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Expression of the Resveratrol Synthase Gene in Transgenic Lettuce

Author: ZhuChunYan
Tutor: YuShouYi
School: Southern Medical University,
Course: Epidemiology and Biostatistics,
Keywords: Wrinkled leaf lettuce Resveratrol synthase Resveratrol Polydatin Transgenic Plants
CLC: S636.2
Type: PhD thesis
Year: 2008
Downloads: 187
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Abstract

Background and Objectives Cardiovascular disease is a serious threat to human health, major diseases, especially in developing countries, assumed the global burden of cardiovascular disease in 86%. For developing countries, cardiovascular disease is not just a threat to health problems, but also on the social and economic development have a serious impact. According to the World Health Organization reported that in 2005 the number of people worldwide die of cardiovascular disease as high as 7.5 million, accounting for 30% of all deaths, of which more than 50% of the deaths were caused by high cholesterol, hypertension and obesity as a result of coronary heart disease and stroke caused, and 80% of these deaths occur in developing countries. By 2015, estimates that the global number of people die each year from cardiovascular disease up to 2 0000000, cardiovascular disease will become the leading cause of death in developing countries. The past 20 years, China's total cardiovascular disease mortality in the proportion of 30% to 40% of urban and rural residents has become a major cause of death. In our country the population aged 35 to 65, 2/3 of people with cardiovascular disease risk factors. Currently, the treatment of cardiovascular disease is to develop and popularize interventional and surgical treatment, but it is for after the onset, even after a serious incident intervention and remediation. To be more effective in reducing the incidence of cardiovascular disease, we must do a good job of a disease control precautions. Resveratrol (Resveratrol, Res) is a plant to resist external stimuli, such as ultraviolet light, fungal, viral infection or mechanical damage and secretion of a phytoalexin (phytoalexin). Resveratrol and resveratrol glucoside biological functions include: cardiovascular protective effects, antagonistic tumor effect, anti-inflammatory, antiviral, neuroprotective role of phytoestrogens. In recent years, Res cardiovascular protective effect has become a research hotspot. Resveratrol and Resveratrol inhibits platelet aggregation, regulate lipid metabolism, protect the vascular endothelium, such as the role Ⅰ endothelin antagonist, thereby preventing coronary heart disease and atherosclerosis and other cardiovascular diseases. Res its glycosides exist only in a limited greenery, such as knotweed, grapes, peanuts, pine and so on. Res natural plant exists, after all, trace, and medicine, such as Polygonum, etc. can not be long-term consumption of food such as grapes, peanuts, high prices, not a wide spread of common food. Therefore, the use of genetic engineering biotechnology to produce a large number of scientists Res much attention. Resveratrol synthase (Resveratrol Synthase, RS) is a stilbene synthase (stilbene synthase, abbreviated STS) in a class. Res biosynthetic pathway which is only required in the key enzyme exists only in a Res synthesis in plants, and therefore lack RS genes most crops. RS Res catalytic precursor (4 - coumaroyl CoA and malonyl-CoA A A) by one step reaction Res, and biosynthetic precursors Res ubiquitous in the plant material. Currently on resveratrol synthase transgenic plant research focuses on aspects of phytoalexin Res qualitative analysis, and its cardiovascular protective effects and other health functions have been few reports of successful research; And there is a foreign gene in the recipient plant the conversion rate and low expression of problems to be solved. Thus the application of genetic engineering techniques in RS heterologous gene expression in plants to synthesize resveratrol and resveratrol glucoside, will raise the level of human health to provide effective way. Use of plant genetic engineering methods, the exogenous gene into the plant expression, thereby producing inexpensive and can be eaten directly functional health food, which is currently the plant genetic engineering and biotechnology research focus is the development trend of disease prevention . Lettuce is a very popular daily consumption of vegetables, and it is important to GM crops. In this study, the cauliflower mosaic virus 35S promoter, plant expression vectors constructed, the grape lettuce gene expression RS Res. Meanwhile, the application of orthogonal design and uniform orthogonal experimental design, statistical design methods, from ① receptor plants (lettuce varieties, explant Ages, drawn parts), ② co-culture conditions (Agrobacterium growth state of bacteria concentration, explant pre-culture time, dip time of incubation time), ③ habitat factors and physical and chemical factors (auxin, cytokinin, AgNO ₃, hormone concentration ratio, acetosyringone etc.) perspective, screening out the optimal recipient plant lettuce vitro regeneration system and Agrobacterium-mediated transformation of lettuce optimal conditions in order to be able to breed a popular expectations of a wide range of easy to use, inexpensive, safe and efficient with the prevention cardiovascular disease and other functions of health vegetables. Research Methods (1) containing resveratrol synthase (RS) gene in the plant expression vector grape extract genomic DNA, RS gene was amplified by PCR, this gene was ligated into a cloning vector pGEM-T Vector, recombinant vector pT-RS; by PCR, restriction enzyme digestion and sequence analysis identified, the RS gene was cloned into the plant expression vector pBI121, recombinant vector pBI-RS, using PCR and restriction enzyme digestion method for identification. Binary expression vector pBI-RS through freeze-thaw method introduced into Agrobacterium tumefaciens strain EHA105, using PCR and restriction enzyme digestion method correctly identified and stored at -70 ℃ spare. (2) lettuce vitro regeneration system and Agrobacterium tumefaciens transformation conditions optimized by orthogonal design, research different lettuce varieties and different sterilization time to cultivate lettuce aseptic effects and different concentrations of plant growth regulators, AgNO < sub> 3 and the different sources of explants lettuce explant bud differentiation frequency and direct Seedling roots to get lettuce high frequency in vitro regeneration system. Dose-response test used to study different kanamycin, carbenicillin concentration on lettuce shoot regeneration effects and carbenicillin, cefotaxime differentiated rooting of shoots. Uniform orthogonal experimental design to explore the concentration of Agrobacterium, explants pre-incubation time of incubation time, dip time and acetosyringone for Agrobacterium transformation efficiency. (3) RS genes in lettuce integration and expression was detected by PCR, RT-PCR method for detection of molecular biology in the RS gene in lettuce integration and expression. High performance liquid chromatography (HPLC) and gas chromatography - mass spectrometry (GC-MS) quantitative detection of transgenic lettuce resveratrol and resveratrol content. (4) Subculture collection by RT-PCR analysis of positive maternal plants (T0) of young leaves, inoculated in sterilized medium inducing regeneration bud. When uncertain shoot to 1 ~ 2 cm, the cut shoots to rooting medium rooting get subculture regenerated. RS was detected by PCR in transgenic lettuce plants subculture stability. Results (1) DNA sequence analysis showed that the grape resveratrol synthase gene is 1 631 bp, and GeneBank accession number AB046374.1 sequence comparison, 99% nucleotide homology, that they have been successful grape resveratrol synthase gene DNA. Recombinant vector pT-RS and pBI-RS of PCR, restriction enzyme digestion and sequence analysis results show that, the plant expression vector pBI-RS constructed correctly. (2) lettuce varieties \Lettuce bud regeneration in vitro differentiation conditions for the optimal combination of MS medium supplemented with BA 0.2 mg / L, NAA 0.1 mg / L, explants of 2 ~ 3 day cotyledons; wherein, BA and NAA interaction on plant regeneration important influence. In this condition, the lettuce bud explants direct differentiation rate was 85.5%. Lettuce regeneration bud containing low concentrations of auxin (NAA 0.05 mg / L, or IAA0.5 mg / L) of 1/2MS rooting medium is better. In Agrobacterium-mediated wrinkled leaf lettuce cotyledons (2 ~ 3 day Ages) transformation assay, using with BA 0.2mg / L, NAA 0.1 mg / L, Kan 100 mg / L, Carb 500 mg / L of MS medium group, the optimal conditions for the conversion of 10-fold dilution of Agrobacterium, the pre-incubation time of 1 day, a total culture time was 4 day, dip time was 15 min, do not add acetosyringone. In this condition, the conversion was 25.2% Agrobacterium. Transformed regenerated shoots containing NAA 0.05 mg / L, Carb 300 mg / L of 1/2MS rooting medium is better. (3) 120 regeneration of transgenic plants, PCR positive rate was 27.5%, RT-PCR positive rate was 25.8%. Using high performance liquid chromatography and gas chromatography - mass spectrometry to identify transgenic lettuce were new compounds resveratrol and resveratrol. Transgenic lettuce leaves resveratrol content of 0.1330 ~ 0.4280μg / g dry weight, with an average content of 0.3050 ± 0.0884μg / g; resveratrol content is 0.5528 ~ 0.8380μg / g dry weight, with an average content of 0.7200 ± 0.0980μg / g. (4) transgenic lettuce plants containing Kan 100 mg / L regeneration medium grew well, and its leaf shape, color and other morphological characteristics and non-transgenic lettuce plants were not observed differences. Randomly selected eight subculture PCR analysis of regenerated plants, of which six to RS gene amplification. Conclusions (1) RS-containing gene was successfully constructed plant expression vector pBI-RS. (2) \(3) to establish a high-frequency regeneration system from lettuce, lettuce cotyledon directly predicted frequency of 85.5% bud differentiation, differentiation frequency of repeated trials of 90%, higher than the current level of foreign conversion (approximately 84%). (4) established the optimal conditions for Agrobacterium-mediated transformation of lettuce, the conversion rate was 25.2%, the conversion rate of regenerated plants PCR-positive 27.5%, RT-PCR positive rate was 25.8%, higher than the current domestic and international level of conversion (approximately 10 %). (5) RS gene in transgenic lettuce subculture can pass relatively stable, but there are still a certain degree of variability. (6) transgenic lettuce resveratrol and resveratrol glucoside average content 0.3050μg / g, 0.7200μg / g, lower than the content of Polygonum cuspidatum and peanuts. Containing CaMV 35S-35S dual promoter expression vector for efficient plant being built in the hope that through this approach can improve the transgenic lettuce resveratrol and its glucoside content.

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CLC: > Agricultural Sciences > Gardening > Vegetable gardening > Green leafy vegetables > Lettuce ( lettuce )
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