棉花农杆菌基因转化体系的优化和转苜蓿抗菌肽基因(alfAFP)植株的获得  

Optimization of Agrobacterium-mediated Cotton Gene Transformation and Obtainment of Transgenic Plants with Alflafa Antifungul Peptide Gene (alfAFP)

作　　者：张海平[1,2] 王学德[1] 邵明彦[1] 袁淑娜[1] 华水金[1] 倪密[1] 

机构地区：[1]浙江大学农业与生物技术学院农学系,杭州310029 [2]山西省农业科学院农作物品种资源研究所,太原030031

出　　处：《农业生物技术学报》2009年第6期1020-1026,共7页Journal of Agricultural Biotechnology

基　　金：国家重点基础研究发展规划(973)项目(No.2004CB11730502(1));浙江省重点科技项目(No.2008C22087);自然科学基金项目(No.Y306093)资助

摘　　要：为了优化根癌农杆菌(Agrobacterium tumefaciens)介导的棉花(Gossypium hirsutumL.)下胚轴切段的基因转化体系,以苜蓿(Medicago sativaL.)抗菌肽基因(alfla faantifungul peptide,alfAFP)为目标,利用β-葡萄糖甘酸酶基因(gus)和潮霉素抗性基因(Hpt)的检测方法,分析了根癌农杆菌菌种、菌液浓度、浸染下胚轴的时间、共培养中乙酰丁香酮(AS)的浓度、共培养温度和时间等因子对基因转化的影响。优化分析表明,用农杆菌菌株LBA4404,当OD600值为0.5～0.7的菌液浸染5～7日龄的棉花无菌苗下胚轴切段10～15min,在含200μmol/LAS的共培养基中21℃暗培养60h,可更有效地提高转化率。经上述条件处理的下胚轴在含50mg/L潮霉素的愈伤诱导培养基上培养3周可产生转化率最高的愈伤组织。愈伤组织经分化培养获得15株再生棉花,经过PCR和PCR-Southern分析,发现其中12株含有外源基因alfAFP。In order to develop a high effective gene transformation protocol of cotton （Gossypium hirsutum L.） hypocotyl pieces medieted via Agrobacterium tumefaciens, an alflafa （Medicago sativa L.） antifungul peptide gene （alfAFP） was used as a foreign gene, and effect factors of transformation,including Agrobacterium strains,bacterium density,infection time,acetosyringone （AS） concen- tration in co-culture medium, co-culture time and co-culture temperature, were evaluated by the expression detection of β-glueuronnidase gene （gus） and hygromycin phosphotransferase gene （Hpt） in transgenie callus. Optimum condition for transformation was that cotton hypocotyl pieces from 5 to 7 days-old sterile seedlings were dipped into Agrobacterium LBA4404 suspension （OD600=0.5 -0.7） for 10-15 min, then transferred on co-culture medium with 200 μmol/L AS and co-cultured at 21℃ for 60 h in the dark. The most amount of transgenie calli was gained by culturing hypocotyl pieces on callus introduction medium with 50 mg/L hygromycin. 15 regenerative plants were developed from somatic embryos differentiated from transgenic calli on embryogenesis medium. PCR and PcR-southem detection proved that the antifungal gene was introduced in 12 regenerative plants.

关 键 词：棉花 农杆菌 基因转化 

分 类 号：S188[农业科学—农业基础科学]