过量表达棉花GhSAP1提高转基因烟草的耐盐性

doi:10.3864/j.issn.0578-1752.2014.08.002

摘要/Abstract

摘要： 【目的】SAP（stress-associated protein）是一类涉及胁迫应答和调控的锌指蛋白。克隆并分析其对逆境胁迫应答的反应，为棉花抗逆育种提供候选基因。【方法】通过电子克隆方法并经RT-PCR验证获得棉花锌指蛋白基因GhSAP1，将带有目的基因的载体质粒通过PEG介导转化棉花叶肉原生质体细胞，瞬时表达分析其编码蛋白的特性及亚细胞定位信息。通过qRT-PCR技术分析目标基因的组织表达特征及非生物胁迫条件下的表达特性。通过农杆菌介导叶盘转化法，经组织培养获得转基因烟草进行异位表达，检测其与抗逆相关的生理指标，证明其提高植物抗逆能力。【结果】GhSAP1 ORF长度为543 bp，编码一条含180个氨基酸残基的多肽。其理论等电点8.97，分子量19.3 kD，具有典型的A20/AN1锌指结构域。亚细胞定位显示该基因编码的蛋白定位于细胞核上。不同组织器官表达分析显示，GhSAP1在根、茎、叶中的表达量高，而在开花后5 d的胚珠中表达量很低。GhSAP1受盐胁迫诱导，高盐处理2 h后表达量最高。利用含100 μg•mL-1 Kan的培养基进行抗性筛选转基因后代，结合目标基因的PCR与RT-PCR验证，获得转GhSAP1的转基因烟草阳性株系10个。目标基因GhSAP1均能在烟草基因组中整合并异位过量表达。随机选择3个转基因烟草株系，盐胁迫处理显示，发芽一周的种子在含200 mmol•L-1 NaCl培养基上胁迫12 d，转基因植株幼苗平均成活率为81.7%，比非转基因对照植株增加近3倍，其平均根长为3.05 cm，极显著高于非转基因对照。利用GhSAP1表达较高的L2纯系进行成株期烟草盐胁迫处理30 d，转基因烟草后代的SOD活性和可溶性总糖含量增幅分别为23.89 U•g-1 FW和8.37 %，均显著高于非转基因对照；与未胁迫处理相比，转基因植株的叶绿素含量降低幅度仅为0.17 mg•g-1 FW，而非转基因对照降低了0.39 mg•g-1 FW；盐胁迫处理后，转基因植株的MDA含量增幅为7.42 nmol•g-1FW，而非转基因对照增幅达20.85 nmol•g-1FW；在盐胁迫下，转基因植株具有更强的K+根部运输到植株绿色组织能力，其地上部的K+/Na+为1.23，显著高于非转基因对照。【结论】GhSAP1在参与植物的逆境应答反应机制中具有重要作用，过量表达GhSAP1可显著提高转基因烟草的耐盐能力。

关键词: 棉花 , GhSAP1 , 超表达 , 转基因烟草 , 盐胁迫

Abstract: 【Objective】Stress associated proteins (SAPs) is a type of novel stress response-inducing zinc finger proteins with A20/AN1 zinc-?nger domain. The objectives of this study are to isolate a member of stress associated protein (SAP) gene family, analyze its responses to abiotic stress, and provide a potential candidate gene for improving abiotic stress tolerance in cotton by biotechnological approaches. 【Method】A cotton zinc protein gene GhSAP1 was isolated based on in silico cloning and confirmed by RT-PCR analysis, further, recombinant plasmid vector with GhSAP1 was transformed into purified cotton mesophyll protoplast mediated with PEG for elucidating the characteristics of encoded proteins and subcellular location by a transient expression system. The expression patterns of GhSAP1 in different tissues and under salt stress were analyzed through real-time RT-PCR. By leaf disc method, Agrobacterium tumefaciens-mediated transformation, and tissue culture, different transgenic tobacco lines with high ectopic overexpression of GhSAP1 were obtained, further, their determination of physiologic index related to abiotic stress was finished and the potential of improving abiotic stress tolerance in plant was elucidated. 【Result】GhSAP1 contained ORF length of 543 bp and encoded a polypeptide containing 180 amino acids with typical A20/AN1 zinc finger structural domain. The theoretical iso-electric point was 8.97 with a calculated molecular weight of 19.3 kD. Subcellular location showed that GhSAP1 was localized in cell nucleus. The expression patterns in different tissues and organs showed that GhSAP1 expressed preferentially in root, stem, and leaf tissues, but the expression level was the lowest in young ovules at five days post anthesis. GhSAP1 was accumulated significantly when induced after 2 h under salt stress treatment. Ectopic overexpression of GhSAP1 in tobacco plants was performed via leaf disc transformation mediated by Agrobacterium tumefaciens, and was confirmed in both genomic and transcriptional level by PCR and RT-PCR in transgenic tobacco seedlings. Ten tobacco transgenic lines were preliminarily obtained through resistance selection with 100 μg•mL-1 Kan culture medium for transgenic progeny, and confirmed by PCR and RT-PCR of GhSAP1. The result showed that GhSAP1 was integrated into tobacco genome validity with high ectopic overexpression. Further, three transgenic tobacco positive lines were selected by random and analyzed under salt stress. Treating the seedlings of germination for seven days using 200 mmol•L-1 NaCl, 12 days later, the average survival rate of transgenic tobacco lines was 81.7%, which was 3 times higher than those of wild-type plants, and the relative root length in transgenic plants was 3.05 cm in average, significantly higher than wild type plants. Further, L2 pure lines with higher GhSAP1 expression was selected to elucidate physiologic index related to abiotic stress. In detail, when treating the plants with 9 to 11 leaves using 200 mmol•L-1 NaCl, 30 days later, the transgenic lines overexpressing GhSAP1 showed better SOD activity and increased total soluble sugar content, with net increase value of 23.89 U•g-1 FW and 8.37 %, respectively, which was significantly higher than non-transgenic plants. Compared with non-stress treatment, the reduction of the chlorophyll content of transgenic plants was only 0.17 mg•g-1 FW, in contrast to the 0.39 mg•g-1 FW of the non-transgenic plants. After treatment under salt stress condition, the MDA content of transgenic plants only increased by 7.42 nmol•g-1 FW, however, the MAD content of the non-transgenic plants increased by 20.85 nmol•g-1 FW. Meanwhile, the transgenic plants had the better transport ability of K+ from plant root to green tissue, the K+/ Na+ ratio of above-ground part was 1.23, with higher transport ability compared with wild type plants.【Conclusion】It was concluded that GhSAP1 plays an important role in response to salinity stress in plants, overexpression of GhSAP1 can improve significantly tolerance to salt stress in transgenic tobacco plants.

Key words: cotton , GhSAP1 , overexpression , transgenic tobacco , salt stress

丁林云, 张微, 王晋成, 田亮亮, 李妮娜, 郭琪, 杨淑明, 何曼林, 郭旺珍. 过量表达棉花GhSAP1提高转基因烟草的耐盐性[J]. 中国农业科学, 2014, 47(8): 1458-1470.

DING Lin-Yun, ZHANG Wei, WANG Jin-Cheng, TIAN Liang-Liang, LI Ni-Na, GUO Qi, YANG Shu-Ming, HE Man-Lin, GUO Wang-Zhen. Overexpression of a Gossypium hirsutum Stress-Associated Protein Gene (GhSAP1) Improves Salt Stress Tolerance in Transgenic Tobacco[J]. Scientia Agricultura Sinica, 2014, 47(8): 1458-1470.

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