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火龙果又名红龙果,属仙人掌科(Cactaceae)量天尺属(Hylocereus),是极具发展前景的热带和亚热带水果[1]。火龙果因具有较高的经济价值和营养价值,集蔬菜、水果、花卉和保健于一体,被誉为“21世纪保健食品和果品珍品”, 受到人们的青睐,具有广阔的国际、国内市场[2]。火龙果20世纪80年代才引入我国台湾,虽然在我国栽培历史较短,但发展迅速,目前主要在台湾、海南、广西、广东、福建、贵州等省区种植。溃疡病是近年来火龙果种植园发生的严重真菌病害之一,发病率高达60%[3-7]。溃疡病造成火龙果枝条腐烂,果实变黑,失去食用价值,有些果园甚至颗粒无收。该病害在夏秋季高温高湿的气候条件下发病尤为严重,尤其台风过后更容易大面积暴发,这也是该病在海南地区盛行的主要原因之一。该病害由新暗色柱节孢(Neoscytalidium dimidiatum (Penz.) Crous & Slippers)侵染引起的[4-11],开展病原菌的致病机理研究可以为病害防治所需新药以及抗性种苗的研发提供相关的理论依据,从而达到对病害长期、有效的防控。病原菌基因功能缺失突变体的构建是研究病原菌致病机理的有效手段,这一方法的关键技术是建立遗传转化体系。农杆菌介导的遗传转化(Agrobaeterium tumefaciens-Meidated Transformation)可快速构建真菌T-DNA(transfer DNA)随机插入突变体,因其操作简单,可转化完整的细胞,比如分生孢子、菌丝、子实体等,免去了制备原生质体的繁琐,广泛应用于多种真菌[12]。目前已有100多种真菌使用该方法进行成功转化[13],比如玉米大斑病菌[14]、稻瘟病菌[15]、尖镰孢菌[16]、玉米灰斑病菌[17]等。本研究拟建立根癌农杆菌介导的火龙果溃疡病菌的遗传转化体系,为火龙果溃疡病菌致病机理的研究奠定技术基础。
Establishment of Agrobacterium tumefaciens-mediated transformation system for Neoscytalidium dimidiatum and Screening of Transformants
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摘要: 为了探究火龙果溃疡病病原菌的致病机理,笔者利用农杆菌介导的遗传转化技术,分析了根癌农杆菌浓度、分生孢子萌发时间以及共培养温度等3个主要因素对遗传转化效率的影响,建立火龙果溃疡病菌的遗传转化技术体系,并对转化子进行筛选。结果表明,当农杆菌浓度OD600=0.5、分生孢子萌发时间24 h、共培养温度25 ℃时遗传转化效率最高(833个转化子/106分生孢子);随机挑选200个转化子进行筛选,获得3株菌落形态与野生型菌株差异较大的转化子,4株产孢量减少的转化子,5株产孢量增加的转化子,6株致病力下降的转化子。Abstract: Pitaya is a nutrient-rich fruit with high economic value and great development potential in tropical and subtropical region. Pitaya stem canker caused by Neoscytalidium dimidiatum is one of the most serious fungal diseases infecting pitaya, which has become a main factor restricting the development of pitaya. In order to control the disease effectively, it is necessary to study the pathogenic mechanism of the pathogen causing pitaya stem canker. Agrobacterium tumefaciens-mediated genetic transformation was hence used to establish a genetic transformation system for N. dimidiatum. The effects of three main factors, concentration of A. tumefaciens, germination time of conidia, and co-culture temperature, on the transformation efficiency were analyzed, and transformants were screened. The results showed that the genetic transformation efficiency of N. dimidiatum was the highest, as high as 833 transformants / 106 conidia, when the OD600 of A. tumefaciens was 0.5, the germination time of conidia was 24 h, and the co-culture temperature was 25 ℃. Two hundred transformants were randomly selected from the genetic transformation for screening, of which 3 transformants with different colony morphology, 4 with decreased spore production, 5 with increased spore production, and 6 with decreased pathogenicity were generated.
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图 2 不同影响因子对遗传转化效率的影响
A. 农杆菌浓度;B. 分生孢子萌发时间;C. 共培养温度。a、b、c代表不同处理间存在显著差异(P<0.05)。
Fig. 2 The effect of different factors on the efficiency of genetic transformation
A. The concentration of A. tumefaciens;B. Germination time of conidia;C. Co-culture temperature Note:Letters a. b and c denote significant differences between the treatments at the P<0.05 level.
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