dGFP-GUS基因标记新疆棉花黄萎病原菌的研究

发布时间：2018-08-30 15:18

【摘要】：【目的】研究棉花黄萎病原菌的入侵机理。【方法】以带有潮霉素抗性筛选基因的PUCCATPH载体以及pCAMBIA1304载体作为骨架,构建trpc启动子驱动的GFP-GUS基因的新疆棉花黄萎病菌表达载体1304-P-ORF,导入农杆菌GV3101和AGL-1中。通过农杆菌介导(ATMT)法分别转入新疆棉花黄萎病原菌VD-1和VD-278中,对表达效果进行检测。【结果】筛选的潮霉素B浓度为30μg/m L,农杆菌AGL-1对黄萎病菌的转化效率比GV3101高40%。T0代经含有潮霉素B的PDA培养基筛选,获得了T1代转基因大丽轮枝菌VD-12株,VD-2 784株。通过GUS组织染色,在转基因VD-1和VD-278的菌丝和孢子中均可观察到GUS基因的表达。对转基因黄萎菌进行PCR分子检测,均可检测到GFP和潮霉素B基因。GUS-GFP和潮霉素B基因已成功转入新疆棉花黄萎菌VD-1和VD-278中。【结论】建立了新疆棉花黄萎菌的遗传转化体系,为进一步利用GFP-GUS基因研究新疆棉花黄萎菌对棉花侵染的过程奠定基础。
[Abstract]:[objective] to study the invasion mechanism of Verticillium wilt in cotton. [methods] PUCCATPH vector with hygromycin resistance screening gene and pCAMBIA1304 vector were used as skeleton. The expression vector 1304-P-ORF of GFP-GUS gene driven by trpc promoter was constructed and introduced into Agrobacterium tumefaciens GV3101 and AGL-1. Agrobacterium tumefaciens mediated (ATMT) was transferred into VD-1 and VD-278 of cotton Verticillium wilt in Xinjiang, respectively. [results] the concentration of hygromycin B was 30 渭 g / mL. The transformation efficiency of Agrobacterium tumefaciens AGL-1 against Verticillium wilt was higher than that of GV3101. The PDA medium containing hygromycin B was used to screen the transformation efficiency of Agrobacterium tumefaciens AGL-1 against Verticillium wilt. The T 1 transgenic VD-12 strain VD-2 784 strains were obtained. The expression of GUS gene was observed in hyphae and spores of transgenic VD-1 and VD-278 by GUS staining. GFP and hygromycin B genes. GUS-GFP and hygromycin B genes were successfully transferred into VD-1 and VD-278 of cotton wilt bacteria in Xinjiang by PCR molecular detection. [conclusion] the genetic transformation system of cotton wilt bacteria in Xinjiang was established. The results laid a foundation for the further study of cotton infection by GFP-GUS gene.
【作者单位】：新疆石河子大学生命科学学院;
【基金】：农业部转基因重大专项“转基因棉花环境安全评价技术”(2016ZX08011)~~
【分类号】：S435.621.2

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