珍稀泌盐植物长叶红砂RtSOD基因的克隆及功能分析

doi:10.11686/cyxb2017227

摘要/Abstract

摘要： 长叶红砂是一种强旱生泌盐盐生植物,对盐渍荒漠环境具有极强的适应性。超氧化物歧化酶(superoxide dismutase, SOD)在植物抵御逆境胁迫过程中发挥着重要作用。本研究利用已有长叶红砂转录组数据库中SOD基因的已知序列设计引物,采用PCR方法克隆得到大小为663 bp、编码220个氨基酸的SOD基因的开放阅读框,并将其定名为RtSOD。预测该基因编码蛋白分子量为55.90 kDa,理论等电点5.11。多重序列比对分析结果显示该蛋白属于Cu/Zn SOD家族,与其他植物中SOD蛋白同源性较高。系统进化分析结果显示RtSOD基因与刚毛柽柳的SOD基因亲缘关系较近。实时荧光定量PCR(qRT-PCR)分析结果显示NaCl、4 ℃、PEG、H₂O₂及ABA处理均能诱导该基因表达。构建RtSOD真核表达载体,将其转化到拟南芥中,结果发现:盐、干旱胁迫条件下,转RtSOD基因拟南芥的生长状况明显优于野生型,转基因株系抗氧化酶活性(SOD、POD、CAT)和脯氨酸含量较野生型显著升高,H₂O₂及MDA含量较野生型显著降低。qRT-PCR检测发现转基因拟南芥中响应逆境胁迫相关基因的表达量均显著高于野生型。上述结果说明RtSOD基因的过表达可提高转基因植物的抗逆性,进一步说明RtSOD参与长叶红砂对非生物胁迫的响应,是该植物抗氧化系统中的重要元件。

关键词: 长叶红砂, 超氧化物歧化酶, 非生物胁迫, 转基因拟南芥

Abstract: Reaumuria trigyna, a highly xerophytic halophyte, shows strong adaption to the high saline and hyper-drought environments. Superoxide dismutase (SOD) plays an important role in withstanding these abiotic stressors. In this study, primers were designed using the known sequence of a SOD gene in the transcription database of R. trigyna. The open reading frame of the SOD gene with a size of 663 bp and encoding 220 amino acids was cloned by PCR and identified as RtSOD. The molecular weight of the protein was predicted to be 55.90 kDa and the theoretical isoelectric point was pH 5.11. The results of multiple sequence alignment analysis showed that the protein belongs to the Cu/Zn SOD family and had high homology with the SOD protein of other plants. Real-time fluorescence quantitative PCR analysis showed that NaCl, 4 ℃ temperature, polyethylene glycol, H₂O₂ and abscisic acid could induce the expression of the gene. Construction and transfer into Arabidopsis thaliana of a eukaryotic expression vector enhanced the growth rate of A. thaliana, compared to that of the wild type. In addition, antioxidant enzyme activities (SOD, peroxidase, catalase) and the content of proline were significantly higher than in wild type A. thaliana, and the contents of H₂O₂ and malondialdehyde were significantly lower than those of wild type A. thaliana under salt and drought stress. qRT-PCR analysis indicated that the expressions of stress-related genes in transgenic Arabidopsis were significantly higher than those of the wild type. The above results indicate that overexpression of RtSOD could increase the stress tolerance of transgenic plants, and further demonstrated that the RtSOD gene is involved in the response to the abiotic stress of R. trigyna and is an important component in the plant antioxidant system.

Key words: Reaumuria trigyna, superoxide dismutase, abiotic stress, transgenic Arabidopsis thaliana

索雅飞,杜超,李宁宁,王燕,王迎春. 珍稀泌盐植物长叶红砂RtSOD基因的克隆及功能分析[J]. 草业学报, 2018, 27(4): 98-110.

SUO Ya-fei, DU Chao, LI Ning-ning, WANG Yan, WANG Ying-chun. Cloning and function analysis of RtSOD gene in the rare recretohalophyte Reaumuria trigyna[J]. Acta Prataculturae Sinica, 2018, 27(4): 98-110.

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