Expression Of The Resveratrol Synthase Gene In Transgenic Lettuce

Background & ObjectiveCardiovascular disease(CVD) is the leading risk factor of health status worldwide,and the developing countries account for 86%of the global CVD disease burden.To developing,low and middle-income countries,CVD is not only a risk factor of health status,but a factor of undermining the socioeconomic development. An estimated 17.5 million people died from cardiovascular disease in 2005, representing 30%of all global deaths.Among these deaths,more than 50%were due to coronary heart disease and stroke caused by hyperlipernia,hypertension and obesity,and around 80%of these deaths occurred in low and middle income countries.By 2015,an estimated 20 million people will die from cardiovascular disease every year,and it is estimated that CVD will be the leading cause of death in developing countries.In the recent twenty years,30%～40%of deaths in our country are due to CVD,and CVD has been the leading cause of death.About 2/3 people 35-65 years old in our country have the risk factors of CVD.At present,the main treatments of CVD are intervention treatment and operation,which are the remedy measures aiming directly at the disease,even severe events.In order to effective prevention of CVD,it is necessary to carry out the primary prevention measures of disease prevention and cure.Resveratrol(Res) is a phytoalexin produced in various plants in response to UV irradiation,fungal infection,viral infection or mechanical damage.Resveratrol and resveratrol glucoside have beneficial effects on human health,known as cardioprotection,anticancer effect,antioxidant activity,anti-inflammatory action, antibacterial action,anti-virus action,neuroprotective effect and phytoestrogen effect, et al.For the past few years,the cardioprotection role of Res has been a research hot topic.Resveratrol and resveratrol glucoside could inhibit platelet aggregation,adjust the lipid metabolism,protect the blood vessel endothelium,rivalry endothelin I,and thereby prevent coronary artery disease and artherosclerosis.Resveratrol is a naturally occurring stilbene found in a limited number of unrelated plant species such as giant knotweed rhizome,grape,peanut or pine.Its content is microcrystalline in the plants,and giant knotweed rhizome couldn't be edible for long time,and grape and peanut are expensive.Therefore,the scientists pay close attention to use biotechnology such as gene engineering and fermentation engineering to produce a lot of Res and its glucoside.Resveratrol Synthase(RS) is a kind of stilbene synthase(STS).The biosynthesis of resveratrol is catalysed by the enzyme resveratrol synthase(RS),which converts one molecule of 4-coumaroyl-CoA and three molecules of malonyl-CoA into resveratrol.Resveratrol synthase is the key,exclusively necessary enzyme in the resveratrol synthetic pathway and it presents only in the plants which can synthetize resveratrol.Most plants are short of resveratrol synthase gene,while the precursor molecules of resveratrol are present throughout the plant kingdom.At present,the researches about resveratrol synthase transgenic plant are mostly on its phytoalexin role,and few are on its cardioprotection role,and the problems such as the low transformation efficiency of exogenous gene and the low expression are urgently to be solved.Therefore,the introduction of the gene encoding resveratrol synthase for resveratrol production in heterologous plant species using genetic engineering will provide a tempting approach to improve human health.Now,to transfer and express exogenous genes in plants and to produce functionality health-care foods which are cheap and direct-edible,is not only the popular topic of plant genetic engineering and biotechnology,but also the development tendency of disease prevention.Lettuce(Lactuca sativa var.crispa L.) is one of the most popular vegetables that is cultivated worldwide,and is an important transgenic plant.In this study,the plant expression vector pBI-RS carrying the CaMV 35S promoter was constructed and transformed to lettuce,and the biologic activity of transgenic lettuce was evaluated.At the same time,in order to obtain a highfrequency regeneration system in vitro culture of lettuce and the optimal condition of Agrobacterium tumefaciens-mediated transformation of lettuce explants,the acceptor plant(such as lettuce genotype,explants),the co-culture conditions(such as the density of Agrobacterium cells,the preincubate duration of explants,the co-culture duration and the inoculation time),habitat factors(such as auxin, cytokinin,AgNO3,cytokinin/auxin molar ratios,acetosyringone) were studied using orthogonal design and uniformly orthogonal design.The objective of this research was to get functional lettuce containing anti-disease or health protection composition, which was popular,convenient,cheap and effective.Methods(1) Construction of plant expression vector containing RS geneThe cloning vector pT-RS was constructed containing RS gene extracted from the leaf tip of Vitis vinifera by PCR amplification and RS sequence was identified by PCR amplification,enzymes digestion and sequence analysis.The plant expression vector containing RS gene was obtained and RS sequence was identified by PCR amplification and double enzymes digestion too.Binary plasmid vector was introduced into Agrobacterium tumefaciens strain EHA105 by freeze-thaw method and was stored at -70℃after identifying by PCR amplification and double enzymes digestion.(2) Optimization of the regeneration system in vitro culture of lettuce and the transformation conditions of Agrobacterium Tumefaciens.To obtain a high-frequency regeneration system in vitro culture of lettuce,the effect of genotype and sterilization time on the sterile seedling of lettuce was studied, and the effect of different plant growth regulators,AgNO₃ and explants on induction of direct adventitious buds and adventitious roots was also studied using orthogonal experiment.The effect of kanamycin(Kan) and carbenicillin(Carb) on regeneration shoots,carbenicillin and cefradium(Cef) on root,were tested using the dose-response tests.To establish the optimal condition of Agrobacterium tumefaciens-mediated transformation of lettuce cotyledons,the density of Agrobacterium cells,the preincubate duration of explants,the co-culture duration, the inoculation time and AS were studied using uniformly orthogonal design.(3) Integration and expression detection of RS in transgenic lettuceThe integration information and expression level of RS in transgenic lettuce were detected by PCR amplification and RT-PCR amplification.The content of Res and glucoside was measured by high performance liquid chromatography(HPLC) and gas chromatogram-mass spectrography(GC-MS).(4) Subculture analysisThe young leaves from TO plants which were RT-PCR-positive were cultured on regeneration medium after sterilized.The adventitious shoots were carefully excised from the explants when they were 1～2 cm high,and were rooted in the root media and obtained regeneration plants from subculture.RS gene was amplified by PCR in the regeneration plants of subculture.Results(1) The results of sequence analysis showed that the RS gene had a length of 1631 bp.The RS gene sequences were 99%in consistent with the sequences that reported by Gene Bank(accession number:AB046374.1).The results of PCR and restriction enzyme digestion of the recombinant vector pT-RS and pBI-RS showed that the plant expression vector pBI-RS had expected length of the target fragment.(2) The germination rate reached 80%and the sterile seedlings grew well when the lettuce seeds were treated for 60 sec with 70%ethanol,and then with 2%sodium hypochlorite for 15 min.The optimal condition of regeneration system in vitro culture of lettuce was Murashige and Skoog's medium(MS) supplemented with 0.2 mg/L 6-benzylaminopurine(BA),0.1 mg/Lα-naphthaleneacetic acid(NAA),and using the cotyledons of seedling with 2～3 days-old as explants.The interaction of BA×NAA played an important role in the expression of regeneration responses. Under this condition,the predicted frequency of shoots regeneration was 85.5%.The adventitious roots grew well in half-strength basal MS(1/2 MS) medium containing 0.05 mg/L NAA or 0.5 mg/L indole-3-acetic acid(IAA).The greatest efficiency in Agrobacterium-mediated transformation of lettuce cotyledons(2～3 days-old) was obtained by 10-time dilution of Agrobacterium cells, 15-min inoculation time,1-day pre-culture,4-day co-culture and without AS using the MS(Murashige and Skoog's,1962) medium supplemented with 0.2 mg/L 6-benzylaminopurine(BA),0.1 mg/Lα-naphthaleneacetic acid(NAA),100 mg/L Kan and 500 mg/L Carb,.Under this condition,the shoot rate of transformation explants was 25.2%.Roots of transformation regeneration shoots grown well on 1/2 MS medium supplemented with 0.05mg/L NAA and 300 mg/L Carb.(3) PCR-positive rate was 27.5%and RT-PCR-positive rate was 25.8%among 120 transgenic Kan-resistant regeneration plants.In transgenic lettuce plants,two additional compounds were identified as resveratrol and glucoside by HPLC and GC-MS.Quantitative analysis showed that the average content ofresveratrol reached 0.3050±0.0884μg/g leaf dry weight,and glucoside was 0.7200±0.0980μg/g leaf dry weight.(4) The transgenic lettuce plants could grow in regeneration medium containing 100 mg/L Kan,no difference was observed between transgenic versus non-transgenic lettuce seedlings in morphological assays.RS gene was found in 6 among 8 randomly selected regeneration plants of subculture.Conclusions(1) The plant expression vector pBI-RS containing RS gene was successfully constructed.(2) The germination capacity of Lactuca sativa var.crispa L.,cultivar "MGDSS" had an advantage on cultivar "Italian Yearly Very Late Bolt Lettuce".(3) The high-frequency regeneration system in vitro culture of lettuce was established and the predicted frequency of shoots regeneration was 85.5%.(4) The optimization transformation condition of Agrobacterium-mediated transformation of lettuce explants was obtained,and the shoot rate of transformation explants was 25.2%,PCR-positive rate was 27.5%and RT-PCR-positive rate was 25.8%,which was higher than that of other studies(about 10%).(5) RS gene could transmit in subculture of the transgenic lettuces,but the gene variation appeared.(6) In transgenic lettuce plants,the average content of resveratrol and Rglucoside were 0.3050 +/- 0.0884μg/g and 0.7200 +/- 0.0980μg/g leaf dry weight, respectively.The effective plant expression vector carrying CaMV 35S-35S double promoter is being constructed,which is expected to improve the content of resveratrol and R- glucoside in transgenic lettuce.