板栗非胚性愈伤组织DsRed基因瞬时转化方法建立和优化

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英文篇名：Establishment and optimization of transient transformation method of non-embryonic callus with DsRed gene in Chinese chestnut (Castanea mollissima Blume) 作者：李志 ; 邢宇 ; 房克凤 ; 曹庆芹 ; 秦岭 ; 张卿 英文作者：LI Zhi;XING Yu;FANG Kefeng;CAO Qingqin;QIN Ling;ZHANG Qing;College of Plant Science and Technology,Beijing Key Laboratory of New Technology in Agricultural Application;Beijing University of Agriculture;Beijing Collaborative Innovation Center for Eco-Environmental Improvement with Forestry and Fruit Tree;Beijing University of Agriculture;College of Landscape Architecture;Beijing University of Agriculture;College of Bioscience and Resource Environment;Beijing University of Agriculture; 关键词：‘燕山红栗’ ; 非胚性愈伤组织 ; 瞬时转化 ; 正交试验 ; 方法优化 英文关键词：chestnut;;Non-embryonic callus;;transient transformation;;orthogonal test;;method optimization 中文刊名：北京农学院学报 英文刊名：Journal of Beijing University of Agriculture 机构：北京农学院植物科学技术学院/农业应用新技术北京市重点实验室;北京农学院北京林果业生态功能提升协同创新中心;北京农学院园林学院;北京农学院生物与资源环境学院; 出版日期：2019-01-21 15:37 出版单位：北京农学院学报 年：2019 期：03 基金：国家自然科学基金项目(31870671);; 2016年北京市教委科研计划一般项目(PXM2016-014207-000008);; 2017年北京市农委菜篮子工程提升项目 语种：中文; 页：113-116 页数：4 CN：11-2156/S ISSN：1002-3186 分类号：S664.2

摘要

【目的】为建立并优化板栗非胚性愈伤组织瞬时转化技术体系。【方法】以‘燕山红栗’幼胚根尖诱导的非胚性愈伤组织为材料,参照美洲栗胚性愈伤组织转化方法,转化DsRed红色荧光蛋白基因,建立板栗非胚性愈伤组织瞬时转化技术体系。针对影响转化率的重要因子,设计菌液浓度、超声处理时间、共培养时间、干燥时间四因素五水平正交试验优化该方法。【结果】成功诱导板栗非胚性愈伤组织并观察到红色荧光,表明成功建立并优化了板栗非胚性愈伤组织瞬时转化方法:菌液浓度OD600=1.50,超声处理时间90s,共培养时间1h,干燥时间3d。【结论】该方法对于在细胞水平上研究糖类和淀粉代谢的分子机理具有重要意义。
        【Objective】The experiment is to establish and optimize the transient transformation technology system of non-embryonic callus in Chinese chestnut(Castanea mollissima Blume).【Methods】The Chinese chestnut non-embryonic callus was induced by the young embryonic root tip of Chinese chestnut cultivar'Yanshanhongli',and then the DsRed gene was transferred into non-embryonic callus based on the transient transformation method of American chestnut embryogenic callus.The Chinese chestnut non-embryonic callus transformation efficiency was determined by four factors:Agrobacterium tumefaciens concentration,sonication duration,co-culture duration and desiccation duration,therefore,the experiment was optimized by designing a four-factor and five-level orthogonal experiments.【Results】We successfully observed red fluorescence from Chinese chestnut non-embryonic callus transferred DsRedgene.The results showed that the optimal transient transformation technology system of non-embryonic callus in Chinese chestnut was as the following:the concentration of culture was OD600=1.50,the sonication time was 90 s,and the co-culture time was 1 h,the drying time was 3 d.【Conclusion】This method is significant for studying the molecular mechanism of carbohydrate and starch metabolism at the cellular level.

引文

[1]王广鹏,孔德军,刘庆香.板栗支链淀粉含量与坚果糯性的定量关系研究[J].河北果树,2008(4):6-7
    [2]陈良珂.板栗坚果淀粉积累规律及淀粉分支酶的基因克隆与分析[D].北京:北京农学院,2017
    [3]Chen L,Lu D,Wang T,Li Z,Zhao Y,Jiang Y,Zhang Q,Cao Q,Fang K,Xing Y,Qin L.Identification and expression analysis of starch branching enzymes involved in starch synthesis during the development of chestnut(Castanea mollissima Blume)cotyledons[J].PLoS ONE,2017,12(5):e0177792
    [4]王猛,梁雪.板栗颗粒型淀粉结合酶基因cDNA的克隆与生物信息学分析[J].北方园艺,2016(16):99-103
    [5]Zhang B,Newhouse A,McGuigan L,Maynard C,Powell W.Agrobacterium-mediated co-transformation of American Chestnut(Castanea dentata)somatic embryos with a wheat oxalate oxidase gene[J].BMC Proceedings,2011(Suppl7):43
    [6]Corredoira E,San JoséM C,Vieitez A M,Allona I,Aragoncillo C,Ballester A.Agrobacterium-mediated transformation of European chestnut somatic embryos with a Castanea sativa(Mill.)endochitinase gene[J].New Forests,2016,47(5):669-684
    [7]Lu D,Wei W,Zhou W,McGuigan L,Ji F,Li X,Xing Y,Zhang Q,Fang K,Cao Q,Qin L.Establishment of a somatic embryo regeneration system and expression analysis of somatic embryogenesis-related genes in Chinese chestnut(Castanea mollissima Blume.)[J].Plant Cell,Tissue and Organ Culture,2017,130(3):601-616
    [8]Newhouse AE,Polin-McGuigan LD,Baier KA,Valletta KE,Rottmann WH,Tschaplinski TJ,Maynard CA,Powell WA.Transgenic American chestnuts show enhanced blight resistance and transmit the trait to T1progeny[J].Plant Science,2014,228:88-97
    [9]王栋鑫,彭棣,张爽.农杆菌介导木本植物遗传转化的研究进展[J].北方园艺,2018(2):181-185
    [10]王建军,冯莉赟,申虎飞,刘佼.超声波辅助的植物转化方法的研究进展[J].农学学报,2017(11):6-13
    [11]孙华军,李国瑞,陈永胜,黄凤兰,李跃,邢超,赵永,陈晓凤,包长春,张智勇.农杆菌介导的植物遗传转化影响因素研究进展[J].安徽农业科学,2015(24):26-27,77
    [12]叶兴国,王新敏,王轲,杜丽璞,林志珊,徐惠君.提高植物农杆菌转化效率辅助策略研究进展[J].中国农业科学,2012(15):3007-3019