巴哈雀稗PnDREB2基因克隆及时序表达图谱构建
|
摘要
脱水反应元件结合蛋白(DREBs)在植物非生物逆境胁迫中可调节下游一系列抗逆基因的表达。为探究巴哈雀稗DREB2基因在逆境胁迫下的功能,本研究采用RNA-Seq结合RT-PCR技术从巴哈雀稗中获得一个DREB2基因CDS区全序列,命名为PnDREB2(GenBank登录号:MH150946)。核苷酸序列分析表明,该基因开放阅读框全长774 bp,编码257个氨基酸,相对分子质量为28.09 kD,理论等电点为5.25;该植物蛋白中具有DREB类基因家族典型的保守域AP2结构域,属于DREB2类转录因子A类中A2亚类的亚型1。进化和聚类分析结果表明,巴哈雀稗PnDREB2基因与高粱、割手密、玉米、谷子及牛鞭草亲缘关系较近,氨基酸序列同源性分别为91.10%、89.50%、88.20%、87.60%和87.60%。RT-qPCR和生物信息学分析表明,PnDREB2基因在茎中表达量最高,幼穗次之,叶最低;表达产物定位于细胞质中,具有26个磷酸化位点,并具有结构和功能的特异性。PnDREB2表达量受干旱(PEG-6000,20%)和高温(40℃)的强烈诱导,同时也受到高盐(NaCl,300 mmol·L~(-1))、低温(4℃)、脱落酸(ABA,100μmol·L~(-1))和赤霉素(GA,100μmol·L~(-1))等非生物胁迫和激素的不同程度诱导,随着处理时间的延长,整体上均呈先增加后降低的趋势。本试验为巴哈雀稗抗逆分子机理研究与抗性增强的转基因材料培育奠定了理论基础。
DREBs play important roles in regulating the expression of downstream genes in response to a variety of abiotic stresses. In order to study the function and mechanism of DREB2 gene in Paspalum notatum and further enrich the database of Paspalum notatum genome, monoclonal technique was used to clone the CDS region of PnDREB2 gene(GenBank No. MH150946) from the genome of Paspalum notatum. The analysis of nucleotide sequence indicated that PnDREB2 gene contained a 774 bp intact open reading frame, which encoded a peptide of 257 amino acids, and the predicted molecular weight and isoelctric point of the polypeptide were 28.09 kD and 5.25, respectively. The amino acid sequence analysis indicated that the predicted protein contained a typical AP2 structural domain, belonging to the subtype 1 of Subgroup A-2 in transcription factors of DREB2. Evolution and clustering analysis revealed that PnDREB2 gene of Paspalum notatum has the closest genetic relationship with Sorghum bicolor, Saccharum spontaneum, Setaria italica, Hemarthria compressa and Zea mays, and the homologies up to 91.10%, 89.50%, 88.2%, 87.6% and 87.6%, respectively. The results of realtime quantitative PCR and bioinformatics analysis suggested that PnDREB2 gene was constitutively expressed in various tissues of Paspalum notatum with higher level in stem, and then in young ear, but lower in leaf. Its expression product was located in cytoplasm, with 26 phosphorylation sites, representing the structural and functional specificity. Meanwhile, the expression of PnDREB2 gene was induced by drought(PEG-6000, 20%) and high temperature(40℃), andalso induced by high-salt(NaCl, 300 mmol·L~(-1)), low temperature(4℃), abscisic acid(ABA, 100 μmol·L~(-1)) and gibberellic acid(GA, 100 μmol·L~(-1)), with the increase of treatment time, the expression trend was first increasing and then decreasing. This work could lay a theoretical foundation for the study of resistance molecular mechanism of Paspalum notatum and for the develepment of genetically modified materials with enhanced stress resistance.
DREBs play important roles in regulating the expression of downstream genes in response to a variety of abiotic stresses. In order to study the function and mechanism of DREB2 gene in Paspalum notatum and further enrich the database of Paspalum notatum genome, monoclonal technique was used to clone the CDS region of PnDREB2 gene(GenBank No. MH150946) from the genome of Paspalum notatum. The analysis of nucleotide sequence indicated that PnDREB2 gene contained a 774 bp intact open reading frame, which encoded a peptide of 257 amino acids, and the predicted molecular weight and isoelctric point of the polypeptide were 28.09 kD and 5.25, respectively. The amino acid sequence analysis indicated that the predicted protein contained a typical AP2 structural domain, belonging to the subtype 1 of Subgroup A-2 in transcription factors of DREB2. Evolution and clustering analysis revealed that PnDREB2 gene of Paspalum notatum has the closest genetic relationship with Sorghum bicolor, Saccharum spontaneum, Setaria italica, Hemarthria compressa and Zea mays, and the homologies up to 91.10%, 89.50%, 88.2%, 87.6% and 87.6%, respectively. The results of realtime quantitative PCR and bioinformatics analysis suggested that PnDREB2 gene was constitutively expressed in various tissues of Paspalum notatum with higher level in stem, and then in young ear, but lower in leaf. Its expression product was located in cytoplasm, with 26 phosphorylation sites, representing the structural and functional specificity. Meanwhile, the expression of PnDREB2 gene was induced by drought(PEG-6000, 20%) and high temperature(40℃), andalso induced by high-salt(NaCl, 300 mmol·L~(-1)), low temperature(4℃), abscisic acid(ABA, 100 μmol·L~(-1)) and gibberellic acid(GA, 100 μmol·L~(-1)), with the increase of treatment time, the expression trend was first increasing and then decreasing. This work could lay a theoretical foundation for the study of resistance molecular mechanism of Paspalum notatum and for the develepment of genetically modified materials with enhanced stress resistance.
引文
[1] 罗涵夫, 宋华伟, 刘天增, 张巨明. 铝胁迫条件下镧对百喜草幼苗根系生长的影响[J]. 草业学报, 2017, 26(9): 208-213
[2] 侯晓龙, 蔡丽平, 韩航, 周垂帆, 马祥庆. 铅胁迫对百喜草叶绿素荧光特性及酶活性的影响[J]. 草业学报, 2017, 26(3): 142-148
[3] 张鹍飞, 牟利, 李鹏, 方建, 刘新春, 冯宗云. 青稞OMT1基因克隆及原核表达分析[J]. 核农学报, 2017, 31(12): 2314-2322
[4] 严莉, 陈建伟, 王翠平, 乔改霞, 李健. 黑果枸杞WD40编码基因LrAN11的克隆及表达分析[J]. 核农学报, 2017, 31(11): 2103-2112
[5] Liu Q, Zhao N M, Yamaguchi-Shinozaki K, Shinozaki K. Regulatory role of DREB transcription factors in plant drought, salt and cold tolerance[J]. Science Bulletin, 2000, 45(11): 970-975
[6] 马兴勇, 彭献军, 苏蔓, 张乐新, 周庆源, 陈双燕, 程丽琴, 刘公社. 羊草DREB转录因子的系统发育和功能研究[J]. 草业学报, 2012, 21(6): 190-197
[7] Liu Q, Kasuga M, Sakuma Y, Abe H, Miura S, Yamagu-chi-Shinozaki K, Shinozaki K. Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA-binding domain separate two cellular signal transduction pathways in drought-and low-temperature-responsive gene expression, respectively, in Arabidopsis[J]. Plant Cell, 1998, 10(5): 1391-1406
[8] 冯军, 郑彩霞. DREB转录因子在植物非生物胁迫中的作用及应用研究[J]. 植物生理学报, 2011, 47(5): 437-442
[9] Shinozaki K, Yamaguchishinozaki K. Gene networks involved in drought stress response and tolerance.[J]. Journal of Experimental Botany, 2007, 58(2): 221-234
[10] Haake Ⅴ, Cook D, Riechmann J L, Pineda O, Thomashow M F, Zhang J Z. Transcription factor CBF4 is a regulator of drought adaptation in Arabidopsis[J]. Plant Physiology, 2002, 130(2): 639-648
[11] Qin F, Sakuma Y, Li J, Liu Q, Li Y Q, Shinozaki K, Yamaguchi K. Cloning and functional analysis of a novel DREB1/CBF tran-scription factor involved in cold-responsive gene expression in Zea mays L.[J]. Plant Cell Physiol, 2014, 45(8): 1042-1052
[12] Kume S, Kobayashi F, Ishibashi M, Ohno R, Nakamura C, Ta-kumi S. Differential and coordinated expression of Cbf and Cor/Lea genes during long-term cold acclimation in two Triticum aestivum cultivars showing distinct levels of freezing tolerance[J]. Genes Gene Syst, 2015, 80(3): 185-197
[13] Dubouzet J G, Sakuma Y, Ito Y, Kasuga M, Dubouzet E G. OsDREB genes in rice, Oryza sativa L., encode transcription activators that function in drought high-salt and cold-responsive gene expression [J]. The Plant Journal, 2003, 33(4): 751-763
[14] 杨凤萍. 利用抗逆调节转录因子(DREB1B和CBF1)基因转化多年生黑麦草提高其抗旱性[D]. 北京: 中国农业大学, 2005: 49-51
[15] Li R, Wei J, Wang H, He J, Sun Z. Development of highly regenerable callus lines and Agrobacterium-mediated transformation of Chinese lawngrass (Zoysia sinica Hance) with a cold inducible transcription factor, CBF1[J]. Plant Cell Tissue & Organ Culture, 2006, 85(3): 297-305
[16] 谢登雷. 高粱转录因子SbDREB2基因的克隆与功能分析[D]. 保定: 河北农业大学, 2013: 45-61
[17] 丁咚, 陈亚娟, 崔进荣, 魏建华, 张玉红, 张杰伟. 毛果杨CBF/DREB1基因家族生物信息学分析[J]. 西南农业学报, 2018, 31(3): 457-461
[18] 史松昌, 张如月, 赵振廷, 杨磊, 葛伟娜. 谷子DREBs亚家族的全基因组学分析[J]. 基因组学与应用生物学, 2018, 37(2): 827-835
[19] 马刘峰, 陈芸, 任羽, 司马义·巴拉提, 李玲. 棉花CBF2基因克隆和超表达CBF2棉花增强抗冷性[J]. 植物生理学报, 2018, 54(2): 255-264
[20] 肖雄. 紫大麦草(Hordeum viloaceum)中DREB类转录因子基因的克隆、原核表达及序列分析[D]. 保定: 河北农业大学, 2012: 31-33
[21] Lata C, Bhutty S, Bahadur R P. Association of an SNP in a novel DREB2-like gene SiDREB2 with stress tolerance in foxtail millet [Setaria italica (L.)][J]. Journal of Experimental Botany, 2011, 62(10):3387-3399
[22] 张志飞, 杨知建, 周倩, 赵志丽. DREB2s转录因子基因的表达调控机制研究进展[J]. 植物科学学报, 2014, 32(3): 297-303
[23] Upadhyay R K, Gupta A, Soni D, Rashmi G, Uday Ⅴ P, Pravendra N, Aniruddha P S. Ectopic expression of a tomato DREB gene affects several ABA processes and influences plant growth and root architecture in an age-dependent manner[J]. Journal of Plant Physiology, 2017, 214(7): 97-107
[24] 苏世超, 唐益苗, 徐磊, 王伟伟, 高世庆, 马锦绣, 孙辉, 王永波, 乔亚科, 赵昌平. 普通小麦TaSEC14p-5基因的克隆及表达分析[J]. 农业生物技术学报, 2016, 24(8): 1129-1137
[25] 肖瑞霞, 王新国, 夏国军, 李永春, 牛洪斌, 王翔, 尹钧, 任江萍. 小麦逆境胁迫相关基因TaC2DP1的克隆及表达分析[J]. 中国农业科学, 2015, 48(8):1463-1472
[26] 魏晓玲, 程龙军, 窦锦青, 徐凤华. 巨桉EgrDREB2A基因结构及表达特性分析[J]. 林业科学, 2015, 51(2): 80-89
[27] 谢登雷, 崔江慧, 常金华. 高粱中SbDREB2基因的克隆与表达分析[J]. 作物学报, 2013, 39(8): 1352-1359
[28] Chen J, Xia X, Yin W, Chen J, Xia X, Yin W. Expression profiling and functional characterization of a DREB2-type gene from Populus euphratica[J]. Biochemical & Biophysical Research Communications, 2008, 378(3): 483-487
[29] 吴国栋, 修宇, 王华芳. 优化子叶节转化法培育大豆MtDREB2A转基因植株[J]. 植物学报, 2018, 53(1): 59-71
[30] 李章磊, 高飞, 曹玉震, 张至玮, 王宁, 李华云, 周宜君. 蒙古沙冬青AmDREB2.1基因的克隆及表达分析[J]. 生物技术通报, 2015, 33(3): 108-114
[31] Eftekhari A, Baghizadeh A, Yaghoobi M M, Abdolshahi R. Differences in the drought stress response of DREB2 and CAT1 genes and evaluation of related physiological parameters in some bread wheat cultivars[J]. Biotechnology & Biotechnological Equipment, 2017, 54(2): 1-8
[32] Zhao H, Zhao X, Li M, Jiang Y, Xu J Q, Jin J J, Li K L. Ectopic expression of Limonium bicolor, (Bag.) Kuntze DREB, (LbDREB) results in enhanced salt stress tolerance of transgenic Populus ussuriensis, Kom[J]. Plant Cell Tissue & Organ Culture, 2018, 132(1): 123-136
[2] 侯晓龙, 蔡丽平, 韩航, 周垂帆, 马祥庆. 铅胁迫对百喜草叶绿素荧光特性及酶活性的影响[J]. 草业学报, 2017, 26(3): 142-148
[3] 张鹍飞, 牟利, 李鹏, 方建, 刘新春, 冯宗云. 青稞OMT1基因克隆及原核表达分析[J]. 核农学报, 2017, 31(12): 2314-2322
[4] 严莉, 陈建伟, 王翠平, 乔改霞, 李健. 黑果枸杞WD40编码基因LrAN11的克隆及表达分析[J]. 核农学报, 2017, 31(11): 2103-2112
[5] Liu Q, Zhao N M, Yamaguchi-Shinozaki K, Shinozaki K. Regulatory role of DREB transcription factors in plant drought, salt and cold tolerance[J]. Science Bulletin, 2000, 45(11): 970-975
[6] 马兴勇, 彭献军, 苏蔓, 张乐新, 周庆源, 陈双燕, 程丽琴, 刘公社. 羊草DREB转录因子的系统发育和功能研究[J]. 草业学报, 2012, 21(6): 190-197
[7] Liu Q, Kasuga M, Sakuma Y, Abe H, Miura S, Yamagu-chi-Shinozaki K, Shinozaki K. Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA-binding domain separate two cellular signal transduction pathways in drought-and low-temperature-responsive gene expression, respectively, in Arabidopsis[J]. Plant Cell, 1998, 10(5): 1391-1406
[8] 冯军, 郑彩霞. DREB转录因子在植物非生物胁迫中的作用及应用研究[J]. 植物生理学报, 2011, 47(5): 437-442
[9] Shinozaki K, Yamaguchishinozaki K. Gene networks involved in drought stress response and tolerance.[J]. Journal of Experimental Botany, 2007, 58(2): 221-234
[10] Haake Ⅴ, Cook D, Riechmann J L, Pineda O, Thomashow M F, Zhang J Z. Transcription factor CBF4 is a regulator of drought adaptation in Arabidopsis[J]. Plant Physiology, 2002, 130(2): 639-648
[11] Qin F, Sakuma Y, Li J, Liu Q, Li Y Q, Shinozaki K, Yamaguchi K. Cloning and functional analysis of a novel DREB1/CBF tran-scription factor involved in cold-responsive gene expression in Zea mays L.[J]. Plant Cell Physiol, 2014, 45(8): 1042-1052
[12] Kume S, Kobayashi F, Ishibashi M, Ohno R, Nakamura C, Ta-kumi S. Differential and coordinated expression of Cbf and Cor/Lea genes during long-term cold acclimation in two Triticum aestivum cultivars showing distinct levels of freezing tolerance[J]. Genes Gene Syst, 2015, 80(3): 185-197
[13] Dubouzet J G, Sakuma Y, Ito Y, Kasuga M, Dubouzet E G. OsDREB genes in rice, Oryza sativa L., encode transcription activators that function in drought high-salt and cold-responsive gene expression [J]. The Plant Journal, 2003, 33(4): 751-763
[14] 杨凤萍. 利用抗逆调节转录因子(DREB1B和CBF1)基因转化多年生黑麦草提高其抗旱性[D]. 北京: 中国农业大学, 2005: 49-51
[15] Li R, Wei J, Wang H, He J, Sun Z. Development of highly regenerable callus lines and Agrobacterium-mediated transformation of Chinese lawngrass (Zoysia sinica Hance) with a cold inducible transcription factor, CBF1[J]. Plant Cell Tissue & Organ Culture, 2006, 85(3): 297-305
[16] 谢登雷. 高粱转录因子SbDREB2基因的克隆与功能分析[D]. 保定: 河北农业大学, 2013: 45-61
[17] 丁咚, 陈亚娟, 崔进荣, 魏建华, 张玉红, 张杰伟. 毛果杨CBF/DREB1基因家族生物信息学分析[J]. 西南农业学报, 2018, 31(3): 457-461
[18] 史松昌, 张如月, 赵振廷, 杨磊, 葛伟娜. 谷子DREBs亚家族的全基因组学分析[J]. 基因组学与应用生物学, 2018, 37(2): 827-835
[19] 马刘峰, 陈芸, 任羽, 司马义·巴拉提, 李玲. 棉花CBF2基因克隆和超表达CBF2棉花增强抗冷性[J]. 植物生理学报, 2018, 54(2): 255-264
[20] 肖雄. 紫大麦草(Hordeum viloaceum)中DREB类转录因子基因的克隆、原核表达及序列分析[D]. 保定: 河北农业大学, 2012: 31-33
[21] Lata C, Bhutty S, Bahadur R P. Association of an SNP in a novel DREB2-like gene SiDREB2 with stress tolerance in foxtail millet [Setaria italica (L.)][J]. Journal of Experimental Botany, 2011, 62(10):3387-3399
[22] 张志飞, 杨知建, 周倩, 赵志丽. DREB2s转录因子基因的表达调控机制研究进展[J]. 植物科学学报, 2014, 32(3): 297-303
[23] Upadhyay R K, Gupta A, Soni D, Rashmi G, Uday Ⅴ P, Pravendra N, Aniruddha P S. Ectopic expression of a tomato DREB gene affects several ABA processes and influences plant growth and root architecture in an age-dependent manner[J]. Journal of Plant Physiology, 2017, 214(7): 97-107
[24] 苏世超, 唐益苗, 徐磊, 王伟伟, 高世庆, 马锦绣, 孙辉, 王永波, 乔亚科, 赵昌平. 普通小麦TaSEC14p-5基因的克隆及表达分析[J]. 农业生物技术学报, 2016, 24(8): 1129-1137
[25] 肖瑞霞, 王新国, 夏国军, 李永春, 牛洪斌, 王翔, 尹钧, 任江萍. 小麦逆境胁迫相关基因TaC2DP1的克隆及表达分析[J]. 中国农业科学, 2015, 48(8):1463-1472
[26] 魏晓玲, 程龙军, 窦锦青, 徐凤华. 巨桉EgrDREB2A基因结构及表达特性分析[J]. 林业科学, 2015, 51(2): 80-89
[27] 谢登雷, 崔江慧, 常金华. 高粱中SbDREB2基因的克隆与表达分析[J]. 作物学报, 2013, 39(8): 1352-1359
[28] Chen J, Xia X, Yin W, Chen J, Xia X, Yin W. Expression profiling and functional characterization of a DREB2-type gene from Populus euphratica[J]. Biochemical & Biophysical Research Communications, 2008, 378(3): 483-487
[29] 吴国栋, 修宇, 王华芳. 优化子叶节转化法培育大豆MtDREB2A转基因植株[J]. 植物学报, 2018, 53(1): 59-71
[30] 李章磊, 高飞, 曹玉震, 张至玮, 王宁, 李华云, 周宜君. 蒙古沙冬青AmDREB2.1基因的克隆及表达分析[J]. 生物技术通报, 2015, 33(3): 108-114
[31] Eftekhari A, Baghizadeh A, Yaghoobi M M, Abdolshahi R. Differences in the drought stress response of DREB2 and CAT1 genes and evaluation of related physiological parameters in some bread wheat cultivars[J]. Biotechnology & Biotechnological Equipment, 2017, 54(2): 1-8
[32] Zhao H, Zhao X, Li M, Jiang Y, Xu J Q, Jin J J, Li K L. Ectopic expression of Limonium bicolor, (Bag.) Kuntze DREB, (LbDREB) results in enhanced salt stress tolerance of transgenic Populus ussuriensis, Kom[J]. Plant Cell Tissue & Organ Culture, 2018, 132(1): 123-136