radiation.Protoplasts were treated with UV-B irradiation for 40s and incubated for 1-6h, TailDNA% value of protoplasts in UV-B+MEL (1μM or 10μM) treated group was lower than that in the group irradiated by UV-B alone at the same culture time. It showed that exogenous melatonin enhanced the capacity of DNA repair of protoplasts reduced DNA damage induced by UV-B radiation. This effect was closely related to the concentration of exogenous melatonin, i.e. higher concentration of melatonin (10.0μM) expressed better affects than low concentration of melatonin (1.0μM) for protecting cells from UV-B irradiation.
Keywords/Search Tags:melatonin, Nicotiana sylvestris, AANATgene, HIOMTgene, Genetic Transformation, HPLC, comet assay
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