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通过含有pCAMBIA1300载体(抗潮霉素基因(hpt)作为选择标记基因)的根癌农杆菌菌株EHA105的介导,将326个银合欢I型碱性壳多糖酶基因(KB3,GenBank登录号为:AAM49597)的氨基酸导入到籼稻Khao Dawk Mali 105(KDML 105)植株中,获得了KDML 105籼稻的愈伤组织。pCAMBIA1300载体包含有CaMV35S启动子、银合欢壳多糖酶基因和NOS终止子。在NB培养基的水稻秧苗中愈伤组织的转化率为94%.在培养温度为28℃,以含有4 mg/l BAP和7 g/l植物凝胶再生培养基的条件下,愈伤组织的再生频率高达80%.为了建立一个KDML 105农杆菌转化方法,考察了各方面的因素。共培养期间,在培养基中添加50μM的乙酰丁香酮对提高瞬时转化率非常重要。4周龄的盾片衍生的愈伤组织是最佳的起始材料。添加40 mg/l潮霉素和400 mg/l头孢霉素的NB培养基是水稻愈伤组织选择性转化的最佳培养基,其转化率达到78%.采用PCR、Northern Blot杂交凝胶、Southern Blot杂交凝胶和GUS分析技术研究了愈伤组织和转基因水稻中的壳多糖酶和gus基因的表达。壳多糖酶和gus基因在水稻的多个部位被检测到,包括愈伤组织、叶、根、茎和秧苗,但是它们在水稻根部的含量较少。抗真菌的水稻壳多糖酶表现出抗串珠镰刀菌的性质。
326 Agrobacyl-type I basic chitinase genes (KB3, GenBank accession number: 1) were introduced into Agrobacterium tumefaciens strain EHA105 containing the pCAMBIA1300 vector (hyptomycin hpt as a selectable marker gene) AAM49597) was introduced into indica rice Khao Dawk Mali 105 (KDML 105) plants to obtain callus of KDML 105 indica rice. The pCAMBIA1300 vector contains the CaMV35S promoter, the silver flavanase gene and the NOS terminator. The callus transformation rate was 94% in the NB medium of rice seedlings.At the culture temperature of 28 ° C, with 4 mg / l BAP and 7 g / l plant gel regeneration medium, the callus The frequency of regeneration is as high as 80% .In order to establish a KDML 105 Agrobacterium transformation method, various factors were examined. During co-cultivation, adding 50 μM of acetosyringone to the medium is important for improving transient conversion. 4-week-old scutella-derived callus is the best starting material. NB medium supplemented with 40 mg / l hygromycin and 400 mg / l cefotaxine was the best medium for the selective transformation of rice callus with a conversion rate of 78% .Using PCR, Northern Blot Hybridization Gel, Southern Blot hybridization gel and GUS analysis of callus and transgenic rice chitinase and gus gene expression. Chitinase and gus genes are detected at multiple sites in rice, including callus, leaves, roots, stems and seedlings, but they are less abundant in rice roots. Antifungal rice chitinase exhibits Fusarium-resistant properties.