核桃JrGRAS2基因响应热胁迫的表达及功能分析
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摘要
GRAS转录因子在植物响应逆境中起重要作用。为更好的了解核桃(Juglans regia)在逆境胁迫下的适应机制,本研究从‘香玲’核桃转录组中克隆获得一条GRAS基因(命名为Jr GRAS2),对其在不同高温胁迫下的表达进行分析,并将该基因插入酵母表达载体p YES2中构建重组载体p YES2-Jr GRAS2,将p YES2-Jr GRAS2转入酿酒酵母(Saccharomyces cerevisiae)INVSCI,同时以转化p YES2的重组酵母作为阴性对照,在酵母表达系统中研究该基因的抗热胁迫功能。结果显示,该基因开放读码框(ORF)全长1 296 bp,拟推导的蛋白分子量为47 405.83 Da,含有氨基酸数为431,理论等电点为5.66。在热胁迫下,Jr GRAS2基因被显著诱导,特别是在36℃胁迫0.5 h的茎内,其表达相对于对照被上调了335.5倍。对两种酵母进行热胁迫,发现转Jr GRAS2基因酵母表现出较对照更高的生存活性。表明Jr GRAS2基因具有响应热胁迫的能力,且能提高酵母的抗性,Jr GRAS2基因可作为核桃逆境应答的重要候选基因。
The GRAS transcription factor is important for plant response to abiotic stress.To well understand the adaptive mechanism to adverse environment of walnut tree,a GRAS gene was cloned from the transcriptome of Juglans regia(Named as Jr GRAS2).The expression of Jr GRAS2 was analyzed under heat stress,and Jr GRAS2 was inserted into the yeast expression vector p YES2 for constructing recombinant plasmid p YES2-Jr GRAS2,which was transformed into a Saccharomyces cerevisiae strain(INVSCI).The yeast transformed with empty p YES2 was used as a negative control.The yeast expression system was used for analysis the heat stress tolerance.The full length open reading frame(ORF) of Jr GRAS2 was 1 296 bp,the deduced protein was47 405.83 Da with 431 amino acids,and the theoretical isoelectric point(p I) was 5.66.Under heat stress,Jr GRAS2 was highly induced,especially exposed to 36℃ for 0.5 h in the stems,it was induced to 335.5-fold of control.When both recombinant yeasts were treated with 53℃,the Jr GRAS2 expressed yeast displayed higher vitality and survival rate than the control yeast.Therefore,Jr GRAS2 gene could effectively response to heatstress and improve heat tolerance of transgenic yeasts,Jr GRAS2 may be an important candidate gene for walnut response to adverse stimulus.
The GRAS transcription factor is important for plant response to abiotic stress.To well understand the adaptive mechanism to adverse environment of walnut tree,a GRAS gene was cloned from the transcriptome of Juglans regia(Named as Jr GRAS2).The expression of Jr GRAS2 was analyzed under heat stress,and Jr GRAS2 was inserted into the yeast expression vector p YES2 for constructing recombinant plasmid p YES2-Jr GRAS2,which was transformed into a Saccharomyces cerevisiae strain(INVSCI).The yeast transformed with empty p YES2 was used as a negative control.The yeast expression system was used for analysis the heat stress tolerance.The full length open reading frame(ORF) of Jr GRAS2 was 1 296 bp,the deduced protein was47 405.83 Da with 431 amino acids,and the theoretical isoelectric point(p I) was 5.66.Under heat stress,Jr GRAS2 was highly induced,especially exposed to 36℃ for 0.5 h in the stems,it was induced to 335.5-fold of control.When both recombinant yeasts were treated with 53℃,the Jr GRAS2 expressed yeast displayed higher vitality and survival rate than the control yeast.Therefore,Jr GRAS2 gene could effectively response to heatstress and improve heat tolerance of transgenic yeasts,Jr GRAS2 may be an important candidate gene for walnut response to adverse stimulus.
引文
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18.周莲洁,杨中敏,张富春,等.新疆盐穗木GRAS转录因子基因克隆及表达分析[J].西北植物学报,2013,33(6):1091-1097.Zhou L J,Yang Z M,Zhang F C,et al.Expression analysis and cloning of GRAS transcription factor gene from Halostachys caspica[J].Acta Botanica Boreali-Occidentalia Sinica,2013,33(6):1091-1097.
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20.陈裕坤,林玉玲,田奇琳,等.龙眼胚性愈伤组织DlGRAS4与Dl GRAS54基因的克隆及表达分析[J].西北植物学报,2014,34(2):215-224.Chen Y K,Lin Y L,Tian Q L,et al.Cloning and expression analysis of Dl GRAS4 and Dl GRAS54 from embryogenic callus of Dimocarpus longan Lour[J].Acta Botanica BorealiOccidentalia Sinica,2014,34(2):215-224.
21.Maser P,Hosoo Y,Goshima S,et al.Glycine residues in potassium channel-like selectivity filters determine potassium selectivity in four-loop-per-subunit HKT transporters from plants[J].Proceedings of the National Academy of Sciences of the United States of America,2002,99(9):6428-6433.
22.杨桂燕,于丽丽,赵玉琳,等.刚毛柽柳The IF1A基因转入酵母的抗逆能力分析[J].南京林业大学学报:自然科学版,2014(5):62-66.Yang G Y,Yu L L,Zhao Y L,et al.Stress tolerance analysis of a Tamarix hispida The IF1A in Saccharomyces cerevisiae[J].Journal of Nanjing Forestry University:Natural Sciences Edition,2014(5):62-66.
2.Liu W,Kohlen W,Lillo A,et al.Strigolactone Biosynthesis in Medicago truncatula and rice requires the symbiotic GRAS-Type transcription factors NSP1 and NSP2[J].The Plant Cell,2011,23(10):3853-3865.
3.Wu N N,Zhu Y,Song W L,et al.Unusual tandem expansion and positive selection in subgroups of the plant GRAStranscription factor superfamily[J].BMC Plant Biology,2014,14:373.
4.汪莹,孔俊花,陈微微,等.番茄果实成熟相关转录因子的研究进展[J].园艺学报,2014,41(9):1811-1820.Wang Y,Kong J H,Chen W W,et al.Fruit ripening-related transcription factors in tomato[J].Acta Horticulturae Sinica,2014,41(9):1811-1820.
5.张利娟,梁卫红,刘月霞,等.两种水稻OsRho GDIs基因启动子的克隆及分析[J].中国农业科学,2008,41(10):2916-2922.Zhang L J,Liang W H,Liu Y X,et al.Cloning and analysis of the promoters of two OsRho GDIs genes in rice[J].Scientia Agricultura Sinica,2008,41(10):2916-2922.
6.孙欣,王晨,房经贵,等.葡萄GRAS基因家族生物信息学分析[J].江西农业学报,2011,23(7):1-8.Sun X,Wang C,Fang J G,et al.Bioinformatics analysis of GRAS gene family in grapevine[J].Acta Agriculture Jiangxi,2011,23(7):1-8.
7.Li J H,Yu C Y,Wu H,et al.Knockdown of a Jmj C domain-containing gene JMJ524 confers altered gibberellin responses by transcriptional regulation of GRAS protein lacking the DELLA domain genes in tomato[J].Journal of Experimental Botany,2015,66(5):1413-1426.
8.Hessam S,Georges D,Sand M,et al.Comparison of lipidocolloid and chlorhexidine-impregnated tulle gras dressings following microscopically controlled surgery[J].Journal of Wound Care,2015,24(3):135-138.
9.郭华军,焦远年,邸超,等.拟南芥转录因子GRAS家族基因群响应渗透和干旱胁迫的初步探索[J].植物学报,2009,44(3):290-299.Guo H J,Jiao Y N,Di C,et al.Discovery of Arabidopsis GRAS family genes in response to osmotic and drought stresses[J].Bulletin of Botany,44(3):290-299.
10.Xu K,Chen S J,Li T F,et al.Os GRAS23,a rice GRAStranscription factor gene,is involved in drought stress response through regulating expression of stress-responsive genes[J].BMC Plant Biology,2015,15:141.
11.Czikkel B E,Maxwell D P.Nt GRAS1,a novel stress-induced member of the GRAS family in tobacco,localizes to the nucleus[J].Journal of Plant Physiology,2007,164(9):1220-1230.
12.Yang G,Zhang W,Liu Z,et al.Both Jr WRKY2 and JrWRKY7 of Juglans regia mediate responses to abiotic stresses and abscisic acid through formation of homodimers and interaction[J].Plant biology(Stuttgart,Germany),2017,19(2):268-278.
13.Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the2-ΔΔCTMethod[J].Methods,2001,25(4):402-408.
14.张甜,王玛丽,赵鹏.基于核基因序列JRD5680的核桃群体遗传多样性和遗传结构研究[J].植物研究,2016,36(2):232-241.Zhang T,Wang M L,Zhao P.Sequence analysis of nuclear DNA JRD5680 for determining genetic diversity and genetic structure analysis of common walnut(Juglans regia L.)[J].Bulletin of Botanical Research,2016,36(2):232-241.
15.Dogramaci M,Foley M E,Horvath D P,et al.Glyphosate’s impact on vegetative growth in leafy spurge identifies molecular processes and hormone cross-talk associated with increased branching[J].BMC Genomics,2015,16:395.
16.Busov V,Meilan R,Pearce D W,et al.Transgenic modification of gai or rgl1 causes dwarfing and alters gibberellins,root growth,and metabolite profiles in Populus[J].Planta,2006,224(2):288-299.
17.Torres-galea P,Hirtreiter B,Bolle C.Two GRAS proteins,SCARECROW-LIKE21 and PHYTOCHROME A SIGNALTRANSDUCTION1,function cooperatively in phytochrome A signal transduction[J].Plant Physiology,2013,161(1):291-304.
18.周莲洁,杨中敏,张富春,等.新疆盐穗木GRAS转录因子基因克隆及表达分析[J].西北植物学报,2013,33(6):1091-1097.Zhou L J,Yang Z M,Zhang F C,et al.Expression analysis and cloning of GRAS transcription factor gene from Halostachys caspica[J].Acta Botanica Boreali-Occidentalia Sinica,2013,33(6):1091-1097.
19.石瑞,曹诣斌,陈文荣,等.佛手GRAS基因的克隆及表达分析[J].浙江师范大学学报:自然科学版,2011,34(4):446-451.Shi R,Cao Y B,Chen W R,et al.On c DNA cloning and expression analysis of GRAS gene in fingered citron[J].Journal of Zhejiang Normal University:Natural Sciences E-dition,2011,34(4):446-451.
20.陈裕坤,林玉玲,田奇琳,等.龙眼胚性愈伤组织DlGRAS4与Dl GRAS54基因的克隆及表达分析[J].西北植物学报,2014,34(2):215-224.Chen Y K,Lin Y L,Tian Q L,et al.Cloning and expression analysis of Dl GRAS4 and Dl GRAS54 from embryogenic callus of Dimocarpus longan Lour[J].Acta Botanica BorealiOccidentalia Sinica,2014,34(2):215-224.
21.Maser P,Hosoo Y,Goshima S,et al.Glycine residues in potassium channel-like selectivity filters determine potassium selectivity in four-loop-per-subunit HKT transporters from plants[J].Proceedings of the National Academy of Sciences of the United States of America,2002,99(9):6428-6433.
22.杨桂燕,于丽丽,赵玉琳,等.刚毛柽柳The IF1A基因转入酵母的抗逆能力分析[J].南京林业大学学报:自然科学版,2014(5):62-66.Yang G Y,Yu L L,Zhao Y L,et al.Stress tolerance analysis of a Tamarix hispida The IF1A in Saccharomyces cerevisiae[J].Journal of Nanjing Forestry University:Natural Sciences Edition,2014(5):62-66.