胡萝卜与黄粉虫抗冻融合基因在拟南芥中的表达与抗冻性分析

抗冻蛋白(antifreeze protein, AFP)是一类能控制冰晶生长和抑制冰晶之间发生重结晶的蛋白质，能在低温结冰条件下保护生物体不受伤害。抗冻蛋白具有2种明显不同的抗冻活性--热滞(thermal hysteresis, TH)活性和重结晶抑制(recrystallizationinhibition, RI)活性。鱼类和昆虫抗冻蛋白的特征是高TH低RI活性，而植物抗冻蛋白的特征是低TH高RI活性。为了获得高TH、高RI活性的抗冻蛋白(antifreeze protein, AFP)，克隆得到胡萝卜抗冻蛋白基因(DcAFP)和黄粉虫抗冻蛋白基因(TmAFP)cDNA全序列。通过重叠延伸PCR，将2个基因前后串联构建融合基因Dc-TmAFP和Tm-DcAFP，并构建了植物表达载体，通过农杆菌介导转化模式植物拟南芥，成功获得4个基因的转基因拟南芥植株。对转基因拟南芥植株进行抗冻试验处理，并进行了存活率统计和叶片黄化率统计分析。分析结果表明，不同功能的抗冻蛋白对植物的抗冻性有不同的帮助，热滞效应高效的黄粉虫抗冻蛋白对植物降低冰点方面效果大，而在植物遭受较长时间冻害时，抑制重结晶效应高效的胡萝卜抗冻蛋白对植物质外体中流体保持稳定性作用较大，从而提高植物的耐冻能力。

Expression and antifreeze feature analysis of the Daucus carota and Tentbrio molitor fusion antifreeze protein (AFP) gene in Arabidopsis thaliana

Antifreeze proteins（AFPs） are a family of proteins capable of protecting organisms from damage in freezing or sub-freezing conditions by controlling the growth of ice and inhibiting the recrystallization between ice granules,which are termed thermal hysteresis（TH） activity and recrystallization inhibition（RI） activity respectively.In comparison with the characteristic of high TH and low RI of fish AFPs and insect AFPs,antifreeze proteins of plants was low TH and high RI activity.In order to obtain antifreeze proteins with high TH and high RI activity,the complete cDNA of antifreeze protein genes（DcAFP and TmAFP） were cloned from Daucus carota and Tentbrio molitor.The two fusion antifreeze protein genes,Dc-TmAFP and Tm-DcAFP,were constructed series before and after by overlap extension PCR.The four genes were successfully transferred into Arabidopsis thaliana via Agrobacterium tumefaciens,and then the transgenic plants were coldtreated.The frost-resistance of the transgenic plants was studied and analyzed.The results from statistical analysis of mortality and blade etiolation rate indicated that the AFPs had different functions which improved the cold resistance in many different ways.Hyperactive thermal hysteresis protein（THP） like TmAFP was a great help to depress the freezing point.However,when plant suffered from freeze injury a little longer,hyperactive ice re-crystallization inhibition protein（IRIP） like DcAFP kept stabilization of the fluid in the apoplast.