利用酵母建立植物抗逆基因快速筛选体系

宋红改; 蒋晶; 乔桂荣; 杨晔; 周婧; 潘銮银; 卓仁英

doi:10.11833/j.issn.2095-0756.2010.06.014

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利用酵母建立植物抗逆基因快速筛选体系

宋红改 , 蒋晶 , 乔桂荣 , 杨晔 , 周婧 , 潘銮银 , 卓仁英

文章导航 > 浙江农林大学学报 > 2010 > 27(6): 890-895

宋红改, 蒋晶, 乔桂荣, 杨晔, 周婧, 潘銮银, 卓仁英. 利用酵母建立植物抗逆基因快速筛选体系[J]. 浙江农林大学学报, 2010, 27(6): 890-895. doi: 10.11833/j.issn.2095-0756.2010.06.014

引用本文:

SONG Hong－gai, JIANG Jing, QIAO Gui－rong, YANG Ye, ZHOU Jing, PAN Luan－yin, ZHUO Ren－ying. Plant stress－resistant gene cloning for Na and Cd using an INVSc1 yeast[J]. Journal of Zhejiang A&F University, 2010, 27(6): 890-895. doi: 10.11833/j.issn.2095-0756.2010.06.014

Citation:

SONG Hong－gai, JIANG Jing, QIAO Gui－rong, YANG Ye, ZHOU Jing, PAN Luan－yin, ZHUO Ren－ying. Plant stress－resistant gene cloning for Na and Cd using an INVSc1 yeast[J]. Journal of Zhejiang A&F University, 2010, 27(6): 890-895. doi: 10.11833/j.issn.2095-0756.2010.06.014

利用酵母建立植物抗逆基因快速筛选体系

doi: 10.11833/j.issn.2095-0756.2010.06.014

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,

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

详细信息

通信作者: 卓仁英

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

1.
Biotechnology Research Center/Hubei Province Key Laboratory of Natural Products Research and Development， China Three Gorges University，Yichang 443002，Hubei，China;
2.
The Research Institute of Subtropical Forestry，The Chinese Academy of Forestry，Fuyang 311400，Zhejiang，China

More Information

Corresponding author: ZHUO Ren－ying

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 5end 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1.709 molL－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0.123 mmolL－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（1 molL－1氯化钠）和高氯化镉（150 molL－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。图9参12
- 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
Abstract: 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，full－length 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 molL-1 NaCl），or high cadmium（150 molL-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 molL-1 NaCl and two positive transformants with a tolerance of 0.123 mmolL-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.［Ch，9 fig. 12 ref.］
- forest tree breeding /
- Salix matsudana /
- Sedum alfredii /
- cDNA library /
- yeast /
- salt tolerance /
- cadmium tolerance /
- grouth curves

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利用酵母建立植物抗逆基因快速筛选体系

doi: 10.11833/j.issn.2095-0756.2010.06.014

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

通信作者: 卓仁英

刊出日期: 2010-12-20

关键词:

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 5end 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1.709 molL－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0.123 mmolL－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（1 molL－1氯化钠）和高氯化镉（150 molL－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。图9参12

English Abstract

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

1. Biotechnology Research Center/Hubei Province Key Laboratory of Natural Products Research and Development， China Three Gorges University，Yichang 443002，Hubei，China;

2. The Research Institute of Subtropical Forestry，The Chinese Academy of Forestry，Fuyang 311400，Zhejiang，China

Corresponding author: ZHUO Ren－ying

Publish Date: 2010-12-20

Keywords:

Abstract: 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，full－length 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 molL-1 NaCl），or high cadmium（150 molL-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 molL-1 NaCl and two positive transformants with a tolerance of 0.123 mmolL-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.［Ch，9 fig. 12 ref.］

引用本文: