Acta Agrestia Sinica

›› 2010, Vol. 18 ›› Issue (3): 352-357.DOI: 10.11733/j.issn.1007-0435.2010.03.008

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Lysozyme-GFP Dual Gene in the Transformation in Callus of Alfalfa

Han Bin1,2,3, MA Hui-ling1,2,3, Li Yun-Xia1,2,3   

  1. 1. College of Prataculture, Gansu Agricultural University, Lanzhou, 730070, China;
    2. Key Laboratory of Grassland Ecosystem, Ministry of Education/Sino-U.S., Lanzhou, 730070, China;
    3. Centers for Grazingland Ecosystem Sustainability, Lanzhou, 730070, China
  • Received:2009-12-09 Revised:2010-01-28 Online:2010-06-15 Published:2010-06-15

Lyz-GFP双元基因在苜蓿愈伤组织中的转化

韩斌1,2,3, 马晖玲1,2,3, 李云霞1,2,3   

  1. 1. 甘肃农业大学草业学院, 甘肃, 兰州, 730070;
    2. 草业生态系统教育部重点实验室, 甘肃, 兰州, 730070;
    3. 中-美草地畜牧业可持续研究中心, 甘肃, 兰州, 730070
  • 通讯作者: 马晖玲,E-mail:mahl@g sau.edu.cn
  • 作者简介:韩斌(1984- ),男,河北人,硕士研究生,研究方向为牧草种质资源及育种,E-mail:hanbin-david@163.com
  • 基金资助:
    甘肃省农业生物技术研究与应用开发项目(GNSW-2007-03);国家科技支撑计划(2006BAD04A04-01-08)资助

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Abstract: Hypocotyls of 2 varieties of alfalfa(Gan-nong No.1 and Gan-nong No.3) were used as receptors respectively for transformation of Lyz-GFP genes by Agrobacterium tumefaciens mediated since December 2007.The callus containing dual genes of Lyz-GFP genes were harvested in this research.The results of fluorescence observation and PCR amplification showed that the Lys-GFP genes could express their function in alfalfa callus successfully.The effects of transformation of Lys-GFP dual genes in Gan-nong No.1 and Gan-nong No.3 were also studied in this paper,and better transformation conditions were established.A large number of resistant callus had been received.The results indicated that 75 mg·L-1 Kanamycin could restrain growth of alfalfa callus,300 mg·L-1 Cefotaxine could inhibit growth of Agrobacterium tumefaciens LBA4404.The optimum conditions for gene transformation in alfalfa were that hypocotyls were conducted to be precultured for 3-4 days,Agrobacterium tumefaciens whose concentration was OD600 0.4-0.5 was needed to inoculate alfalfa explants for 10 minutes.And MS medium supplemented with 2.0 mg·L-1 2,4-D,0.5 mg·L-1 KT,0.6% agar and 2.5% sucrose was needed to induce production of callus which resist Kanamycin from hypocotyls of alfalfa after these explants being incubated with Agrobacterium tumefaciens for 3 days.The induction rates of callus which resist Kanamycin of Gan-nong No.1 and Gan-nong No.3 were 35% and 32%,respectively.

Key words: Alfalfa, lysozyme gene, green fluorescent protein(GFP) gene, Callus, Agrobacterium tumefaciens mediated

摘要： 利用农杆菌介导法将溶菌酶(Lyz)与绿色荧光蛋白(GFP)基因转入"甘农1号"杂花苜蓿(Medicago sativa Martyn)和"甘农3号"紫花苜蓿(Medicagosativa L.)品种中,获得含有该双元基因的苜蓿转基因愈伤组织;采用荧光检测和PCR扩增技术,检测到溶菌酶Lyz和绿色荧光蛋白GFP基因在苜蓿愈伤细胞中的表达;研究该双元基因在苜蓿品种"甘农1号"和"甘农3号"中转化的若干影响因素,确定适宜的转化条件以期提高基因转化效率。结果表明:75 mg·L-1的卡那霉素对苜蓿愈伤组织的生长具有显著抑制效应;300mg·L-1头孢霉素能够有效地抑制农杆菌LBA4404的生长;OD600值为0.4-0.5的农杆菌适宜的侵染时间是10min;经预培养3-4d的材料侵染后再共培养3d之后,在愈伤组织诱导培养基MS+2,4-D 2.0mg·L-1+KT0.5 mg·L-1+0.6%琼脂+2.5%蔗糖的培养基上诱导出抗性愈伤组织;"甘农1号"和"甘农3号"的抗性愈伤组织诱导率分别为35%和32%。

关键词: 苜蓿, 溶菌酶Lyz基因, 绿色荧光蛋白GFP基因, 愈伤组织, 农杆菌介导

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