转基因HaBADH马铃薯无性一代耐盐性及农艺性状分析
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摘要
采用分子生物学、形态学及生理检测等方法对转化HaBADH马铃薯无性一代2个株系目的基因整合情况,耐盐性鉴定及农艺性状进行了检测与分析。经PCR、RT-PCR及Southern blot分析,验证HaBADH基因稳定存在于转基因无性一代株系基因组中。当120 mmol/L NaCl持续胁迫20 d后,转基因无性一代株系表现出较强的抗盐能力,B10及B22株高平均增幅均达到15 cm,冠径平均增幅均达到12 cm,对照株高和冠径平均增幅分别达到5 cm和7 cm,平均单株结薯重量分别较对照重9 g和2 g,平均单株结薯数量较对照高0.53个和12.28个,Pro含量显著上升,MDA含量显著下降,进一步表明了转入HaBADH基因可稳定提高马铃薯的耐盐性。
The molecular integration, salt tolerance identification and agronomic traits of two lines of HaBADH potato asexual generation were detected and analyzed by approaches of molecular biology, morphology and physiological detection. By PCR, RT-PCR and Southern blot analysis, it was verified that the HaBADH gene was stably present in the genome of the transgenic asexual generation line. When 120 mmol/L NaCl was continuously treated for 20 days, the transgenic asexual generation showed strong salt tolerance. The average increase of B10 and B22 plants reached 15 cm, and the average increase of crown diameter reached 12 cm. The average increase of crown diameter reached 5 cm and 7 cm respectively. The average weight of single potato was 9 g and 2 g,respectively. The average number of single potato was 0.53 and 12.28 higher than that of the control. The Pro content increased significantly, and the MDA content was significant. The decline further indicates that the transfer of the HaBADH gene can stably increase the salt tolerance of the potato.
The molecular integration, salt tolerance identification and agronomic traits of two lines of HaBADH potato asexual generation were detected and analyzed by approaches of molecular biology, morphology and physiological detection. By PCR, RT-PCR and Southern blot analysis, it was verified that the HaBADH gene was stably present in the genome of the transgenic asexual generation line. When 120 mmol/L NaCl was continuously treated for 20 days, the transgenic asexual generation showed strong salt tolerance. The average increase of B10 and B22 plants reached 15 cm, and the average increase of crown diameter reached 12 cm. The average increase of crown diameter reached 5 cm and 7 cm respectively. The average weight of single potato was 9 g and 2 g,respectively. The average number of single potato was 0.53 and 12.28 higher than that of the control. The Pro content increased significantly, and the MDA content was significant. The decline further indicates that the transfer of the HaBADH gene can stably increase the salt tolerance of the potato.
引文
Agarwal S.,and Pandey V.,2004,Antioxidant enzyme responses to NaCl stress in Cassia angustifolia,Biol.Plant.,48(4):555-560
Cui X.Y.,Yong W.,and Guo J.X.,2008,Osmotic regulation of betaine content in Leymus chinensis under saline-alkali stress and cloning and expression of betaine aldehyde dehydrogenase(BADH)gene,Chemical Research in Chinese Universities,24(2):204-209
Guptab B.,and Huang B.,2014,Mechanism of salinity tolerance in plants:physiological,biochemical,and molecular characterization,Int.J.Genomics,2014:701596
Hanson A.D.,May A.M.,Grumet R.,Bode J.,and Jamieson G.C.,1985,Betaine synthesis in chenopods:localization in chloroplasts,Proc.Natl.Acad.Sci.USA,82(11):3678-3682
Ishitani M.,Nakamura T.,Han S.Y.,and Takabe T.,1995,Expression of the betaine aldehyde dehydrogenase gene in barley in response to osmotic stress and abscisic acid,Plant Mol.Biol.,27(2):307-315
McCue K.F.,and Hanson A.D.,1992,Salt-inducible betaine aldehyde dehydrogenase from sugar beet:cDNA cloning and expression,Plant Mol.Biol.,18(1):1-11
Meloni D.A.,Oliva M.A.,Martinez C.A.,and Cambraia J.,2003,Photosynthesis and activity of superoxide dismutase,peroxidase and glutathione reductase in cotton under salt stress,Environ.Exp.Bot.,49(1):69-76
Rathinasabapathi B.,McCue K.F.,Gage D.A.,and Hanson A.D.,1994,Metabolic engineering of glycine betaine synthesis:plant betaine aldehyde dehydrogenases lacking typical transit peptides are targeted to tobacco chloroplasts where they confer betaine aldehyde resistance,Planta,193(2):155-162
Roy S.J.,Negr觔o S.,and Tester M.,2014,Salt resistant crop plants,Curr.Opin.Biotechnol.,26(4):115-124
Shang Q.M.,Song S.Q.,Zhang Z.G.,and Guo S.R.,2006,Exogenous brassinosteroid induced the salt resistance of cucumber(Cucumis sativus L.)seedlings,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),39(9):1872-1877(尚庆茂,宋士清,张志刚,郭世荣,2006,外源BR诱导黄瓜(Cucumis sativus L.)幼苗的抗盐性,中国农业科学,39(9):1872-1877)
Shi L.,Zhou X.Y.,Gan X.Y.,Ma H.A.,and Song Y.X.,2012,Construction and transformation of binary plant expression vector pCAMBIA2300-HaBADH-HaCMO,Xibei Nonglin Keji Daxue Xuebao(Journal of Northwest A&F University),40(6):210-216,223(石磊,周晓燕,甘晓燕,马洪爱,宋玉霞,2012,双元植物表达载体p CAMBIA2300-HaBADH-HaCMO的构建及转化,西北农林科技大学学报,40(6):210-216,223)
Tian X.,Wei Q.R.,Liang X.L.,Qi C.H.,and Shi Y.,2015,Effects of nitrogen on yield and quality of potato variety Dongnong311,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(9):1975-1980(田洵,魏峭嵘,梁晓丽,祁驰恒,石瑛,2015,氮素对马铃薯品种东农311产量及品质的影响,基因组学与应用生物学,35(9):1975-1980)
Wang X.L.,2014,Rocovery of transgenic maize plants with salt-tolerance gene by pollen-mediated and salt-tolerance evaluation of transgenic maize progenies,Thesis for M.S.,Shanxi University,Supervisor:Sun Y.,pp.1-58(王小丽,2014,花粉介导法获得转耐盐基因玉米植株及转化后代的耐盐性鉴定,硕士学位论文,山西大学,导师:孙毅,pp.1-58)
Weigel P.,Weretilnyk E.A.,and Hanson A.D.,1986,Betaine aldehyde oxidation by spinach chloroplasts,Plant Physiol.,82(3):753-759
Weretilnyk E.A.,Bednarek S.,McCue K.F.,Rhodes D.,and Hanson A.D.,1989,Comparative biochemical and immunological studies of the glycine betaine synthesis pathway in diverse families of dicotyledons,Planta,178(3):342-352
Wood A.J.,Saneoka H.,Rhodes D.,Joly R.J.,and Goldsbrough P.B.,1996,Betaine aldehyde dehydrogenase in Sorghum,Plant Physiol.,110(4):1301-1308
Xiao W.,Jia H.X.,and Pu L.M.,Studies on physiological index of some halophytes,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),20(5):818-825(肖雯,贾恢先,蒲陆梅,2000,几种盐生植物抗盐生理指标的研究,西北植物学报,20(5):818-825)
Zhao K.F.,and Fan H.,2000,Comparative study on osmotic adjustment substances and their contribution of true salt plants and salt-salting plants under salt stress,Yingyong Yu Huanjing Shengwu Xuebao(Chinese Journal of Applied and Environmental Biology),6(2):99-105(赵可夫,范海,2000,盐胁迫下真盐植物与泌盐植物的渗透调节物质及其贡献的比较研究,应用与环境生物学报,6(2):99-105)
Cui X.Y.,Yong W.,and Guo J.X.,2008,Osmotic regulation of betaine content in Leymus chinensis under saline-alkali stress and cloning and expression of betaine aldehyde dehydrogenase(BADH)gene,Chemical Research in Chinese Universities,24(2):204-209
Guptab B.,and Huang B.,2014,Mechanism of salinity tolerance in plants:physiological,biochemical,and molecular characterization,Int.J.Genomics,2014:701596
Hanson A.D.,May A.M.,Grumet R.,Bode J.,and Jamieson G.C.,1985,Betaine synthesis in chenopods:localization in chloroplasts,Proc.Natl.Acad.Sci.USA,82(11):3678-3682
Ishitani M.,Nakamura T.,Han S.Y.,and Takabe T.,1995,Expression of the betaine aldehyde dehydrogenase gene in barley in response to osmotic stress and abscisic acid,Plant Mol.Biol.,27(2):307-315
McCue K.F.,and Hanson A.D.,1992,Salt-inducible betaine aldehyde dehydrogenase from sugar beet:cDNA cloning and expression,Plant Mol.Biol.,18(1):1-11
Meloni D.A.,Oliva M.A.,Martinez C.A.,and Cambraia J.,2003,Photosynthesis and activity of superoxide dismutase,peroxidase and glutathione reductase in cotton under salt stress,Environ.Exp.Bot.,49(1):69-76
Rathinasabapathi B.,McCue K.F.,Gage D.A.,and Hanson A.D.,1994,Metabolic engineering of glycine betaine synthesis:plant betaine aldehyde dehydrogenases lacking typical transit peptides are targeted to tobacco chloroplasts where they confer betaine aldehyde resistance,Planta,193(2):155-162
Roy S.J.,Negr觔o S.,and Tester M.,2014,Salt resistant crop plants,Curr.Opin.Biotechnol.,26(4):115-124
Shang Q.M.,Song S.Q.,Zhang Z.G.,and Guo S.R.,2006,Exogenous brassinosteroid induced the salt resistance of cucumber(Cucumis sativus L.)seedlings,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),39(9):1872-1877(尚庆茂,宋士清,张志刚,郭世荣,2006,外源BR诱导黄瓜(Cucumis sativus L.)幼苗的抗盐性,中国农业科学,39(9):1872-1877)
Shi L.,Zhou X.Y.,Gan X.Y.,Ma H.A.,and Song Y.X.,2012,Construction and transformation of binary plant expression vector pCAMBIA2300-HaBADH-HaCMO,Xibei Nonglin Keji Daxue Xuebao(Journal of Northwest A&F University),40(6):210-216,223(石磊,周晓燕,甘晓燕,马洪爱,宋玉霞,2012,双元植物表达载体p CAMBIA2300-HaBADH-HaCMO的构建及转化,西北农林科技大学学报,40(6):210-216,223)
Tian X.,Wei Q.R.,Liang X.L.,Qi C.H.,and Shi Y.,2015,Effects of nitrogen on yield and quality of potato variety Dongnong311,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(9):1975-1980(田洵,魏峭嵘,梁晓丽,祁驰恒,石瑛,2015,氮素对马铃薯品种东农311产量及品质的影响,基因组学与应用生物学,35(9):1975-1980)
Wang X.L.,2014,Rocovery of transgenic maize plants with salt-tolerance gene by pollen-mediated and salt-tolerance evaluation of transgenic maize progenies,Thesis for M.S.,Shanxi University,Supervisor:Sun Y.,pp.1-58(王小丽,2014,花粉介导法获得转耐盐基因玉米植株及转化后代的耐盐性鉴定,硕士学位论文,山西大学,导师:孙毅,pp.1-58)
Weigel P.,Weretilnyk E.A.,and Hanson A.D.,1986,Betaine aldehyde oxidation by spinach chloroplasts,Plant Physiol.,82(3):753-759
Weretilnyk E.A.,Bednarek S.,McCue K.F.,Rhodes D.,and Hanson A.D.,1989,Comparative biochemical and immunological studies of the glycine betaine synthesis pathway in diverse families of dicotyledons,Planta,178(3):342-352
Wood A.J.,Saneoka H.,Rhodes D.,Joly R.J.,and Goldsbrough P.B.,1996,Betaine aldehyde dehydrogenase in Sorghum,Plant Physiol.,110(4):1301-1308
Xiao W.,Jia H.X.,and Pu L.M.,Studies on physiological index of some halophytes,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),20(5):818-825(肖雯,贾恢先,蒲陆梅,2000,几种盐生植物抗盐生理指标的研究,西北植物学报,20(5):818-825)
Zhao K.F.,and Fan H.,2000,Comparative study on osmotic adjustment substances and their contribution of true salt plants and salt-salting plants under salt stress,Yingyong Yu Huanjing Shengwu Xuebao(Chinese Journal of Applied and Environmental Biology),6(2):99-105(赵可夫,范海,2000,盐胁迫下真盐植物与泌盐植物的渗透调节物质及其贡献的比较研究,应用与环境生物学报,6(2):99-105)