利用酵母建立植物抗逆基因快速筛选体系  

Plant stress-resistant gene cloning for Na and Cd using an INVSc1 yeast

作　　者：宋红改[1,2] 蒋晶[2] 乔桂荣[2] 杨晔[1,2] 周婧[2] 潘銮银[2] 卓仁英[2] 

机构地区：[1]三峡大学生物技术研究中心湖北省天然产物研究与利用重点实验室,湖北宜昌443002 [2]中国林业科学研究院亚热带林业研究所,浙江富阳311400

出　　处：《浙江林学院学报》2010年第6期890-895,共6页Journal of Zhejiang Forestry College

基　　金：国家自然科学基金资助项目(30972340);浙江省自然科学基金杰出青年团队项目(R3090070)

摘　　要：以旱柳Salix matsudana及东南景天Sedum alfredii为实验材料,利用改进的SMART(switching mechanism at 5’end of RNA transcript)技术分别构建了旱柳盐胁迫和东南景天氯化镉胁迫后的全长cDNA文库,把文库包装到酵母表达载体中,转化酿酒酵母感受态细胞,通过在尿嘧啶缺陷型培养基上添加不同浓度梯度的筛选压来筛选酵母高抗转化子,并对其基因进行鉴定。结果从旱柳盐胁迫全长cDNA文库中得到了2个能耐受1.709mol·L-1氯化钠的阳性转化子,从东南景天氯化镉胁迫全长cDNA文库中得到了2个能耐受0.123mmol·L-1氯化镉的阳性转化子。分别测定对照菌(转空载体重组菌INV/pYES2.1)和转cDNA重组菌在无胁迫、高盐(1mol·L-1氯化钠)和高氯化镉(150μmol·L-1)胁迫下的生长曲线。结果表明高耐盐/耐镉相关基因的表达对酵母正常生长没有影响。在含高盐或者高镉的培养基里,2个转基因重组菌的延滞期短于对照菌。这表明高耐盐/耐镉相关基因的表达可以提高酵母细胞的耐盐/耐镉能力。对6号耐盐转化子序列进行生物信息学和表达模式分析,发现该基因为一盐胁迫应答相关基因SMpla6(暂定名)。用酿酒酵母建立了一个高效快速筛选植物抗逆基因的体系,研究结果为快速克隆植物抗逆相关基因奠定基础。To provide a basis for studying a new stress-related gene of Salix matsudana and Sedum alfredii,the salt-stress induced full-length cDNA Library of Salix matsudana and the cadmium-stress induced,fulllength cDNA Library of Sedum alfredii were constructed using an improved switching mechanism at 5 end of RNA transcript （SMART） method.The full-length cDNA fragment was linked to the yeast vector pYES2.1,and then the linked product was transferred onto the yeast INVSc1.Concentrations of NaCl and cadmium stress were utilized to construct yeast transformants library.Growth performance of yeast recombinants and their control with an empty vector were tested under the control （0）,high salinity （1 mol·L-1 NaCl）,or high cadmium（150 μmol·L-1 cadmium） conditions.Also,a bioinformatics analysis and real-time polymerase chain reaction （PCR） were conducted.Results for the yeast transformants library revealed two positive transformants with a tolerance of 1.709 mol·L-1 NaCl and two positive transformants with a tolerance of 0.123 mmol·L-1 cadmium.Growth rates of the recombinants with cDNA protein under the non-stress condition indicated that the expression of cDNA was not deleterious to yeast growth.Compared to the control,yeast cells expressing cDNA showed a shorter lag period when transferred to a medium containing high salinity and high cadmium.The results of a bioinformatics analysis and real-time PCR showed that the No.6 salt-tolerant transformant gene was a salt-resistant related gene.This study showed that the expression of cDNA could confer salt and cadmium tolerance in yeast and provided an important tool for establishing an efficient system of stress-resistant gene cloning using the yeast INVSc1.

关 键 词：林木育种学 旱柳 东南景天 CDNA文库 酵母 耐盐性 耐镉性 生长曲线 

分 类 号：S722.3[农业科学—林木遗传育种]