超声波介导Mz_2NHX_1基因遗传转化提高珠美海棠耐盐性The research on ultrasonic mediated genetic transformation of the Mz_2NHX_1 gene enhancing salt tolerance of Malus zumi
孙婷梅;王璐平;张永利;秦家慧;于梦楠;李爱;彭立新;
摘要(Abstract):
【目的】提高珠美海棠的耐盐性,建立更高效的珠美海棠遗传转化体系。【方法】在转化液中利用超声波直接转化无菌条件下横切主脉的珠美海棠试管苗幼叶,通过愈伤组织培养诱导植株再生、抗性筛选,GUS染色鉴定,RT-PCR分析转基因植株的耐盐性。【结果】较利于珠美海棠愈伤组织诱导分化及抗性筛选的培养基为MS+6-BA 2.0 mg·L~(-1)+NAA 0.1 mg·L~(-1)+Kana 50 mg·L~(-1)。超声波处理的最优工作条件为:处理6 s,间歇10 s,工作重复20次,功率80 W,转化效率为31.3%。GUS染色与RT-PCR鉴定结果一致率为100%,RT-PCR结果分析显示,Mz2NHX1基因在转化苗中的表达量约为对照的3倍。荧光定量PCR结果显示转化苗Mz2NHX1基因的表达量为对照组的6.21倍,盐处理试验结果显示转化苗耐盐性得到了显著地提高。【结论】建立了超声波介导的珠美海棠高效遗传转化体系,获得珠美海棠耐盐新材料,为该耐盐基因的研究和盐碱地的改良奠定了基础。
关键词(KeyWords): 珠美海棠;遗传转化体系;超声波;植株再生;耐盐基因;过表达
基金项目(Foundation): 国家自然科学基金(30671440,31300564)
作者(Author): 孙婷梅;王璐平;张永利;秦家慧;于梦楠;李爱;彭立新;
Email:
DOI: 10.13925/j.cnki.gsxb.20160063
参考文献(References):
- [1]王玉珍,冯雪赞.多用途耐盐碱经济树种——珠美海棠[J].林业科技,1999,24(2):53-55.WANG Yuzhen,FENG Xuezan.Multi-purpose and salt-toleranteconomic tree—Malus zumi[J].The Forestry Science and Technol-ogy,1999,24(2):53-55.
- [2]赵慧祥.苹果砧木——珠美海棠茎尖培养简报[J].中国果树,1981(3):41-42.ZHAO Huixiang.The report of stem tip cultivation of Malus zumi—a kind of apple rootstock[J].China Fruits,1981(3):41-42.
- [3]张永利,孟晓烨,孙婷梅,李爱,彭立新.珠美海棠Mz2NHX1基因的克隆和序列分析[J].江苏农业科学,2015,43(9):20-24.ZHANG Yongli,MENG Xiaoye,SUN Tingmei,LI Ai,PENG Lix-in.Coloning and sequence analysis of the Na+/H+antiporter gene(Mz2NHX1)of Malus zumi[J].Jiangsu Agricultural Sciences,2015,43(9):20-24.
- [4]孟晓烨.盐胁迫下珠美海棠Na+/H+逆向转运蛋白基因(Mz NHX1)的分离及表达特性研究[D].天津:天津农学院,2010.MENG Xiaoye.Isolation and expression of the Na+/H+antiportergene of Malus zumi(Mz NHX1)under salt stress[D].Tianjin:Tian-jin Agriculture University,2010.
- [5]XU J,WANG Y Z,YIN H X,LIU X J.Efficient Agrobacterium tumefaciens-mediated transformation of Malus zumi(Matsumura)Rehd.using leaf explant regeneration system[J].Electronic Jour-nal of Biotechnology,2009,12(1):288-289.
- [6]陶建敏,章镇,王三红,乔玉山,徐长宝,王红霞.一种利用超声波直接转化苹果属植物获得转基因苗木的方法:200610085395.5[P].2007-04-11.TAO Jianmin,ZHANG Zhen,WANG Sanhong,QIAO Yushan,XU Changbao,WANG Hongxia.A method of obtaining transfor-mants of plant by ultrosonic mediated transformation:200610085395.5[P].2007-04-11.
- [7]王旭明,何道一,陈丽萍.超声波辅助农杆菌介导的小麦遗传转化初步研究[J].激光生物学报,2012,21(2):176-180.WANG Xuming,HE Daoyi,CHEN Liping.Sonication-assisted Agrobacterium-mediated transformation of wheat[J].Acta LaserBiology Sinica,2012,21(2):176-180.
- [8]柴慈江,孙世海,王玉,彭立新.珠美海棠叶片离体培养与植株再生[J].果树学报,2011,28(1):124-128.CHAI Cijiang,SUN Shihai,WNAG Yu,PENG Lixin.Plant regen-eration from in vitro cultured leaves of Malus zumi[J].Journal ofFruit Science,2011,28(1):124-128.
- [9]吴瑞刚,杨洪强,沙广利.苹果砧木‘青砧一号’离体高效再生体系的建立[J].植物生理学报,2013,49(10):1053-1056.WU Ruigang,YANG Hongqiang,SHA Gangli.Establishment ofefficient in vitro regeneration system of apple rootstock‘QZ-1’(Malus hupehensis×M.domestica)[J].Plant Physiology Journal,2013,49(10):1053-1056.
- [10]肖波,廖尔华,胡开治.植物生长物质诱导芦荟叶片愈伤组织效果的研究[J].中国农学通报,2006,22(1):163-165.XIAO Bo,LIAO Erhua,HU Kaizhi.Study on callus induction ofaloe leaves with plant growth substances[J].Chinese AgriculturalScience Bulletin,2006,22(1):163-165.
- [11]KENNETH J,LIVAK T D.Analysis of relative gene expressiondata using real-time quanti-tative PCR and the 2-△△Ctmethod[J].Method,2001,25:402-408.
- [12]刘佳,李擎天,孔瑾.苹果砧木珠眉海棠盐胁迫应答基因的微列阵研究[J].中国农业大学学报,2009,14(5):61-67.LIU Jia,LI Qingtian,KONG Jin.Microarray analysis ofsalt2stress responsive genes in Malus zumi Mats[J].Journal ofChina Agricultural University,2009,14(5):61-67.
- [13]LI Q T,LIU J,TAN D X.A genome-wide expression profile ofsalt-responsive genes in the apple rootstock Malus zumi[J].Inter-national Journal of Molecular Sciences,2013,14:21053-21070.
- [14]黄黎芳,李擎天,蒋玉妆,陈浩.珠眉海棠盐应答Mz DREBb基因的克隆与功能研究[J].中国农业大学学报,2011,16(6):76-82.HUANG Lifang,LI Qingtian,JIANG Yuzhuang,CHEN Hao.Clon-ing and characterization of salt-responding Mz DREBb gene from Malus zumi Mats[J].Journal of China Agricultural University,2011,16(6):76-82.
- [15]YANG X L,JI J,WANG G.Over-expressing Salicania europaea(Se NHX1)gene in tobacco improves tolerance to salt[J].AfricanJournal of Biotechnology,2011,10(73):16452-16460.
- [16]YAMAGUCHI T,AHARON G S,SOTTOSANTO J B.VacuolarNa+/H+antiporter cation selectivity is regulated by calmodulinfrom within the vacuole in a Ca2+and p H dependent manner[J].Proceedings of the National Academy of Science USA,2005,102(44):16107-16112.
- [17]YIN C M,ZHENG L S,ZHU J H,CHEN L G,MA A M.Enhanc-ing stress tolerance by overexpression of a methionine sulfoxidereductase A(Msr A)gene in Pleurotus ostreatus[J].Applied Mi-crobiology and Biotechnology,2015,99:3115-3126.
- [18]SU X H,ZHOU P,WANG R,LUO Z P,XIA Z L.Overexpressionof the maize psb A gene enhances sulfur dioxide tolerance in trans-genic tobacco[J].Plant Cell Tissue and Organ Culture,2015,120:303-311.
- [19]XU X,WANG C,MA X,PAN Y,YING Q.Overexpression of Dn-WRKY29 in tobacco impaired plants tolerance to salt anddrought stresses[J].Russian Journal of Plant Physiology,2015,62(2):262-269.
- [20]SU Q,RAN K,MEN X J,ZHANG W W.Response of vacuolarprocessing enzyme in Malus hupehensis and Mh VPEc-overex-pressing Arabidopsis to high temperature stress[J].Acta Physiolo-giae Plantarum,2015,37(4):1-11.
- [21]刘会超,刘孟刚,郭丽娟.农杆菌介导的Se NHX1基因转化月季愈伤组织的研究[J].林业科学研究,2010,23(4):617-621.LIU Huichao,LIU Menggang,GUO Lijuan.Studies on Agrobacterium-mediated transformation of rose callus with Se NHX1 gene[J].Forest Research,2010,23(4):617-621.