生菜低温胁迫转录因子LsICE1的克隆、特征分析及其转化水稻

doi:10.3864/j.issn.0578-1752.2011.21.002

Abstract

Abstract: 【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.

Key words: Lactuca sativa L., rice, LsICE1 gene, gene cloning, SON-PCR, sequence analysis

XIANG Dian-Jun, YIN Kui-De, MAN Li-Li, XU Zheng-Jin. Cloning and Characteristic Analysis of Cold Stress Transcription Factor LsICE1 from Lettuce and Transformation into Rice[J].Scientia Agricultura Sinica, 2011, 44(21): 4340-4349.

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

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