《超表达MfCOR1提高转基因烟草抗寒性和生物量》3.docVIP

超表达MfCOR1提高转基因烟草抗寒性和生物量 摘 要：黄花苜蓿（Medicago falcata）是一种非常耐寒的豆科牧草，是苜蓿抗性育种的重要基因库。在本实验室已构建了黄花苜蓿响应低温的cDNA消减文库基础上，从中选择一个cDNA片段克隆cDNA全长，含有579 bp的开放阅读框，编码一个由192个氨基酸组成的蛋白。在GenBank中尚无与该蛋白同源性高的序列，将编码该蛋白的基因暂命名为MfCOR1（Medicago falcalta cold responsive gene 1）。该蛋白高度亲水，N-端含有叶绿体信号肽，推测存在于叶绿体基质中。利用半定量RT-PCR技术，分析了该基因在低温胁迫下的表达规律，在低温处理2-4 h时表达已明显受到诱导，低温处理8-96 h后表达量一直维持在高水平，初步证明MfCOR1是低温诱导基因。构建了超表达载体pBI-MfCOR1，通过农杆菌介导法转化烟草获得了转基因植株，经过PCR检测筛选出51株阳性植株，挑选其中15株进行Southern杂交分析，结果表明这些转化植株来自不同的转化事件。T1代Southern杂交RT-PCR检测，结果表明转基因能稳定遗传并且能表达。进一步比较测定低温胁迫转基因T1代及其野生型对照，表明显著提高了抗寒性，充分证明MfCOR1是一个耐寒基因。转基因株系还表现为正常生长条件下苗期生物量显著增大，这与具有更高的光合速率有关。研究结果表明，MfCOR1是一个新的耐寒基因，可能参与叶绿体光合作用，提高植物的光合速率，促进苗期的生物量积累，是一个很有应用潜力的新基因，可应用于改良农作物的抗寒性和提高生物产量。关键词：耐寒基因 克隆 转基因烟草 抗寒性 生物量Overexpression of MfCOR1 in Transgenic Tobacco Improves Cold Resistance and Biomass Abstract: Medicago falcata is a kind of Leguminosae forage grasses which has strong tolerance to cold stress. Because of its great resistance to cold, drought and leanness, Medicago falcate serve an important gene pool for crop resistance breeding. A SSH cDNA library of cold-responsive gene in Medicago falcata has been established in our laboratory, in which MfCOR1 gene has been shown to be cold-induced. The full length of MfCOR1 cDNA is 605 bp, which contains an open reading frame (ORF) of 579 bp and encodes a peptide of 192 amino acids, but no obvious homological sequence has been found in GenBank. The bioinformatics analysis indicats that the hydrophibic domain is dominant in the structure of MfCOR1 protein which has a predicted signal peptide of chloroplast and might be localized in stroma of chloroplast. The expression pattern of MfCOR1 was analyzed by semi-quantitative RT-PCR. The expression of MfCOR1 is rapidly induced in response to low temperature, and sustained at high expression level up to 96 h after treatment. The over-expression vector of MfCOR1, pBI-MfCOR1, was constructed. The transgenic tobaccos were generated using Agrobacterium-mediated transformati