生菜低温胁迫转录因子LsICE1的克隆、特征分析及其转化水稻

Cloning and Characteristic Analysis of Cold Stress Transcription Factor LsICE1 from Lettuce and Transformation into Rice

【目的】克隆生菜(Lactuca sativa L.)低温胁迫转录因子LsICE1,对其进行序列分析和水稻遗传转化,研究超表达LsICE1基因对水稻耐低温能力的影响。【方法】设计简并引物,利用RT-PCR技术获得生菜LsICE1基因保守区域,再通过SON-PCR技术获得LsICE1基因的5′端和3′端,拼接得到全长的cDNA。对该序列进行生物信息学分析,采用半定量RT-PCR研究LsICE1的低温表达模式。最后构建植物表达载体,利用农杆菌介导法对水稻进行遗传转化。通过比较低温处理后对照和转基因株系的存活率和生理指标,鉴定超表达LsICE1基因对水稻耐低温能力的调控作用。【结果】测序结果显示,拼接后的cDNA片段长1 622 bp,包含一个1 497 bp完整的开放阅读框,编码498个氨基酸残基,命名为LsICE1,GenBank登录号为HQ848932。半定量RT-PCR研究表明,LsICE1基因是冷诱导条件下差异表达的基因。进化树分析表明,LsICE1蛋白与葡萄的ICE1蛋白亲缘关系最近,处于同一进化分枝。PCR和RT-PCR分子检测证明,LsICE1基因已经整合到水稻基因组中。与对照相比,低温处理后超表达LsICE1基因的转基因株系存活率和脯氨酸含量明显增加,相对电导率和丙二醛含量积累速率明显下降。【结论】首次从生菜中克隆了低温胁迫转录因子LsICE1,超表达LsICE1基因水稻株系提高了抗低温胁迫能力。

【Objective】 Cloning,sequence analysis and transformation into rice with cold stress transcription factor LsICE1 from lettuce were conducted to elucidate the effects of overexpression of LsICE1 on low temperature stress tolerant capacities in rice.【Method】The highly conserved cDNA region of LsICE1 gene from Lactuca sativa L.was isolated by reverse transcription-PCR（RT-PCR） with a pair of degenerate primers,3′-end and 5′-end were amplified by single oligonucleotide nested PCR（SON-PCR） and then the three cDNA segments were combined and constituted a full-length cDNA sequence of LsICE1 gene.Bioinformatics methods were used to analyze the obtained cDNA sequences and the deduced amino acid sequences.The gene expression levels at 4℃ were analyzed by semi-quantitative RT-PCR（s-qRT-PCR）.With constructed plant expressing vector,LsICE1 gene was successfully transferred into rice by Agrobacterium-mediated transgenic technique.The regulation effects of LsICE1 gene with high expression levels on low temperature stress tolerant capacities were evaluated by the comparisons of survival rate and physiological parameters among the transgenic lines and control（CK） after low temperature treatment.【Result】 Sequence analysis showed that the combined cDNA（designated as LsICE1,GenBank Accession No.HQ848932） fragment length was 1 622 bp containing a full coding region of 1 497 bp encoding 498 amino acid residues.The qRT-PCR result suggested that LsICE1 was differential expression gene under low temperature stress.The homology tree demonstrated that LsICE1 was at the same evolutionary branch with VvICE1.PCR and RT-PCR detection confirmed that LsICE1 gene had been integrated into rice genome.After low temperature stress treatment,compared with control,the transgenic lines with high expression levels of the LsICE1 gene had obvious higher survival rates and proline content.In the meantime,the accumulation rate of relative conductivity and malondialdehyde（MDA） content were much lower.【Conclusion】 The cold stress transcription factor LsICE1 was firstly cloned from leaf of Lactuca sativa L.with cold tolerance.Overexpression of LsICE1 gene improved low temperature stress tolerant capacities in transgenic rice lines.