盐胁迫应答基因GmWRKY6的克隆及转基因百脉根的抗盐分析

doi:10.11686/cyxb2017522

摘要/Abstract

摘要： 根据大豆盐胁迫RNA-Seq数据,筛选并克隆得到1个大豆WRKY类转录因子GmWRKY6基因。序列分析表明,该基因含有1 个1674 bp的开放阅读框,编码557 个AA,编码蛋白属于IIb 类WRKY 家族成员,含有1个WRKY 保守结构域和1个C₂H₂锌指结构基序。基因表达模式分析表明,GmWRKY6基因在大豆根中表达量较高,并且该基因受盐胁迫诱导表达。构建GmWRKY6基因的植物超表达载体,获得7株T₀代阳性转GmWRKY6基因百脉根。进一步对T₁代转基因株系进行抗盐分析发现,在200 mmol·L^-1 NaCl 处理14 d后转基因株系生长状态良好,而野生型对照生长矮小,叶片干枯。此外,对相关生理指标的测定发现,转基因株系叶片中脯氨酸和叶绿素含量显著高于野生型对照,而丙二醛含量和相对质膜透性显著低于野生型对照。此外,盐胁迫下转基因百脉根叶片中钠离子含量显著低于对照,而钾离子含量显著高于对照。以上结果表明大豆GmWRKY6基因的超表达能够显著增强百脉根的抗盐能力。

关键词: 大豆, WRKY转录因子, 子叶节转化, 百脉根, 耐盐性

Abstract: A GmWRKY6 gene from WRKY transcription factor family members from soybean was cloned from salt stress RNA-Seq data. Sequence analysis showed that the gene contained an open reading frame (ORF) of 1674 bp and 557 encoding amino acids. The GmWRKY6 protein contained a conserved WRKY domain and a C2H2 zinc finger motif, belonged to group IIb WRKY transcription factor. Analysis of gene expression pattern showed that the expression level of GmWRKY6 gene is highest in the root and was induced by salt stress. Plant overexpression vector of the GmWRKY6 gene was constructed and 7 positive T₀ transgenic Lotus japonicus plants were obtained. The salt resistance of T₁ transgenic lines was further analyzed, after exposure to 200 mmol·L^-1 NaCl for 14 days; transgenic lines grew well while the wild type were stunted with dry foliage. Proline and chlorophyll content in transgenic lines were significantly higher than wild plants, while the MDA content and the relative permeability of plasma membrane were significantly lower than that of wild plants. In addition, the sodium ion content in transgenic lines was significantly lower than wild plants under salt stress, while the potassium content was significantly higher than that of wild plants. These results showed that over expression of the soybean GmWRKY6 gene can significantly enhance the salt resistance of L. japonicus.

Key words: soybean, WRKY transcription factor, cotyledonary node transformation, Lotus japonicus, salt tolerance

柯丹霞, 彭昆鹏, 夏远君, 朱玉莹, 张丹丹. 盐胁迫应答基因GmWRKY6的克隆及转基因百脉根的抗盐分析[J]. 草业学报, 2018, 27(8): 95-106.

KE Dan-xia, PENG Kun-peng, XIA Yuan-jun, ZHU Yu-ying, ZHANG Dan-dan. Cloning of salt-stressed responsive gene GmWRKY6 and salt resistance analysis of transgenic Lotus japonicus[J]. Acta Prataculturae Sinica, 2018, 27(8): 95-106.

参考文献

[1] Ulker B, Somssich I E. WRKY transcription factor: from DNA binding towards biological function. Current Opinion in Plant Biology, 2004, 7(5): 491-498.
[2] Jue D W, Sang X L, Shu B, et al . Expression analysis of maize WRKY transcription factor genes under abiotic stress. Guangdong Agricultural Sciences, 2017, 44(1): 15-22.
决登伟, 桑雪莲, 舒波, 等. 玉米WRKY转录因子非生物胁迫的表达分析. 广东农业科学, 2017, 44(1): 15-22.
[3] Jue D W, Sang X L, Shu B, et al . Functional analysis for maize WRKY gene. Journal of Southern Agriculture, 2017, 48(6): 945-951.
决登伟, 桑雪莲, 舒波, 等. 玉米WRKY基因功能分析. 南方农业学报, 2017, 48(6): 945-951.
[4] Jue D W, Sang X L, Shu B, et al . Expression of maize ZmWRKY 53 -like and ZmWRKY 67 -like gene under abiotic stresses. Guizhou Agricultural Sciences, 2017, 45(6): 15-20.
决登伟, 桑雪莲, 舒波, 等. 玉米 ZmWRKY 53 -like 与 ZmWRKY 67 -like 基因在非生物胁迫下的表达. 贵州农业科学, 2017, 45(6): 15-20.
[5] Lü Q X, Gao S, He H, et al . Expression analysis of the maize ZmWRKY 45 gene under the saline-alkali adversity stress. Journal of Maize Sciences, 2017, 25(3): 28-32.
吕庆雪, 高嵩, 何欢, 等. 玉米 ZmWRKY 45基因克隆及盐碱逆境胁迫下的表达分析. 玉米科学, 2017, 25(3): 28-32.
[6] Sun X C, Gao Y F, Li H R, et al . Over-expression of SlWRKY 39 leads to enhanced resistance to multiple stress factors in tomato. Journal of Plant Biology, 2015, 58(1): 52-60.
[7] Su Y, Zhen J B, Zhang X, et al . Cloning and analysis of a salt stress responsive gene GhWRKY 41 in upland cotton ( Gossypium hirsutum L.). Journal of China Agricultural University, 2016, 21(12): 1-11.
苏莹, 甄军波, 张曦, 等. 陆地棉盐胁迫应答基因 GhWRKY 41的克隆与分析. 中国农业大学学报, 2016, 21(12): 1-11.
[8] Shi W N, Hao L L, Li J, et al . The Gossypium hirsutum WRKY gene GhWRKY 39-1 promotes pathogen infection defense responses and mediates salt stress tolerance in transgenic Nicoti-ana benthamiana. Plant Cell Reports, 2014, 33(3): 483-498.
[9] Dou L L, Li G L, Pang C Y, et al . Cloning and function analysis of GhWRKY 11 in cotton ( Gossypium hirsutum ). Journal of Agricultural Biotechnology, 2016, 24(5): 625-636.
窦玲玲, 李光雷, 庞朝友, 等. 棉花转录因子 GhWRKY 11的克隆及功能分析. 农业生物技术学报, 2016, 24(5): 625-636.
[10] Chen X L, Yan D, Sun L N, et al . Cloning and expression analysis of HbWRKY 75 gene in leaf from Hevea brasiliensis . Plant Physiology Communications, 2016, 52(3): 250-258.
陈晓丽, 闫栋, 孙丽娜, 等. 橡胶树WRKY家族转录因子 HbWRKY 75基因的克隆及表达分析. 植物生理学报, 2016, 52(3): 250-258.
[11] Yao H R, Zeng B S, Fan C J, et al . Cloning and expression analysis of EgrWRKY 70 in Eucalyptus grandis . Chinese Journal of Tropical Crops, 2016, 37(7): 1341-1348.
姚海荣, 曾炳山, 范春节, 等. 巨桉 EgrWRKY 70基因克隆和初步表达分析. 热带作物学报, 2016, 37(7): 1341-1348.
[12] Wei X C, Yao Q J, Yuan Y X, et al . Cloning and expression analysis of CaWRKY 13 gene from Capsicum annuum L. under abiotic stress. Molecular Plant Breeding, 2016, 14(10): 2582-2588.
魏小春, 姚秋菊, 原玉香, 等. 辣椒 CaWRKY 13基因克隆及非生物胁迫下表达分析. 分子植物育种, 2016, 14(10): 2582-2588.
[13] Guo J H, Wang W D, Gu X, et al . Cloning and expression analysis of WRKY transcription factor gene CsWRKY 57 in tea plant ( Camellia sinensis ). Journal of Tea Science, 2017, 37(4): 411-419.
郭俊红, 王伟东, 谷星, 等. 茶树WRKY转录因子基因 CsWRKY 57的克隆及表达分析. 茶叶科学, 2017, 37(4): 411-419.
[14] Tang L L. Transformation of GsZFP 1 and GsWRKY 20 genes into Medicago sativa L. and salt and drought tolerance analysis in transgenic plants. Harbin: Northeast Agricultural University, 2013.
唐立郦. GsZFP 1和 GsWRKY 20基因转化苜蓿及耐盐耐旱性分析. 哈尔滨: 东北农业大学, 2013.
[15] Zhu P H, Chen R R, Yu Y, et al . Cloning of gene GsWRKY 15 related to alkaline stress and alkaline tolerance of transgenic plants. Acta Agronomica Sinica, 2017, 43(9): 1319-1327.
朱娉慧, 陈冉冉, 于洋, 等. 碱胁迫相关基因 GsWRKY 15的克隆及其转基因苜蓿的耐碱性分析. 作物学报, 2017, 43(9): 1319-1327.
[16] Ke D X, Li X Y, Han Y P, et al . ROP6 is involved in root hair deformation induced by Nod factors in Lotus japonicus . Plant Physiology and Biochemistry, 2016, 108: 488-498.
[17] Ke D X, Fang Q, Chen C F, et al . The small GTPase ROP6 interacts with NFR5 and is involved in nodule formation in Lotus japonicus . Plant Physiology, 2012, 159(1): 131-143.
[18] Ke D X, Zhang W, Li X Y, et al . Polyclonal antibody preparation and application of Lotus japonicus Rac1 protein. Acta Botanica Boreali-Occidentalia Sinica, 2016, 36(4): 655-660.
柯丹霞, 张伟, 李祥永, 等. 百脉根Rac1蛋白多克隆抗体的制备与应用. 西北植物学报, 2016, 36(4): 655-660.
[19] Ke D X, Li X Y, Wang J J, et al . Functions of small GTPase Rac 1 in nodulation process in Lotus japonicus . Acta Botanica Boreali-Occidentalia Sinica, 2015, 35(12): 2365-2372.
柯丹霞, 李祥永, 王静静, 等. 百脉根小G蛋白 Rac 1基因的克隆与功能分析. 西北植物学报, 2015, 35(12): 2365-2372.
[20] Ke D X, Li X Y, Peng K P, et al . Cloning and expression analysis of the promoter region of Rac 1 gene of Lotus japonicus . Journal of Xinyang Normal University (Natural Science Edition), 2018, (1): 1-5.
柯丹霞, 李祥永, 彭昆鹏, 等. 百脉根 Rac 1基因启动子的克隆与表达分析. 信阳师范学院学报(自然科学版), 2018, (1): 1-5.
[21] Ke D X, Li X Y. Research progress of key regulatory proteins in nodulation pathway. Journal of Xinyang Normal University (Natural Science Edition), 2015, (4): 621-626.
柯丹霞, 李祥永. 结瘤信号途径中相关调控蛋白的研究进展. 信阳师范学院学报, 2015, (4): 621-626.
[22] Ke D X, Li X Y, Wang L, et al . Isolation of GmHAT 5 from Glycine max and analysis of saline tolerance for transgenic Lotus japonicus . Scientia Agricultura Sinica, 2017, 50(9): 1559-1570.
柯丹霞, 李祥永, 王磊, 等. 大豆 GmHAT 5的克隆及其转基因百脉根的抗盐分析. 中国农业科学, 2017, 50(9): 1559-1570.
[23] Ke D X, Xiong W Z, Peng K P, et al . Study on genetic transformation of salt resistant gene Gm 01 g 04890 in soybean. Journal of Xinyang Normal University (Natural Science Edition), 2017, (1): 46-51.
柯丹霞, 熊文真, 彭昆鹏, 等. 抗盐基因 Gm 01 g 04890大豆子叶节遗传转化研究. 信阳师范学院学报(自然科学版), 2017, (1): 46-51.
[24] Song H, Wang P F, Hou L, et al . Global analysis of WRKY genes and their response to dehydration and salt stress in soybean. Frontiers in Plant Science, 2016, 7: 9.
[25] Yu Y C, Wang N, Hu R B, et al . Genome-wide identification of soybean WRKY transcription factors in response to salt stress. Springer Plus, 2016, 5: 920.
[26] Marquez A J. Lotus japonicus handbook. The Netherlands: Springer, 2005: 279-284.
[27] Ishiguro S, Nakamura K. Characterization of a cDNA encoding a novel DNA-binding protein, SPF1, that recognizes SP8 sequences in the 5' upstream regions of genes coding for sporam in and β-amylase from sweet potato. Molecular and General Genetics MGG, 1994, 244(6): 563-571.
[28] Rushton P J, Somssich I E, Ringler P, et al . WRKY transcription factors. Trends in Plant Science, 2010, 15(5): 247-258.
[29] Eulgem T, Somssich I E. Networks of WRKY transcription factors in defense signaling. Current Opinion in Plant Biology, 2007, 10: 366-371.
[30] Pandey S P, Somssich I E. The role of WRKY transcription factors in plant immunity. Plant Physiology, 2009, 150(4): 1648-1655.
[31] Eulgem T, Rushton P J, Robatzek S, et al . The WRKY superfamily of plant transcription factors. Trends in Plant Science, 2000, 5(5): 199-206.
[32] Zhou Q Y, Tian A G, Zou H F, et al . Soybean WRKY-type transcription factor genes, GmWRKY 13, GmWRKY 21, and GmWRKY 54, confer differential tolerance to abiotic stresses in transgenic Arabidopsis plants. Plant Biotechnology Journal, 2008, 6(5): 486-503.
[33] Wang T T, Cong Y H, Liu J G, et al . Cloning and functional analysis of a WRKY 28 -like gene in soybean. Acta Agronomica Sinica, 2016, 42(4): 469-481.
王婷婷, 丛亚辉, 柳聚阁, 等. 大豆中一个 WRKY 28 -like 基因的克隆与功能分析. 作物学报, 2016, 42(4): 469-481.
[34] Li D H, Wang C H, Liu X P, et al . Expression of GmWRKY 35, a soybean WRKY gene, in transgenic tobacco confers drought stress tolerances. Soybean Science, 2017, 36(5): 685-691.
李大红, 王春弘, 刘喜平, 等. 大豆 GmWRKY 35基因的克隆及其增强烟草耐旱能力研究. 大豆科学, 2017, 36(5): 685-691.