棉花GhNCED2基因表达载体构建及转基因烟草表达分析

目的]9-顺式环氧类胡萝卜素双加氧酶(NCED)是高等植物脱落酸(ABA)生物合成途径中的关键限速酶.为了进一步研究GhNCED2基因的功能.方法]构建该基因植物超表达载体PZP35S - GhNCED2,通过农杆菌介导法转入野生烟草,并利用卡那霉素筛选、PCR扩增验证和RT - PCR表达分析.对转基因烟草T1代分别进行2; NaCl、4℃、20; PEG逆境胁迫处理,利用实时荧光定量RT-PCR分析表明.结果](1)GhNCED2基因已整合入烟草基因组中,并能在T1代中稳定表达.(2)随着时间的推移其表达量变化规律基本一致呈现先上升后下降的趋势,2; NaCl、20; PEG胁迫后GhNCED2基因相对表达量到达峰值的时间稍落后于4℃胁迫,20; PEG处理后基因的相对表达量要高于其他处理.结论]干旱、盐碱及低温胁迫均都能诱导GhNCED2基因的大量表达,且该基因对干旱胁迫的响应更加积极,而对冷害更加迅速.

Construction of Expression Vector onGhNCED2 Gene from Gossypuum hirsutum and Its Genetic Transformation and Expression Analysis

【Objective】9-cis-epoxycarotenoid dioxygenase(NCED) is a key limited enzyme for ABA biosynthesis in higher plants.The purpose of this project was to carry out a further study of the function of GhNCED2 gene.【Method】The plant transformation vector PZP35S-GhNCED2 was constructed in this experiment.Leaves of wild tobacco were transformed by Agrobacterium tumefacien and selected by kanamycin.T1 generation of transgenic tobacco were stress treated by 2%NaCl,4℃,and expressed by 20% PEG,RT-PCR analysis.【Result】(1)GhNCED2 gene had been inserted into the genome of tobacco and stably expressed in the T1 generation.(2) The variation of expression of GhNCED2 gene was almost identical in different stress treatments.2% NaCl,20% PEG stress for GhNCED2 gene expression relative to the peak arrival time came later than 4℃ stress.Under the stress of 20% PEG,the relative expression of GhNCED2 gene was higher than other treatments.【Conclusion】 Drought,salinity and low temperature stress were all capable of inducing the expression of GhNCED2 gene.And this genetic response to drought stress was more intense and rapid to chilling injury.