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作为一种典型的土壤习居丝状真菌,淡紫紫孢菌(Purpureocillium lilacinum (Thom) Luangsa-ard, Houbraken, Hywel-Jones & Samson)是最具潜力的植物寄生线虫生防真菌之一[1-2],但它也同其它生防真菌制剂一样,存在着受环境影响较大、防治效果不稳定以及自身抗逆性较差等缺点[3-4]。目前无论是研究通过对生防菌株关键基因进行改造,达到提升生防菌制剂致病性和环境稳定性的目的[5],还是研究生防菌对病原物的致病机制及它们之间的相互作用机制,都需要建立高效的遗传转化系统。
农杆菌(Agrobacterium)介导的真菌转化已经在许多丝状真菌中得到广泛应用[6-15],农杆菌介导的生防真菌转化也已有不少成功的实例,例如已成功实现了昆虫病原真菌白僵菌(Beauveria bassiana ) [10] 、绿僵菌(Metarhizium anisopliae) [12]和蜡蚧轮枝菌(Lecanicillium lecanii)[15] 等生防真菌的遗传转化。真菌菌种的不同可以导致遗传转化效率不同 [16],另外转化载体和农杆菌菌株也可以影响遗传转化效率。G418作为筛选标记建立的遗传转化体系达到了1 000~2 400个转化子/106分生孢子,遗传转化效率和抗性转化子比率偏低[17]。基于此,针对不同真菌的遗传转化体系其具体参数必须进行优化。 beta-tubulin基因是多菌灵的抗性基因,淡紫紫孢菌对多菌灵高度敏感,可用于抗性筛选。
本文在前期研究的基础上,以beta-tubulin基因为筛选标记,对影响转化体系的一些参数,如诱导培养乙酰丁香酮(AS)的浓度、诱导培养时间、农杆菌终浓度 OD660值、共培养 AS 的浓度、共培养时间和共培养温度等因子进行优化,以期建立了农杆菌介导淡紫紫孢菌高效遗传转化体系,并从中突变体库中筛选鉴定出致病力变异的突变体,从而为淡紫紫孢菌的致病机制研究及优良菌株选育奠定基础。
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构建质粒pBI-G3C-BN的示意图见图1,将原有质粒pBI-G3C中的hph基因置换为beta-tubulin基因,beta-tubulin基因从质粒pCPXBN-1上扩增获得。
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淡紫紫孢菌菌株20-7菌落生长与PDA培养基中苯菌灵浓度密切相关,浓度越大,生长越受抑制。当苯菌灵浓度达到300 µg·mL−1以上时,完全抑制菌落生长(表1)。
表 1 苯菌灵浓度对淡紫紫孢菌20-7菌落生长的影响
Table 1. Effects of different concentrations of benomyl on the growth of P. lilacinum 20-7
浓度
Concentration/
(µg·mL−1)20-7菌落直径/cm
The colony diameter of P. lilacinum /cm48 h 72 h 96 h 108 h 0 2.11 5.13 7.57 9.12 100 0.34 0.83 1.46 1.94 200 0.01 0.03 0.04 0.06 300 0 0 0 0 400 0 0 0 0 500 0 0 0 0 1 000 0 0 0 0 -
淡紫紫孢菌孢子新鲜程度不同,对其遗传转化率有所影响。当AS浓度200 µg·mL−1,苯菌灵的筛选浓度为300 µg·mL−1时,新鲜孢子的转化率比保存一个月后的孢子转化率约增长15倍(表2)。
表 2 淡紫紫孢菌20-7菌株孢子新鲜程度对转化效率的影响
Table 2. Effects of different degree of spore freshness on transformation efficiency of P. lilacinum 20-7
孢子新鲜程度
Spores freshness转化子个数
Transformants /(×10−6)保存一个月的孢子
Spores saved for a month187 新鲜孢子
Fresh spores3 019 -
乙酰丁香酮AS浓度对淡紫紫孢菌转化效率影响明显,当AS浓度为200 µg·mL−1到1 000 µg·mL−1时,转化效率均较高(图2)。从节约成本出发,选用AS浓度在200 µg·mL−1到500 µg·mL−1为宜。
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农杆菌菌株EHA105和AGL-1在相同的试验条件下,对淡紫紫孢菌20-7菌株进行遗传转化的效率存在较大差异。农杆菌菌株AGL-1的所有生长浓度(OD660)均能对淡紫紫孢菌进行有效转化,而农杆菌菌株EHA105的生长浓度(OD660)在0.4以上时,遗传转化率较高。因此,农杆菌菌株及浓度的不同均能影响淡紫紫孢菌遗传转化率。EHA105生长浓度0.6时,转化效率最高,明显高于AGL-1的转化率(图3)。
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在相同的试验条件下,共培养培养基的酸碱度、共培养时间和温度都对转化效率有影响。酸碱度、时间和温度分别为pH5.5、48 h和25 ℃时转化效率最高(图4、图5和图6)。
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不同的共培养方式,转化效率不同, EHA105菌液和20-7孢子悬浮液混合振荡培养的转化效率约是混合液直接涂在滤纸上转化效率的1.6倍(表3)。
表 3 共培养方法对淡紫紫孢菌20-7转化效率的影响
Table 3. Effects of different co-culture methods on transformation efficiency of P. lilacinum 20-7
共培养方法
Co-culture method转化子个数
Transformants/ (×10−6)振荡培养 Shake the medium 3 208 涂于滤纸片上 Coat on filter paper 1 948 -
提取随机选择100个的转化子、质粒pBI-G3C-BN和原始菌株基因组DNA,并将质粒pBI-G3C-BN和原始菌株基因组DNA用作PCR研究的参考。使用beta-tubulin基因特异性引物进行扩增,从96个转化子和阳性对照中获得了约2.2 kb的序列,证明苯菌灵基因已整合到20-7转化子的基因组中;而同时观察到4个转化子没有条带,可能为假阳性,图7为随机选取的8个T20-7菌落PCR结果。
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从根癌农杆菌介导转化体系突变体库中随机选取的12个转化子中,每个转化子都有不一致的Southern杂交带,说明T-DNA是随机整合的。有10个转化子是单一杂交带,表明这10个转化子T-DNA插入是单拷贝的,占83.3%。另外2个转化子(6号和9号)有两条杂交带,说明这2个转化子其T-DNA插入为多拷贝,占16.7%(图8)。
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在PDA(苯菌灵 300 µg·mL−1)平板上接种原始菌株20-7和转化子72 h后,转化子T20-7菌落扩展2 cm左右,而20-7菌落没有扩展,被苯菌灵抑制。T20-7连续培养共5代,仍然不被苯菌灵抑制,从而表明转化子T20-7对苯菌灵的抗性稳定遗传。
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随机选取的20个转化子对南方根结线虫卵寄生率测定表明:仅1个转化子对南方根结线虫卵的寄生率显著增大,卵寄生率为86.67%,比野生型菌株20-7的卵寄生率67.64%提高了19.03%;而15个突变体侵染卵的效果明显下降,转化子最低寄生率为20.12%,与野生型菌株20-7相比下降了47.52%,结果最为显著(P=0.05);仅有4个突变体的致病力与20-7相比并无显著性差异(图9)。
以beta-tubulin基因为选择标记的淡紫紫孢菌遗传转化
Efficient Transformation System of Agrobacterium tumefaciens Mediated Transformation of Purpureocillium lilacinum by Using beta-tubulin as Selectable Marker
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摘要:
目的 建立稳定的食线虫真菌淡紫紫孢菌遗传转化体系,并获得插入突变体。 方法 介导的方法,以淡紫紫孢菌20-7的分生孢子为受体,将新构建的携带beta tubulin基因的质粒转化进入淡紫紫孢菌的细胞中,通过优化诱导乙酰丁香酮(AS)的浓度、诱导培养时间、农杆菌终浓度 OD660值、共培养 AS 的浓度、共培养时间和共培养温度等因子,建立高效遗传转化体系,获得致病力不同的突变体。 结果 共培养过程中使用萌发孢子是成功建立淡紫紫孢菌遗传转化体系的必要条件;淡紫紫孢菌萌发的孢子与农杆菌EHA105在25 ℃共振荡培养48 h时,且在共培养阶段当乙酰丁香酮浓度为 200 µg·mL−1(pH5.5)时转化效率最高,转化效率为1 200~3 200个转化子/106分生孢子, 阳性抗性转化子比率为96%;转化子PCR表明,T-DNA已整合到淡紫紫孢菌的基因组中;Southern 杂交验证表明,83.3% 的转化子为T-DNA 单拷贝插入;成功建立了可靠的淡紫紫孢菌的遗传转化体系,并从20个转化子中筛选到16个致病力变异的突变体。 结论 本研究成功构建了农杆菌介导的、以beta-tubulin基因为选择标记的淡紫紫孢菌高效遗传转化体系,并获得致病力变异的插入突变体,为淡紫紫孢菌的基因功能、致病机制研究及优良菌株选育奠定了基础。 -
关键词:
- 淡紫紫孢菌
- / 根癌农杆菌
- / beta-tubulin
- / 插入突变
- / 致病力
Abstract:Purpose To establish an efficient transformation system of the nematopathogenic fungus Purpureocillium lilacinum and obtain its insertional mutagenesis. Methods The benomyl resistance gene beta-tubulin being as the selective marker, Agrobacterium tumefaciens-mediated transformation technique was developed to screen different pathogenicity mutants in P. lilacinum. PCR amplification and Southern hybridization were used to verify the transformation events, and Southern blotting of beta-tubulin gene and cloning of transforming DNA (T-DNA) flanking sequences were used to determine insert number and site of T-DNA in the fungal genome, respectively. Results A reliable transformation method was established for P. lilacinum. Specifically, pre-germinating spores of P. lilacinum used at co-cultivated period was a prerequisite.P. lilacinum germinating spores co-cultivated with A. tumefaciens EHA105 at 25 ℃ for 48 h achieved the highest transformation efficiency, which was 1 200-3 200 transformants per 106 spores, and the ratio of positive resistant transformants was 96%. The transformants were cultivated up to 5 generations on beta-tubulin-containing medium and confirmed by PCR and those genetic traits remained stable. Southern hybridization showed that 83.3% of the transformants were single copy insertions of T-DNA, and 16 mutants with virulence variants were screened from 20 transformants. Conclusion This study successfully constructed an efficient genetic transformation system mediated by A. tumefaciens with beta-tubulin gene as a selective marker, and obtained an insertion mutant with pathogenicity variation, which was P. lilacinum. It provides insights into studying gene function, pathogenic mechanism and breeding excellent strains. -
表 1 苯菌灵浓度对淡紫紫孢菌20-7菌落生长的影响
Table 1. Effects of different concentrations of benomyl on the growth of P. lilacinum 20-7
浓度
Concentration/
(µg·mL−1)20-7菌落直径/cm
The colony diameter of P. lilacinum /cm48 h 72 h 96 h 108 h 0 2.11 5.13 7.57 9.12 100 0.34 0.83 1.46 1.94 200 0.01 0.03 0.04 0.06 300 0 0 0 0 400 0 0 0 0 500 0 0 0 0 1 000 0 0 0 0 表 2 淡紫紫孢菌20-7菌株孢子新鲜程度对转化效率的影响
Table 2. Effects of different degree of spore freshness on transformation efficiency of P. lilacinum 20-7
孢子新鲜程度
Spores freshness转化子个数
Transformants /(×10−6)保存一个月的孢子
Spores saved for a month187 新鲜孢子
Fresh spores3 019 表 3 共培养方法对淡紫紫孢菌20-7转化效率的影响
Table 3. Effects of different co-culture methods on transformation efficiency of P. lilacinum 20-7
共培养方法
Co-culture method转化子个数
Transformants/ (×10−6)振荡培养 Shake the medium 3 208 涂于滤纸片上 Coat on filter paper 1 948 -
[1] KERRY B R. An assessment of progress towards microbial control of plant parasitic nematodes[J]. Journal of Nematology, 1990, 22: 621-631. [2] ZHANG S W, GAN Y T, XU B L. Biocontrol potential of a native species of Trichoderma longibrachiatum against Meloidogyne incognita[J]. Applied Soil Ecology, 2015, 94: 21-29. doi: 10.1016/j.apsoil.2015.04.010 [3] GINE A, SORRIBAS F J. Effect of plant resistance and BioAct WG (Purpureocillium lilacinum strain 251) on Meloidogyne incognita in a tomato-cucumber rotation in a greenhouse[J]. Pest Management Science, 2017, 73(5): 880-887. doi: 10.1002/ps.4357 [4] KOOLIYOTTIL R, DANDURAND L M, KNUDSEN G R. Prospecting fungal parasites of the potato cyst nematode Globodera pallida using a rapid screening technique[J]. Journal of Basic Microbiology, 2017, 5(57): 386-392. [5] 柯心如, 刘登艳, 谭建彬, 等. 一株淡紫紫孢菌的分离、鉴定及生物学特性研究[J]. 广东农业科学, 2022, 49(6):108-117. [6] GROOT M D, BUNDOCK P, HOOYKAAS P, et al. Agrobacterium tumefaciens-mediated transformation of filamentous fungi[J]. Nature Biotechnology, 1998, 16(9): 839-42. doi: 10.1038/nbt0998-839 [7] FURLANETO M C, PAIÃO F G, PINTO F G S, et al. Transformation of the entomopathogenic fungus Metarhizium flavoviride to high resistance to benomyl[J]. Canadian Journal of Microbiology, 1999, 45: 875-878. doi: 10.1139/w99-074 [8] MULLINS E D, CHEN X, ROMAINE P, et al. Agrobacterium-mediated transformation of Fusarium oxysporum: an efficient tool for insertional mutagenesis and gene transfer[J]. Phytopathology, 2001, 91: 173-180. doi: 10.1094/PHYTO.2001.91.2.173 [9] ROGERS C W, CHALLEN M P, GREEN J R, et al. Use of REMI and Agrobacterium-mediated transformation to identify pathogenicity mutants of the biocontrol fungus Coniothyrium minitans[J]. FEMS Microbiology Letters, 2004, 241: 207-214. doi: 10.1016/j.femsle.2004.10.022 [10] REIS M C, PELEGRINELLI F M H, DELGADO R T, et al. Agrobacterium tumefaciens-mediated genetic transformation of the entomopathogenic fungus Beauveria bassiana[J]. Journal of Microbiological Methods, 2004, 58: 197-202. doi: 10.1016/j.mimet.2004.03.012 [11] LI M X, GONG X Y, ZHENG J, et al. Transformation of Coniothyrium minitans, a parasite of Sclerotinia sclerotiorum, with Agrobacterium tumefaciens[J]. FEMS Microbiology Letters, 2005, 243: 323-329. doi: 10.1016/j.femsle.2004.12.033 [12] FANG W, PEI Y, BIDOCHKA M J. Transformation of Metarhizium anisopliae mediated by Agrobacterium tumefaciens[J]. Canadian Journal of Microbiology, 2006, 52(7): 623-626. doi: 10.1139/w06-014 [13] DUARTE R T D, STAATS C C, FUNGARO M H P, et al. Development of a simple and rapid Agrobacterium tumefaciens-mediated transformation system for the entomopathogenic fungus Metarhizium anisopliae var. acridum[J]. Letters in Applied Microbiology, 2007, 44: 248-254. doi: 10.1111/j.1472-765X.2006.02092.x [14] 孙文良, 胡晓璐, 吴萌章, 等. 根癌农杆菌介导的深绿木霉菌T23遗传转化研究[J]. 上海交通大学学报:农业科学版, 2009, 27(5):489-493. [15] 赵津津. 蜡蚧轮枝菌“Lecanicillium lecanii (Zimmerman)Viegas”苯菌灵抗性基因转化的研究[D]. 北京: 中国农业科学院, 2011. [16] DNURM A, BAILEY A M, CAIRNS T C, et al. A silver bullet in a golden age of functional genomics: the impact of Agrobacterium-mediated transformation of fungi[J]. Fungal Biology and Biotechnology, 2017, 4(1): 6. doi: 10.1186/s40694-017-0035-0 [17] 王曦茁, 朴春根, 李 虹, 等. 根癌农杆菌介导的淡紫拟青霉遗传转化体系的建立[J]. 林业科学, 2010, 46(10):95-102. [18] 王关林. 植物基因工程原理与技术[M]. 北京: 科学出版社, 1998. [19] 黄培堂, 王嘉玺, 朱厚础. 分子克隆实验指南[M]. 北京: 科学出版社, 2002. [20] AUSUBEL F, BRENT R, KINGSTON R E, et al. Short Protocols in Molecular Biology. 3rd edition[M]. John Wiley & Sons, 1995: 228-244. [21] 汪来发, 杨宝君, 李传道. 寄生真菌对根结线虫的致病力评价[J]. 林业科学, 1999, 35(3):41-47. [22] MICHIELSE C B, HOOYKAAS P, HONDEL C, et al. Agrobacterium-mediated transformation as a tool for functional genomics in fungi[J]. Current Genetics, 2005, 48: 1-17. doi: 10.1007/s00294-005-0578-0 [23] 胡 懋, 曾杨璇, 苗华彪, 等. 根癌农杆菌介导真菌遗传转化的研究及应用[J]. 微生物学通报, 2021, 48(11):4344-4363. doi: 10.13344/j.microbiol.china.210156 [24] 赵培静, 任文彬, 缪承杜, 等. 2007. 根癌农杆菌介导的淡紫拟青霉遗传转化[C]//中国植物病理学会. 中国植物病理学会2007年学术年会论文集. 北京: 391-397.