马倩倩;宋跃;孙毅;张绍铃;崔贵梅;吴俊;

• 1:南京农业大学梨工程技术研究中心
• 2:山西省农业科学院生物技术研究中心

摘要(Abstract)：

【目的】建立超声波处理‘黄花’梨花粉介导插入外源基因的技术体系。【方法】以‘黄花’梨花粉为试材,检测不同蔗糖溶液中梨花粉破损率;通过中心组合设计方法,对超声波处理功率、处理时间、间隙时间、处理次数4个因子进行优化;利用含荧光蛋白基因的表达载体pCAMBIA 1304检测转化效率。【结果】梨花粉的适宜等渗溶液为15g.L-1蔗糖溶液。超声波处理的梨花粉受体最佳参数为:超声波处理功率160~170 W,处理时间6~7 s,间隙时间7 s,处理次数7次。采用加入含GFP(green fluorescent protein)基因的pCAMBIA 1304表达载体质粒检测优化的超声波处理组合,梨花粉外源基因的插入效率为3%。【结论】研究确定了超声波处理‘黄花’梨花粉遗传转化体系。

关键词(KeyWords)： ‘黄花’梨;花粉介导;超声波处理;转化体系;GFP基因

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(31171928);; 江苏省科技支撑(BE2011320)

作者(Author): 马倩倩;宋跃;孙毅;张绍铃;崔贵梅;吴俊;

Email:

DOI: 10.13925/j.cnki.gsxb.2013.04.012

参考文献(References)：

• [1]WANG Fei,FANG Cheng-quan,LIN Sheng-hua.AFLP technique and its application in fruit tree genetics breeding[J].China Fruit,2006(3):51-53.王斐,方成泉,林盛华.AFLP技术及其在果树遗传育种上的应用[J].中国果树,2006(3):51-53.
• [2]LIU Cui-qiong,TANG Hao-ru.An overview of progress on shoot re-generation from pear leaves and genetic transformation[J].Journal of Fruit Science,2003,20(5):374-378.刘翠琼,汤浩茹.梨叶片培养与转基因研究进展[J].果树学报,2003,20(5):374-378.
• [3]ABU-QAOUDT H,SKIRVIN R M,BELOW F E.Influence of nitro-gen form and NH 4+-N:NO 3-—N ratio on adventious shoot formation from pear(Pyrus communis L.)leaf explant in vitro[J].Plant Cell,Tissue and Organ Culture,1991,27:315-319.
• [4]LEBLY C,CHEVRAU E,RABOIN L M.Adventitious shoot regen-eration from in vitro leaves of several pear cultivars(Pyrus communisL.)[J].Plant Cell,1991,25:99-105.
• [5]ZHU L H,WELANDER M.Adventitious shoot regeneration of two dwarfing pear rootstocks and the development of a transformation pro-tocol[J].Journal of Horticultural Science and Biotechnology,2000,75(6):745-752.
• [6]MENG Hao-guang,ZHANG Chao-hong,WANG Yue-jin.Trans-forming diseases-resistant gene VpPR10into pear‘Dangshan Suli’and optimization of the transformation system[J].Acta Horticulturae Sinica,2010,37(10):1567-1574.孟颢光,张朝红,王跃进.抗病基因VpPR10转化‘砀山酥梨’及转化条件的优化[J].园艺学报.2010,37(10):1567-1574.
• [7]ZHAO Rui-hua,LIU Qing-zhong,SUN Qing-rong,ZHANG Xian-sheng.Obtaining transgenic fertility pear(Pyrus communis L.)plants with antifungalγ-thionin Rs-afp 1 Gene[J].Journal of Agricultural Biotechnology,2004,12(6):729-730.赵瑞华,刘庆忠,孙清荣,张宪省.抗真菌γ-硫堇蛋白Rs-afp 1基因导入丰产梨获得转基因植株[J].农业生物技术学报,2004,12(6):729-730.
• [8]ZHANG Qing-zhu,HAN Tian-fu.Non-tissue culture transformation of plants[J].Molecular Plant Breeding,2004,2(1):85-91.张庆祝,韩天富.植物非组培遗传转化方法研究的进展[J].分子植物育种,2004,2(1):85-91.
• [9]ZENG Jun-zhi,WANG Dong-jiang,WU You-qiang,ZHANG Jian,ZHOU Wen-juan,ZHU Xiao-ping,XU Nai-zheng.Trans-genic wheat plants obtained with pollen tube pathway method[J].Science in China(Series B),1994,37(3):319-326.
• [10]DUAN Xiao-lan,CHEN Shan-bao.Variation of the characters in rice(oryza sativa)induced by foreign DNA uptake[J].Scientia Agri-cultura Sinica,1985(3):6-10.段晓岚,陈善葆.外源DNA导入水稻引起性状变异[J].中国农业科学,1985(3):6-10.
• [11]ZHOU Guang-yu,WENG Jian,GONG Zhen-zhen,ZENG Yi-shen,YANG Wan-xia,SHEN Wei-fang,WANG Zi-fen,TAO Quan-zhou,HUANG Jun-qi,QIAN Si-ying LIU Gui-ling,YING Miao-cheng,XUE Da-yuan,HONG Ai-hua,XU Ying-jun,CHEN Shan-bao,DUAN Xiao-lan.Molecular breeding of agriculture a technique for introducing exogenous DNA into plants after self polli-nation[J].Scientia Agricultura Sinica,1998,21(3):1-6.周光宇,翁坚,龚蓁蓁,曾以申,杨晚霞,沈慰芳,王自芬,陶全洲,黄骏麒,钱思颖,刘桂玲,应苗成,薛达元,洪爱华,徐英俊,陈善葆,段晓岚.农业分子育种授粉后外源DNA导入植物的技术[J].中国农业科学,1998,21(3):1-6.
• [12]HUANG Jun-qi,QIAN Si-ying,LIU Gui-ling,WENG Jian,ZENG Yi-shen,ZHOU Guang-yu.Variations in the characters of upland Cotton(Gossypium hirsutum)Induced by exotic DNA of sea-island cotton(Gossypium barbadense)[J].Acta Genetica Sinica,1981,8(1):56-62.黄骏麒,钱思颖,刘桂玲,翁坚,曾以申,周光宇.外源海岛棉DNA导致陆地棉性状的变异[J].遗传学报,1981,8(1):56-62.
• [13]LIU Ming,YANG Jun,AN Li-jia.Analysis of the factor impacting the gene transferring with the pollen-tube pathway method[J].Jour-nal of Anhui Agri Sci,2007,35(12):3483,3523.刘明,杨君,安利佳.花粉管通道法转化影响因素的分析[J].安徽农业科学,2007,35(12):3483,3523.
• [14]WANG Jing-xue,SUN Yi,CUI Gui-mei,HU Jing-jing.Transgenic maize plants obtained by pollen-mediated transformation[J].Acta Botanica Sinica,2001,43(3):275-279.
• [15]WANG Jie-zhi,HAO Xiao-fen,ZHENG Xiang-yang,WANG Lu-ying,SUN Yi,WANG Jing-xue.Study on pollen-mediated chitinase gene transformation of millet[J].Biotechnology,2004,14(5):5-6.王节之,郝晓芬,郑向阳,王路英,孙毅,王景雪.谷子花粉介导法转几丁质酶基因的研究[J].生物技术,2004,14(5):5-6.
• [16]LIANG Xue-lian,ZHENG Yi-xiong,ZHUANG Dong-hong,HUANG De-qiang.Peanut antipest gene transformation by pollen-mediation[J].Journal of Peanut Science,2006,35(4):1-5.梁雪莲,郑奕雄,庄东红,黄德强.花生CpTI基因转化—花粉介导法[J].花生学报,2006,35(4):1-5.
• [17]WEI Yu-jie,ZHANG Jin-wen,HE Qing-xiang.Study on the influ-ence factors about transferring radish peptide gene to wild opium poppy using pollen-mediated[J].Chinese Agricultural Science Bul-letin,2010,26(15):32-37.魏玉杰,张金文,何庆祥.花粉介导法将萝卜抗菌肽基因导入野罂粟的影响因素研究[J].中国农学通报,2010,26(15):32-37.
• [18]CAO Qiu-fen,YUE Xin-li,MENG Yu-ping,JI Lan,FAN Xin-ping.Effects of ultrasonication treatment on genetic transformation by pollen-mediated in apple[J].Journal of Nourth China Agricultur-al,2005,20(2):16-18.曹秋芬,岳新丽,孟玉平,季兰,樊新萍.超声波处理对苹果花粉介导转基因的影响[J].华北农学报,2005,20(2):16-18.
• [19]YUE Xin-li,CAO Qiu-fen,JI Lan,MENG Yu-ping,CHEN Jun.Studies on the ultrasonication parameters in pollen-mediated genetic transformation of grape[J].Journal of Nourth China Agricultural,2006,21(1):10-13.岳新丽,曹秋芬,季兰,孟玉平,陈俊.葡萄花粉介导转基因超声波处理参数的选择[J].华北农学报,2006,21(1):10-13.
• [20]ZHANG Shao-ling,CHEN Di-xin,KANG Lang,WANG Ling.Ef-fects of medium components and pH on pollen germination and tube growth in pear(Pyrus pyrifolia)[J].Acta Bot Boreal-occident Sinica,2005,25(2):225-230.张绍铃,陈迪新,康琅,汪玲.培养基组分及pH值对梨花粉萌发和花粉管生长的影响[J].西北植物学报,2005,25(2):225-230.
• [21]LIU Mo-fang,WANG En-duo.Green fluorescent protein[J].Progress in Biochemistry and Biophysics,2000,27(3):238-243.刘默芳,王恩多.绿色荧光蛋白[J].生物化学与生物物理进展,2000,27(3):238-243.
• [22]SHI Hua-zhong,YANG Hong-yuan,ZHOU Chang.Intrinsic GUS activity in various tissues and during pollen development of nicotiana tabacun[J].Acta Botanica Sinica,1995,37(2):134-139.施华中,杨弘远,周嫦.烟草花粉发育过程及不同组织中的内源GUS活性[J].植物学报,1995,37(2):134-139.
• [23]PIAO Yong-hao,QU Bai-hong,DAI Zhi-guo,JIN Jing-nan,PIAO Ri-zi.The research of pear pollen storage characteristics and pollina-tion abilities[J].Northern Horticulture,2002(5):54-55.朴永浩,曲柏宏,代志国,金京南,朴日子.梨花粉贮藏特性与授粉能力的研究[J].北方园艺,2002(5):54-55.