过量表达棉花CBF2基因提高转基因拟南芥抗旱耐盐能力

Overexpression of the Cotton CBF2 Gene Enhances Salt and Drought Tolerance in Arabidopsis thaliana

CBF/DREB是一类植物中特有的转录因子,在植物抵抗逆境胁迫过程中发挥重要功能。本研究从陆地棉(Gossypium hirsutum L.)Coker 312中克隆获得1个棉花CBF/DREB基因,命名为Gh CBF2,该基因编码一个由216个氨基酸组成的CBF蛋白。序列分析结果显示,Gh CBF2与其他植物的CBF蛋白类似,含有AP2转录因子典型的保守结构域。干旱或高盐胁迫处理明显增加了Gh CBF2基因的表达量。亚细胞定位分析结果发现Gh CBF2定位在细胞核中。将Gh CBF2基因构建到由35S启动子调控的植物表达载体p MD上并转化拟南芥(Arabidopsis thaliana L.),结果表明,在干旱和盐胁迫条件下,过量表达Gh CBF2基因拟南芥的成活率显著高于野生型,并且游离脯氨酸和可溶性糖含量也高于野生型,说明转Gh CBF2基因提高了拟南芥的耐盐抗旱能力。采用实时荧光定量PCR方法分析胁迫相关标记基因COR15A、RD29A和ERD6的表达情况,结果显示转基因株系中的表达量显著高于野生型,说明Gh CBF2参与调控拟南芥干旱和盐胁迫相关基因的表达。

CBF / DREB proteins are transcription factors in plants,which play important biological functions in cold,drought,and salt stress resistance. In this study,a CBF / DREB gene,Gh CBF2,was cloned from Gossypium hirsutum L.,coding a protein consisting of 216 amino acids. Sequence analysis indicated that Gh CBF2 contained a typical AP2 conservative domain structure,much like the CBF proteins in other plant species. The transcript of the Gh CBF2 gene in cotton seedlings was upregulated following exposure to drought and salt stress.Subcellular localization of proteins showed that the Gh CBF2-GFP fusion protein was localized to the nucleus. To study the function of the Gh CBF2 gene in drought and salt stress resistance,it was inserted into p MD to construct a Gh CBF2-overexpression vector under the control of Ca MV 35 S promoter. The construct was introduced into Arabidopsis thaliana by the floral dip method. Analysis of resistance to drought and salt stress showed that the survival rate of transgenic A. thaliana plants was improved remarkably compared to that of the wildtype（ WT）plants. The proline and soluble sugar contents in the transgenic plants were also higher than those in the WT plants. These results show that Gh CBF2 can enhance transgenic plant tolerance to drought and salt stress. We selected several stress related genes,including COR15 A,RD29A,and ERD6,and examined their expression by quantitative RT-PCR in both transgenic and WT plants. Remarkably,the expressions of these marker genes in Gh CBF2-overexpressed transgenic plants were substantially higher than those in WT plants,indicating that the Gh CBF2 gene is involved in the regulation of drought and salt related genes.