Lyz-GFP双元基因在苜蓿愈伤组织中的转化  

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

作　　者：韩斌[1,2,3] 马晖玲[1,2,3] 李云霞[1,2,3] 

机构地区：[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

出　　处：《草地学报》2010年第3期352-357,共6页Acta Agrestia Sinica

基　　金：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

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 sativaMartyn）和＂甘农3号＂紫花苜蓿（Medicago sativaL.）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;300 mg.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10 min;经预培养3~4 d的材料侵染后再共培养3 d之后,在愈伤组织诱导培养基MS＋2,4-D 2.0 mg.L-1＋KT 0.5 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%。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.

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 

分 类 号：Q786[生物学—分子生物学] Q814.9