根瘤农杆菌介导玉米ZmHDZIV13和ZmHDZIV14基因的遗传转化及干旱抗性分析
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摘要
同源异型域-亮氨酸拉链(homeodomain-leucine zipper, HD-Zip)转录因子在植物的生长发育、形态建成及抵抗各种逆境胁迫中起着十分重要的调控作用。本研究构建了植物表达载体pCAMBIA3300-Ubi-ZmHDZIV13/14-bar,利用根瘤农杆菌(Agrobacterium tumefaciens)茎尖转化法将玉米ZmHDZIV13和ZmHDZIV14基因转入玉米自交系'郑58',以Basta除草剂筛选阳性植株,以PCR及Southern blot方法对转基因玉米进行检测;在干旱胁迫下对T2代转基因玉米和非转基因玉米株系进行耐旱性分析。结果表明,正常处理下,转基因株系与非转基因株系均能正常生长,根和叶中丙二醛(malondialdehyde, MDA)、H2O2、脯氨酸(proline, Pro)、可溶性糖(soluble sugar, SS)含量、过氧化物酶(peroxidase, POD)、过氧化氢酶(catalase, CAT)及超氧化物歧化酶(superoxide dismutase, SOD)活性无显著差异;但在干旱胁迫条件下,转ZmHDZIV13和ZmHDZIV14基因玉米株系根和叶中的MDA和H2O2含量极显著降低(P<0.01),Pro、SS含量、POD、CAT及SOD活性极显著增高(P<0.01)。上述结果表明,导入ZmHDZIV13和ZmHDZIV14基因在一定程度上提高了玉米植株对干旱胁迫的耐受能力。本研究为深入探讨ZmHDZIV13和ZmHDZIV14基因功能和创制转基因抗旱材料提供了基础资料。
HD-Zip(homeodomain-leucine zipper) is one of the important transcription factors, which acts important roles in regulating plant growth, development, morphogenesis, and resistance to various abiotic stress. In the present study, the plant expression vector pCAMBIA3300-Ubi-ZmHDZIV13/14-bar was succeeded constructed, the ZmHDZIV13 and ZmHDZIV14 genes were transferred into maize inbred line'Zheng 58' by Agrobacterium tumefaciens-mediated stem dip transformation, and the transgenic maize lines were screened by Basta herbicide, and were detected by PCR and Southern blot method. After that, T2 transgenic and non-transgenic maize lines were used to analyze the drought tolerance. Results showed that the non-transgenic/transgenic maize lines grew normal growth without stress, and there was no significant difference in the contents of the malondialdehyde(MDA), H2 O2, proline(Pro), soluble sugar(SS) contents,and the activity of peroxidase(POD), catalase(CAT), and superoxide dismutase(SOD) in roots and leaves between both maize lines; however, under drought stress, the contents of MDA and H2 O2 decreased significantly(P<0.01), whereas, the contents of Pro and SS, and activity of POD, CAT, and SOD increased significantly(P<0.01) in roots and leaves of ZmHDZIV13 and ZmHDZIV14 transgenic maize lines. These results indicated that ZmHDZIV13 and ZmHDZIV14 genes could improve the drought tolerance in maize plants. Therefore, this study could provide the basis data for the further study of ZmHDZIV13 and ZmHDZIV14 genes and creation of transgenic drought-tolerant materials.
HD-Zip(homeodomain-leucine zipper) is one of the important transcription factors, which acts important roles in regulating plant growth, development, morphogenesis, and resistance to various abiotic stress. In the present study, the plant expression vector pCAMBIA3300-Ubi-ZmHDZIV13/14-bar was succeeded constructed, the ZmHDZIV13 and ZmHDZIV14 genes were transferred into maize inbred line'Zheng 58' by Agrobacterium tumefaciens-mediated stem dip transformation, and the transgenic maize lines were screened by Basta herbicide, and were detected by PCR and Southern blot method. After that, T2 transgenic and non-transgenic maize lines were used to analyze the drought tolerance. Results showed that the non-transgenic/transgenic maize lines grew normal growth without stress, and there was no significant difference in the contents of the malondialdehyde(MDA), H2 O2, proline(Pro), soluble sugar(SS) contents,and the activity of peroxidase(POD), catalase(CAT), and superoxide dismutase(SOD) in roots and leaves between both maize lines; however, under drought stress, the contents of MDA and H2 O2 decreased significantly(P<0.01), whereas, the contents of Pro and SS, and activity of POD, CAT, and SOD increased significantly(P<0.01) in roots and leaves of ZmHDZIV13 and ZmHDZIV14 transgenic maize lines. These results indicated that ZmHDZIV13 and ZmHDZIV14 genes could improve the drought tolerance in maize plants. Therefore, this study could provide the basis data for the further study of ZmHDZIV13 and ZmHDZIV14 genes and creation of transgenic drought-tolerant materials.
引文
陈再刚,周大祥,胡廷章.2007.影响农杆菌介导植物遗传转化的因素[J].重庆工学院学报(自然科学版),21(03):106-109.(Chen Z G,Zhou D X,Hu T Z.2007.Factors influencing genetic transformation of plants via Agrobac?terium tumefaciens[J].Journal of Chongqing Institute of Technology(Natural Science Edition),21(03):106-109.)
胡繁荣,夏英武.2004,结缕草农杆菌介导转基因中辅助处理的效果[J].南京林业大学学报(自然科学版),28(5):37-40.(Hu F R,Xia Y W.2004.Efficiency of the assistant method on Agrobacterium-mediated transformation of Japanese lawngrass[J].Journal of Nanjing Forestry University(Natural Sciences Edition),28(5):37-40.)
刘立鸿,许璐,汪凯,等.2008.地高辛标记探针Southern印迹杂交技术要点及改进[J].生物技术通报,(03):57-59.(Liu L H,Xu L,Wang K,et al.2008.The key technological points of Southern blot using Dig-labeled DNA probes and improvement[J].Biotechnology Bulletin,(03):57-59.)
穆延召.2013.玉米耐旱相关基因ZmHDZIV13和ZmH?DZIV14的克隆、分析及表达载体构建[D].硕士学位论文,甘肃农业大学.导师:王汉宁,彭云玲,pp.45-49.(Mu Y Z.2013.Cloning and analysis of ZmHDZIV13and ZmHDZIV14 gene related to drought tolerance in maize and construction of expression vector[D].Thesis for M.S.,Gansu Agricultural University.Suppervisor:Wang H N,Peng Y L,pp.45-49.)
彭立新,李德全,束怀瑞.2002.园艺植物水分胁迫生理及耐旱机制研究进展[J].西北植物学报,22(05):1275-1281.(Peng L X,Li D Q,Su H R.2002.Progress in water stress physiology and drought tolerance mechanism of horticultural plant[J].Acta Botanica Boreali-Occidentalia Sinica,22(5):1275-1281.)
秦永芳,李登弟,李学宝.2009.植物HD-Zip转录因子研究进展[J].细胞生物学杂志,31(04):514-520.(Qin Y F,Li D D,Li X B.2009.Progress in HD-Zip transcription factors of plant[J].Chinese Journal of Cell Biology,31(4):514-520.)
魏强,宋贺玲,向成斌,等.2010.过量表达拟南芥HD-START转录因子AtHDG11提高黑麦草(Lolium perenne)抗旱性的初步研究[J].植物生理学通讯,46(11):1140-1146.(Wei Q,Song H L,Xiang C B,et al.2010.Preliminary study on improving drought tolerance of ryegrass(Lolium perenne)by overexpressing a HD-START transcription factor AtHDG11 from Arabidopsis[J].Plant Physiology Communications,46(11):1140-1146.)
闫慧萍.2016.玉米HD-Zip转录因子Zmhdz13和Zmhdz14的抗旱功能研究[D].硕士学位论文,甘肃农业大学.导师:彭云玲,pp.24-26.(Yan H P.2016.Study on drought resistance functions of two HD-Zip transcription factors Zmhdz13 and Zmhdz14 in maize(Zea mays L.)[D].Thesis for M.S.,Gansu Agricultural University.Suppervisor:Peng Y L,pp.24-26.)
赵桂琴,慕平,张勃.2006.紫花苜蓿基因工程研究进展[J].草业学报,15(06):9-18.(Zhao G Q,Mu P,Zhang B.2006.Research progress on Medicago sativa genetic engineering[J].Acta Prataculturae Sinica,15(06):9-18.)
赵小强.2018.玉米株型相关耐旱遗传机理研究[D].硕士学位论文,甘肃农业大学.导师:张金文;彭云玲,pp.8-9.(Zhao X Q.2018.Genetic mechanisms study of drought tolerance related to plant architecture in maize(Zea mays L.)[D].Thesis for M.S.,Gansu Agricultural University.Suppervisor:Zhang J W;Peng Y L,pp.8-9.)
赵小强,方鹏,彭云玲,等.2018a.基于两个相关群体的玉米6个穗部性状QTL定位[J].农业生物技术学报,26(5):729-742.(Zhao X Q,Fang P,Peng Y L,et al.QTL mapping for six ear-related traits based on two maize(Zea mays)related populations[J].Journal of Agricural Biotechnology,26(5):729-742.)
赵小强,方鹏,彭云玲,等.2018b.基于两个相关群体的玉米7个主要农艺性状遗传分析和QTL定位[J].草业学报,27(9):152-165.(Zhao X Q,Fang P,Peng Y L,et al.2018c.Genetic analysis and QTL mapping for seven agronomic traits in maize(Zea mays)using two connected populations[J].Acta Prataculturae Sinica,27(9):152-165.)
赵小强,彭云玲,方鹏,等.2018c.不同外源调节物质对低温胁迫下玉米的缓解效应分析[J].干旱地区农业研究,36(3):184-193.(Zhao X Q,Peng Y L,Fang P,et al.2018a.Mitigation effect analysis of different exogenous regulatory substances on maize under low-temperature stress[J].Agricultural Research in the Arid Areas,36(3):184-193.)
赵小强,彭云玲,李建英,等.2014.16份玉米自交系的耐盐性评价[J].干旱地区农业研究,32(5):40-45.(Zhao XQ,Peng Y L,Li J Y,et al.2014.Comprehensive evaluation of salt tolerance in 16 maize inbred lines[J].Agricultural Research in the Arid Areas,32(5):40-45.)
Abe M,Katsumata H,Komeda Y,et al.2003.Regulation of shoot epidermal cell differentiation by a pair of homeodomain proteins in Arabidopsis[J].Development,130(4):635-643.
Armengaud P,Thiery L,Buhot N,et al.2004.Transcriptional regulation of proline biosynthesis in Medicago truncatu?la reveals developmental and environmental specific features[J].Physiologia Plantarum,120(3):442-450.
Binns A N.1990.Agrobacterium-mediated gene delivery and the biology of host range limitations[J].Physiologia Plantarum,79(1):135-139.
Cao Y J,Wei Q,Liao Y,et al.2009.Ectopic overexpression of AtHDG11 in tall fescue resulted in enhanced tolerance to drought and salt stress[J].Plant Cell Reports,28(4):579-588.
Dellaporta S L,Wood J,Hicks J B.1983.A plant DNA minipreparation:VersionⅡ[J].Plant Molecular Biology Reporter,1(4):19-21.
Ingram G C,Boisnard-Lorig C,Dumas C,et al.2000.Expression patterns of genes encoding HD-ZipⅣhomeo domain proteins define specific domains in maize embryos and meristems[J].The Plant Journal,22(5):401-414.
Itoh J,Hibara K,Sato Y,et al.2008.Developmental role and auxin responsiveness of ClassⅢhomeodomain leucine zipper gene family members in rice[J].Plant Physiology,147(4):1960-1975.
Kirch H H,Schlingensiepen S,Kotchoni S,et al.2005.Detailed expression analysis of selected genes of the aldehyde dehydrogenase(ALDH)gene superfamily in Arabi?dopsis thaliana[J].Plant Molecular Biology,57(3):315-332.
Liu J,Zhu J K.1997.Proline accumulation and salt-stress-induced gene expression in a salt-hypersensitive mutant of Arabidopsis[J].Plant Physiology,114(2):591-596.
Mittler R,Vanderauwera S,Gollery M,et al.2004.Reactive oxygen gene network of plants[J].Trends in Plant Science,9(10):490-498.
Nakamura M,Katsumata H,Abe M,et al.2006.Characterization of the classⅣhomeodomain-Leucine Zipper gene family in Arabidopsis[J].Plant Physiology,141(4):1363-1375.
Schrick K,Nguyen D,Karlowski W M,et al.2004.STARTlipid/sterol-binding domains are amplified in plants and are predominantly associated with homeodomain transcription factors[J].Genome Biology,5(6):R41.
Song S,Chen Y,Zhao M,et al.2012.A novel Medicago trun?catula HD-Zip gene,MtHB2,is involved in abiotic stress responses[J].Environmental and Experimental Botany,80(4):1-9.
Szekely G,Abraham E,Cseplo A,et al.2008.Duplicated P5CS genes of Arabidopsis play distinct roles in stress regulation and developmental control of proline biosynthesis[J].The Plant Journal,53(1):11-28.
Witcombe J R,Hollington P A,Howarth C J,et al.2008.Breeding for abiotic stresses for sustainable agriculture[J].Philosophical Transactions of The Royal Society of London Series B-Biologic,363(1492):703-716.
Wu L B,Ueda Y,Lai S K,et al.2016.Shoot tolerance mechanisms to iron toxicity in rice(Oryza sativa L.)[J].Plant Cell and Environment,7(9):1-15.
Xiang Y,Huang Y,Xiong L.2007.Characterization of stressresponsive CIPK genes in rice for stress tolerance improvement[J].Plant Physiology,144(3):1416-1428.
Xiong L,Schumaker K S,Zhu J K.2002.Cell Signaling during cold,drought,and salt stress[J].The Plant Cell,14(1):165-183.
Yang J Y,Chung M C,Tu C Y,et al.2002.OSTF1:A HD-GL2family homeobox gene is developmentally regulated during early embryogenesis in rice[J].Plant and Cell Physiology,43(6):628-638.
Yu H,Chen X,Hong Y Y,et al.2008.Activated expression of an Arabidopsis HD-START protein confers drought tolerance with improved root system and reduced stomatal density[J].The Plant Cell,20(4):1134-1151.
Yu L H,Wu S J,Peng Y S,et al.2016.Arabidopsis EDT1/HDG11 improves drought and salt tolerance in cotton and poplar and increases cotton yield in the field[J].Plant Biotechnology Journal,14(1):72-84.
胡繁荣,夏英武.2004,结缕草农杆菌介导转基因中辅助处理的效果[J].南京林业大学学报(自然科学版),28(5):37-40.(Hu F R,Xia Y W.2004.Efficiency of the assistant method on Agrobacterium-mediated transformation of Japanese lawngrass[J].Journal of Nanjing Forestry University(Natural Sciences Edition),28(5):37-40.)
刘立鸿,许璐,汪凯,等.2008.地高辛标记探针Southern印迹杂交技术要点及改进[J].生物技术通报,(03):57-59.(Liu L H,Xu L,Wang K,et al.2008.The key technological points of Southern blot using Dig-labeled DNA probes and improvement[J].Biotechnology Bulletin,(03):57-59.)
穆延召.2013.玉米耐旱相关基因ZmHDZIV13和ZmH?DZIV14的克隆、分析及表达载体构建[D].硕士学位论文,甘肃农业大学.导师:王汉宁,彭云玲,pp.45-49.(Mu Y Z.2013.Cloning and analysis of ZmHDZIV13and ZmHDZIV14 gene related to drought tolerance in maize and construction of expression vector[D].Thesis for M.S.,Gansu Agricultural University.Suppervisor:Wang H N,Peng Y L,pp.45-49.)
彭立新,李德全,束怀瑞.2002.园艺植物水分胁迫生理及耐旱机制研究进展[J].西北植物学报,22(05):1275-1281.(Peng L X,Li D Q,Su H R.2002.Progress in water stress physiology and drought tolerance mechanism of horticultural plant[J].Acta Botanica Boreali-Occidentalia Sinica,22(5):1275-1281.)
秦永芳,李登弟,李学宝.2009.植物HD-Zip转录因子研究进展[J].细胞生物学杂志,31(04):514-520.(Qin Y F,Li D D,Li X B.2009.Progress in HD-Zip transcription factors of plant[J].Chinese Journal of Cell Biology,31(4):514-520.)
魏强,宋贺玲,向成斌,等.2010.过量表达拟南芥HD-START转录因子AtHDG11提高黑麦草(Lolium perenne)抗旱性的初步研究[J].植物生理学通讯,46(11):1140-1146.(Wei Q,Song H L,Xiang C B,et al.2010.Preliminary study on improving drought tolerance of ryegrass(Lolium perenne)by overexpressing a HD-START transcription factor AtHDG11 from Arabidopsis[J].Plant Physiology Communications,46(11):1140-1146.)
闫慧萍.2016.玉米HD-Zip转录因子Zmhdz13和Zmhdz14的抗旱功能研究[D].硕士学位论文,甘肃农业大学.导师:彭云玲,pp.24-26.(Yan H P.2016.Study on drought resistance functions of two HD-Zip transcription factors Zmhdz13 and Zmhdz14 in maize(Zea mays L.)[D].Thesis for M.S.,Gansu Agricultural University.Suppervisor:Peng Y L,pp.24-26.)
赵桂琴,慕平,张勃.2006.紫花苜蓿基因工程研究进展[J].草业学报,15(06):9-18.(Zhao G Q,Mu P,Zhang B.2006.Research progress on Medicago sativa genetic engineering[J].Acta Prataculturae Sinica,15(06):9-18.)
赵小强.2018.玉米株型相关耐旱遗传机理研究[D].硕士学位论文,甘肃农业大学.导师:张金文;彭云玲,pp.8-9.(Zhao X Q.2018.Genetic mechanisms study of drought tolerance related to plant architecture in maize(Zea mays L.)[D].Thesis for M.S.,Gansu Agricultural University.Suppervisor:Zhang J W;Peng Y L,pp.8-9.)
赵小强,方鹏,彭云玲,等.2018a.基于两个相关群体的玉米6个穗部性状QTL定位[J].农业生物技术学报,26(5):729-742.(Zhao X Q,Fang P,Peng Y L,et al.QTL mapping for six ear-related traits based on two maize(Zea mays)related populations[J].Journal of Agricural Biotechnology,26(5):729-742.)
赵小强,方鹏,彭云玲,等.2018b.基于两个相关群体的玉米7个主要农艺性状遗传分析和QTL定位[J].草业学报,27(9):152-165.(Zhao X Q,Fang P,Peng Y L,et al.2018c.Genetic analysis and QTL mapping for seven agronomic traits in maize(Zea mays)using two connected populations[J].Acta Prataculturae Sinica,27(9):152-165.)
赵小强,彭云玲,方鹏,等.2018c.不同外源调节物质对低温胁迫下玉米的缓解效应分析[J].干旱地区农业研究,36(3):184-193.(Zhao X Q,Peng Y L,Fang P,et al.2018a.Mitigation effect analysis of different exogenous regulatory substances on maize under low-temperature stress[J].Agricultural Research in the Arid Areas,36(3):184-193.)
赵小强,彭云玲,李建英,等.2014.16份玉米自交系的耐盐性评价[J].干旱地区农业研究,32(5):40-45.(Zhao XQ,Peng Y L,Li J Y,et al.2014.Comprehensive evaluation of salt tolerance in 16 maize inbred lines[J].Agricultural Research in the Arid Areas,32(5):40-45.)
Abe M,Katsumata H,Komeda Y,et al.2003.Regulation of shoot epidermal cell differentiation by a pair of homeodomain proteins in Arabidopsis[J].Development,130(4):635-643.
Armengaud P,Thiery L,Buhot N,et al.2004.Transcriptional regulation of proline biosynthesis in Medicago truncatu?la reveals developmental and environmental specific features[J].Physiologia Plantarum,120(3):442-450.
Binns A N.1990.Agrobacterium-mediated gene delivery and the biology of host range limitations[J].Physiologia Plantarum,79(1):135-139.
Cao Y J,Wei Q,Liao Y,et al.2009.Ectopic overexpression of AtHDG11 in tall fescue resulted in enhanced tolerance to drought and salt stress[J].Plant Cell Reports,28(4):579-588.
Dellaporta S L,Wood J,Hicks J B.1983.A plant DNA minipreparation:VersionⅡ[J].Plant Molecular Biology Reporter,1(4):19-21.
Ingram G C,Boisnard-Lorig C,Dumas C,et al.2000.Expression patterns of genes encoding HD-ZipⅣhomeo domain proteins define specific domains in maize embryos and meristems[J].The Plant Journal,22(5):401-414.
Itoh J,Hibara K,Sato Y,et al.2008.Developmental role and auxin responsiveness of ClassⅢhomeodomain leucine zipper gene family members in rice[J].Plant Physiology,147(4):1960-1975.
Kirch H H,Schlingensiepen S,Kotchoni S,et al.2005.Detailed expression analysis of selected genes of the aldehyde dehydrogenase(ALDH)gene superfamily in Arabi?dopsis thaliana[J].Plant Molecular Biology,57(3):315-332.
Liu J,Zhu J K.1997.Proline accumulation and salt-stress-induced gene expression in a salt-hypersensitive mutant of Arabidopsis[J].Plant Physiology,114(2):591-596.
Mittler R,Vanderauwera S,Gollery M,et al.2004.Reactive oxygen gene network of plants[J].Trends in Plant Science,9(10):490-498.
Nakamura M,Katsumata H,Abe M,et al.2006.Characterization of the classⅣhomeodomain-Leucine Zipper gene family in Arabidopsis[J].Plant Physiology,141(4):1363-1375.
Schrick K,Nguyen D,Karlowski W M,et al.2004.STARTlipid/sterol-binding domains are amplified in plants and are predominantly associated with homeodomain transcription factors[J].Genome Biology,5(6):R41.
Song S,Chen Y,Zhao M,et al.2012.A novel Medicago trun?catula HD-Zip gene,MtHB2,is involved in abiotic stress responses[J].Environmental and Experimental Botany,80(4):1-9.
Szekely G,Abraham E,Cseplo A,et al.2008.Duplicated P5CS genes of Arabidopsis play distinct roles in stress regulation and developmental control of proline biosynthesis[J].The Plant Journal,53(1):11-28.
Witcombe J R,Hollington P A,Howarth C J,et al.2008.Breeding for abiotic stresses for sustainable agriculture[J].Philosophical Transactions of The Royal Society of London Series B-Biologic,363(1492):703-716.
Wu L B,Ueda Y,Lai S K,et al.2016.Shoot tolerance mechanisms to iron toxicity in rice(Oryza sativa L.)[J].Plant Cell and Environment,7(9):1-15.
Xiang Y,Huang Y,Xiong L.2007.Characterization of stressresponsive CIPK genes in rice for stress tolerance improvement[J].Plant Physiology,144(3):1416-1428.
Xiong L,Schumaker K S,Zhu J K.2002.Cell Signaling during cold,drought,and salt stress[J].The Plant Cell,14(1):165-183.
Yang J Y,Chung M C,Tu C Y,et al.2002.OSTF1:A HD-GL2family homeobox gene is developmentally regulated during early embryogenesis in rice[J].Plant and Cell Physiology,43(6):628-638.
Yu H,Chen X,Hong Y Y,et al.2008.Activated expression of an Arabidopsis HD-START protein confers drought tolerance with improved root system and reduced stomatal density[J].The Plant Cell,20(4):1134-1151.
Yu L H,Wu S J,Peng Y S,et al.2016.Arabidopsis EDT1/HDG11 improves drought and salt tolerance in cotton and poplar and increases cotton yield in the field[J].Plant Biotechnology Journal,14(1):72-84.