| 摘要: |
| 【目的】从野生型拟南芥中克隆抗寒基因ICE1，通过农杆菌介导法将ICE1基因转到烟草中，对其功能进行研究。【方法】以三叶一心期拟南芥幼苗为材料，提取其总RNA，通过RT PCR扩增得到ICE1基因的完整开放阅读框序列；构建以抗除草剂Bar基因为筛选标记的植物表达载体pCAMBIA3301-ICE1，通过农杆菌介导法转入到烟草中，对阳性植株进行PCR检测和生理生化指标测定，并对ICE1基因在转基因烟草植株中的表达特征进行分析。【结果】成功克隆了拟南芥抗寒基因ICE1，其完整开放阅读框长1 485 bp。系统进化树表明，拟南芥ICE1基因与其他物种的ICE1基因差别较大。用农杆菌介导法得到４株阳性烟草植株，Southern blotting检测证明，ICE1基因已经以单拷贝的形式整合到烟草基因组中。实时荧光定量PCR检测表明，ICE1基因在转基因烟草植株的根、茎和叶片中均有表达，且在叶片中的相对表达量最高。4 ℃低温环境下，与未转化植株相比，转ICE1基因烟草植株叶片的相对电导率降低了12.00%~17.65%，脯氨酸含量增加了13.28%~24.10%，丙二醛含量减少了7.09%~14.52%，过氧化物酶活性提高了9.39%~22.54%。【结论】克隆获得了拟南芥ICE1基因的完整开放阅读框序列，在烟草中转入ICE1基因可提高其耐寒性。 |
| 关键词: ICE1基因 植物表达载体 耐寒性 |
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| 基金项目:吉林省省级粮食生产发展专项资金项目（2015001）；吉林农业大学启动基金项目（201242） |
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| Cloning and functional analysis of Arabidopsis thaliana cold resistance gene ICE1 |
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LIU Shuang,CHEN Zhaoting,DONG Zhaoxu,et al
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| Abstract: |
| 【Objective】The cold resistance gene ICE1 from wild type Arabidopsis thaliana was cloned and transferred to tobacco by Agrobacterium mediated method for function analysis.【Method】The Arabidopsis thaliana seedlings were used to extract the total RNA,and the ICE1 gene was amplified by RT-PCR for the development of the reading frame sequence.The recombinant plant expression vector pCAMBIA3301- ICE1 was constructed and transferred into tobacco through Agrobacterium mediated transformation.PCR detection,physiological and biochemical indexes were used to detect the expression characteristics of ICE1 gene in transgenic tobacco plants.【Result】ICE1 was cloned from Arabidopsis thaliana successfully and the length of its complete open reading frame was 1 485 bp.Phylogenetic tree showed that the ICE1 gene of Arabidopsis thaliana was different from other species.A total of 4 positive plants were obtained by Agrobacterium mediated method and the gene was integrated into the tobacco genome in single copy way by Southern blotting.The mRNA of ICE1 existed in root,stem and leaf of the transferred tobacco based on real time fluorescent quantitative PCR assay (qRT-PCR).The expression of ICE1 in leaves was the most abundant.Compared with the non-transformed plants under low temperature environment (4 ℃),and the relative conductivity of transgenic tobacco was reduced by 12.00%-17.65%.The proline content was increased by 13.28%-24.10%,MDA content was reduced by 7.09%-14.52%,and peroxidase activity was increased by 9.39%-22.54%.【Conclusion】The ICE1 gene of Arabidopsis thaliana was cloned and the sequence of open reading frame was obtained.The transfer of ICE1 gene can improve the cold resistance of tobacco. |
| Key words: ICE1 gene plant expression vector cold tolerance |
