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火龙果 (Hylocereus polyrhizus) 属于仙人掌科的热带植物,原产于中美洲和南美洲[1]。火龙果果实颜色亮丽并含有多酚类化合物[2],红肉火龙果含有甜菜红素[3-4]等丰富的生物活性成分,具有抗氧化、清肠通便等多种生理功能,有很高的营养价值和经济价值[5-7]。近年来,火龙果在我国种植面积增长十分迅速,截至2020年,我国火龙果种植面积已经超过6.67万 hm2。随着种植面积的扩大,火龙果的病害问题日趋严重,其中危害最严重的是火龙果溃疡病[8-9]。2012年,火龙果溃疡病在中国台湾首次被报道[10],随后在马来西亚、以色列、墨西哥、美国、尼加拉瓜和中国的广东、广西、云南、贵州、海南等火龙果种植区均有发现[9,11-12],火龙果溃疡病严重危害火龙果的产量和品质,在夏季,火龙果茎部溃疡病病株发病率高达60%,该病具有发病快、传播广、危害大等特点。经研究表明,引起该病的病原菌为新暗色柱节孢菌 (Neoscytalidium dimidiatum)[13],该病菌主要损害火龙果树的茎和果实,导致果实和茎条先出现白色病斑,随着时间的推移转变为棕色,最后表现为茎腐病、果实开裂[14-15]、内部褐腐病和黑腐病[11,14],此病害在高温和潮湿的环境中更具侵袭性[16],对火龙果的产量、质量产生有害影响,进而影响其商业价值[17]。目前,已经分离和鉴定了火龙果溃疡病的致病菌,但关于其遗传转化体系的研究仍为空白,而有效的遗传转化体系对于新暗色柱节孢菌致病机理及相关致病基因的研究将起到至关重要的作用。真菌遗传转化主要分为原生质体转化和农杆菌介导转化2种途径[18]。原生质体法操作简单,但转化效率低、变异率高,而且PEG有一定的毒性,可能会对原生质体的生长有一定影响,制备原生质体程序繁琐,因此,在丝状真菌的转化应用中受限[4,19-21]。2001年MULLINS 等[22]通过构建二元载体,通过农杆菌的介导实现了对镰刀菌的有效遗传转化,到目前为止已有大量的农杆菌介导转化真菌的报道[23],但是因为真菌材料的性质差异,所用农杆菌菌株和真菌分离株及共培养条件的不同,对不同的真菌物种进行优化是一项艰巨的任务。迄今为止,国内外还没有关于农杆菌介导新暗色柱节孢菌转化的相关报道,其在新暗色柱节孢菌中的有效性尚待进一步研究。笔者利用农杆菌介导转化法,将含有潮霉素抗性基因(hyg)和绿色荧光蛋白基因(mGFP)的双元表达载体转化到新暗色柱节孢菌中,并在阳性转化子中检测绿色荧光蛋白的表达,旨在为进一步研究新暗色柱节孢菌的菌丝生长过程、形态特征、致病菌与火龙果之间的互作致病机制等提供技术支撑。
Agrobacterium tumefaciens-mediated Transformation of Neoscytalidium dimidiatumcausing Stem Canker on Pitaya
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摘要: 火龙果是一种新兴的热带水果,随着种植面积的不断增大,病害问题日趋严重,其中危害最大的是火龙果溃疡病,导致火龙果溃疡病的病原菌为新暗色柱节孢菌(Neoscytalidium dimidiatum)。为了研究新暗色柱节孢菌的遗传变异与基因功能,必须建立有效的遗传转化体系,但目前国内外未见相关报道。本研究通过构建含有gpdA启动子、绿色荧光蛋白(green fluorescent protein)和潮霉素(Hygromycin)抗性基因为筛选标记的双元表达载体,对新暗色柱节孢菌的孢子进行根癌农杆菌介导的遗传转化,成功获得了阳性转化子。通过荧光显微镜观察表明,阳性转化子菌丝能够产生绿色荧光,野生型菌丝不能产生绿色荧光;PCR检测证明了转化子基因组中整合了潮霉素抗性基因。因此,本研究成功实现了根癌农杆菌介导下绿色荧光蛋白基因在新暗色柱节孢菌中的稳定遗传表达,为研究新暗色柱节孢菌对火龙果溃疡病的致病机制奠定了技术基础。
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关键词:
- 火龙果溃疡病 /
- 新暗色柱节孢菌 /
- 潮霉素抗性基因 /
- 绿色荧光蛋白 /
- 农杆菌介导的遗传转化
Abstract: In recent years, pitaya (Hylocereus spp.) has gradually become a newly-emerging tropical fruit. With the continuous increase of planting area, diseases infecting pitaya have become more serious. Among them stem canker is the most important disease, which is caused by Neoscytalidium dimidiatum. To explore the genetic variation and gene function of N. dimidiatum, an effective genetic transformation system must be established. However, there have been no reports documented at home or abroad in this aspect. A binary expression vector containing gpdA promoter, green fluorescent protein (mGFP) and hygromycin resistance gene as screening markers was constructed to establish a genetic transformation system for N. dimidiatum through Agrobacterium tumefacien-mediation, and positive transformants from the spores of N. dimidiatumwere successfully generated. Fluorescence microscope observations showed that the positive transformant hyphae could produce green fluorescence, while the wild-type hyphae could not produce green fluorescence. The PCR test confirmed the integration of hygromycin resistance gene in the transformant genome. Therefore, this Agrobacterium tumefaciens mediated transformation system produced stable genetic expression of mGFP gene in N. dimidiatum , which lays a technical foundation for the further study of the pathogenicity mechanism of dragon fruit canker disease. -
图 2 新暗色柱节孢菌野生型与转化子的菌丝生长情况对比
新暗色柱节孢菌野生型(A)和转化子(C)在不含有抗性的PDA平板上28 ℃培养1周情况对比;新暗色柱节孢菌野生型(B)和转化子(D)在含有50 µg·mL−1潮霉素B和200 µg·mL−1 Cef 的PDA平板上28 ℃培养7 d情况对比。
Fig. 2 Comparison of hyphae growth between wild-type and transformant of Neoscytalidium dimidiatum
Comparison of the wild‐type Neoscytalidium dimidiatum (A) and putative transformants (C) cultured on the Potato Dextrose Agar (PDA) medium without presence of antibiotics for 7 days at 28 ℃; comparison of the growth of the wild-type Neoscytalidium dimidiatum (B) and putative transformants (D) cultured on the PDA medium supplemented with 50 µg·mL−1 of hygromycin and 200 µg·mL−1 of cefotaxime for 7 days at 28 ℃.
图 3 mGFP荧光蛋白在新暗色柱节孢菌转化子中的表达情况检测
野生型新暗色柱节孢菌和转化子在明场、荧光场的绿色荧光蛋白 (mGFP) 表达情况。明场成像A、 C; 荧光场成像B、D。
Fig. 3 Detection of the expression of mGFP fluorescent protein in the transformants of Neoscytalidium dimidiatum
The expression of green fluorescent protein (mGFP) in bright field and fluorescent field of wild-type Neoscytalidium dimidiatum and transformant. A, C: Bright-field imaging; B, D: Fluorescence imaging.
图 4 转化子的PCR检测
M:DNA marker;1~3:野生型新暗色柱节孢菌,作为阴性对照;4:含有潮霉素基因的质粒,阳性对照;5~7:随机挑选的新暗色柱节孢菌转化子。
Fig. 4 PCR identification of transformants
M: DNA Marker DL2000; 1~3: Wild type Neoscytalidium dimidiatum as a negative control; 4: Plasmid with hygromycin gene as a positive control; 5~7: Randomly selected transformants of Neoscytalidium dimidiatum.
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