CHINESE JOURNAL OF OIL CROP SCIENCES ›› 2021, Vol. 43 ›› Issue (6): 1115-1125.doi: 10.19802/j.issn.1007-9084.2020256
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Yue-han YANG(), Jing ZHANG, Long YANG, Ming-de WU, Guo-qing LI(
)
Received:
2020-08-26
Online:
2021-12-22
Published:
2021-12-23
Contact:
Guo-qing LI
E-mail:329017660@qq.com;guoqingli@mail.hzau.edu.cn
CLC Number:
Yue-han YANG, Jing ZHANG, Long YANG, Ming-de WU, Guo-qing LI. Optimization of T-DNA insertion-mediated mutagenesis of Leptosphaeria biglobosa and mutant screening[J]. CHINESE JOURNAL OF OIL CROP SCIENCES, 2021, 43(6): 1115-1125.
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URL: http://www.jouroilcrops.cn/EN/10.19802/j.issn.1007-9084.2020256
Table 1
Primers and their oligonucleotide sequences
引物 Primer | 序列(5’-3’) Sequence (5’ to 3’) | 用途 Purpose |
---|---|---|
HYG-F | GACAGCGTCTCCGACCTGA | 检测潮霉素抗性基因,探针 Detection of the hph gene as probe |
HYG-R | GCTCCATACAAGCCAACCAC | |
LAD1-1 | ACGATGGACTCCAGAGCGGCCGCVNVNNNGGAA | hiTAIL-PCR(第一轮) hiTAIL-PCR (the first run) |
LAD1-2 | ACGATGGACTCCAGAGCGGCCGCBNBNNNGGTT | hiTAIL-PCR(第一轮) hiTAIL-PCR (the first run) |
LAD1-3 | ACGATGGACTCCAGAGCGGCCGCVVNVNNNCCAA | hiTAIL-PCR(第一轮) hiTAIL-PCR (the first run) |
LAD1-4 | ACGATGGACTCCAGAGCGGCCGCBDNBNNNCGGT | hiTAIL-PCR(第一轮) hiTAIL-PCR (the first run) |
AC-1 | ACGATGGACTCCAGAG | hiTAIL-PCR(第一、二轮) hiTAIL-PCR (the first and second runs) |
RB-0a | GGCAATAAAGTTTCTTAAGATTGAATCCTGT | hiTAIL-PCR(第一轮) hiTAIL-PCR (the first run) |
RB-1a | ACGATGGACTCCAGTCCGGCCTGTTGCCGGTCTTGCGATGATTATCA | hiTAIL-PCR(第二轮) hiTAIL-PCR (the second run) |
RB-2a | GTAATGCATGACGTTATTTATGAGATGGGTT | hiTAIL-PCR(第三轮) hiTAIL-PCR (the thirsd run) |
Fig. 2
Optimization of the factors affecting the efficiency of Agrobacterium tumeficiens-mediated transformation of L. biglobosa Lb731Note: A: Incubation time for transformation recipients (conidia); B: Concentration of the transformation recipients; C: Co-incubation temperature for A. tumeficiens-L. biglobosa; D: Co-incubation time for A. tumeficiens-L. biglobosa
Fig. 3
Quality evaluation of T-DNA-insertional library for L. biglobosa Lb731Note: A: An electrophoregram showing the PCR-amplified DNA fragments of the hygromycin-resistance gene in the wild type Lb731 and 14 transformants; B: Comparison of the morphology of the first and the fifth generation of the representative transformants (PDA + hygromycin B at 50 μg/mL, 20℃, 15 d); C: Southern blotting of the Sac I-digested DNA from Lb731 and representative transformants using the partial sequence of the hygromycin-resistance gene as probe
Table 2
Comparison of mycelial growth and colony morphology among 35 mutants
突变体类型 Mutation type | 突变体 Mutant | 菌丝生长 Mycelial growth | 色素产生 Pigment production | 分生孢子产生 Conidial production |
---|---|---|---|---|
菌丝生长抑制型 Suppressed mycelial growth | LbT-411 | ─ | + | ─ |
LbT-529 | ─ | + | ND | |
LbT-605 | ─ | + | ─ | |
LbT-667 | ─ | + | ND | |
LbT-671 | ─ | + | ND | |
LbT-1256 | ─ | + | ─ | |
LbT-1709 | ─ | + | ─ | |
LbT-1729 | ─ | + | ─ | |
LbT-1818 | ─ | + | ─ | |
LbT-1854 | ─ | + | ─ | |
LbT-1960 | ─ | ─ | ─ | |
色素产生缺陷型 Deficiency in pigment production | LbT-126 | ND | ─ | ─ |
LbT-574 | ND | ─ | ─ | |
LbT-595 | ND | ─ | ND | |
LbT-754 | ─ | ─ | ─ | |
LbT-1570 | ND | ─ | ─ | |
LbT-1731 | ND | ─ | ND | |
LbT-2233 | ND | ─ | ─ | |
分生孢子产生缺陷型 Deficiency in conidial production | LbT-85 | ND | + | ─ |
LbT-314 | ─ | ND | ─ | |
LbT-340 | ND | ND | ─ | |
LbT-392 | ND | ND | ─ | |
LbT-544 | ─ | ND | ─ | |
LbT-646 | ND | + | ─ | |
LbT-721 | ─ | + | ─ | |
LbT-894 | ND | ND | ─ | |
LbT-999 | ─ | ND | ─ | |
LbT-1208 | ─ | ND | ─ | |
LbT-1361 | ND | ND | ─ | |
LbT-1598 | ─ | ND | ─ | |
LbT-1601 | + | ND | ─ | |
LbT-2153 | ─ | ND | ─ |
1 |
West J S, Kharbanda P D, Barbetti M J, et al. Epidemiology and management of Leptosphaeria maculans (Phoma stem canker) on oilseed rape in Australia, Canada and Europe[J]. Plant Pathol, 2001, 50(1): 10-27. DOI:10.1046/j.1365-3059.2001.00546.x.
doi: 10.1046/j.1365-3059.2001.00546.x |
2 |
Fitt B D L, Brun H, Barbetti M J, et al. World-wide importance of Phoma stem canker (Leptosphaeria maculans and L. biglobosa) on oilseed rape (Brassica napus)[J]. Eur J Plant Pathol, 2006, 114(1): 3-15. DOI:10.1007/s10658-005-2233-5.
doi: 10.1007/s10658-005-2233-5 |
3 |
West J S, Evans N, Liu S, et al. Leptosphaeria maculans causing stem canker of oilseed rape in China[J]. Plant Pathol, 2000, 49(6): 800. DOI:10.1046/j.1365-3059.2000.00503.x.
doi: 10.1046/j.1365-3059.2000.00503.x |
4 |
李强生, 荣松柏, 胡宝成, 等. 中国油菜黑胫病害分布及病原菌鉴定[J]. 中国油料作物学报, 2013, 35(4): 415-423. DOI:10.7505/j.issn.1007-9084.2013.04.011.
doi: 10.7505/j.issn.1007-9084.2013.04.011 |
5 |
宋培玲, 吴晶, 史志丹, 等. 油菜黑胫病的病原、病害循环及其传播危害[J]. 北方农业学报, 2018, 46(2): 88-93. DOI:10.3969/j.issn.2096-1197.2018.02.18.
doi: 10.3969/j.issn.2096-1197.2018.02.18 |
6 |
郝丽芬, 宋培玲, 李子钦, 等. ISSR标记分析油菜黑胫病原菌遗传多样性[J]. 中国油料作物学报, 2014, 36(1): 98-105. DOI:10.7505/j.issn.1007-9084.2014.01.015.
doi: 10.7505/j.issn.1007-9084.2014.01.015 |
7 |
Liu Z, Latunde-Dada A O, Hall A M, et al. Phoma stem canker disease on oilseed rape (Brassica napus) in China is caused by Leptosphaeria biglobosa ‘brassicae’[J]. Eur J Plant Pathol, 2014, 140(4): 841-857. DOI:10.1007/s10658-014-0513-7.
doi: 10.1007/s10658-014-0513-7 |
8 |
Cai X, Zhang J, Wu M D, et al. Effect of water flooding on survival of Leptosphaeria biglobosa ‘brassicae’ in stubble of oilseed rape (Brassica napus) in central China[J]. Plant Dis, 2015, 99(10): 1426-1433. DOI:10.1094/pdis-10-14-1042-re.
doi: 10.1094/pdis-10-14-1042-re |
9 |
Cai X, Huang Y J, Jiang D H, et al. Evaluation of oilseed rape seed yield losses caused by Leptosphaeria biglobosa in central China[J]. Eur J Plant Pathol, 2018, 150(1): 179-190. DOI:10.1007/s10658-017-1266-x.
doi: 10.1007/s10658-017-1266-x |
10 |
荣松柏, 胡宝成, 陈凤祥, 等. 油菜黑胫病对油菜产量及农艺性状的影响[J]. 作物杂志, 2015(6): 159-161, 167. DOI:10.16035/j.issn.1001-7283.2015.06.029.
doi: 10.16035/j.issn.1001-7283.2015.06.029 |
11 |
Rouxel T, Balesdent M H. The stem canker (blackleg) fungus, Leptosphaeria maculans, enters the genomic era[J]. Mol Plant Pathol, 2005, 6(3): 225-241. DOI:10.1111/j.1364-3703.2005.00282.x.
doi: 10.1111/j.1364-3703.2005.00282.x |
12 |
Grandaubert J, Lowe R G, Soyer J L, et al. Transposable element-assisted evolution and adaptation to host plant within the Leptosphaeria maculans-Leptosphaeria biglobosa species complex of fungal pathogens[J]. BMC Genomics, 2014, 15: 891. DOI:10.1186/1471-2164-15-891.
doi: 10.1186/1471-2164-15-891 |
13 |
荣松柏, 李强生, 初明光. 油菜黑胫病病原菌致病力差异分析[J]. 安徽农业科学, 2019, 47(21): 136-138, 159. DOI:10.3969/j.issn.0517-6611.2019.21.040.
doi: 10.3969/j.issn.0517-6611.2019.21.040 |
14 |
Bundock P, den Dulk-Ras A, Beijersbergen A, et al. Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae[J]. EMBO J, 1995, 14(13): 3206-3214. DOI:10.1002/j.1460-2075.1995.tb07323.x.
doi: 10.1002/j.1460-2075.1995.tb07323.x |
15 |
Michielse C B, Hooykaas P J, van den Hondel C A, et al. Agrobacterium-mediated transformation as a tool for functional genomics in fungi[J]. Curr Genet, 2005, 48(1): 1-17. DOI:10.1007/s00294-005-0578-0.
doi: 10.1007/s00294-005-0578-0 |
16 |
Eckert M, Maguire K, Urban M, et al. Agrobacterium tumefaciens-mediated transformation of Leptosphaeria spp. and Oculimacula spp. with the reef coral gene DsRed and the jellyfish gene gfp[J]. FEMS Microbiol Lett, 2005, 253(1): 67-74. DOI:10.1016/j.femsle.2005.09.041.
doi: 10.1016/j.femsle.2005.09.041 |
17 |
Blaise F, Rémy E, Meyer M, et al. A critical assessment of Agrobacterium tumefaciens-mediated transformation as a tool for pathogenicity gene discovery in the phytopathogenic fungus Leptosphaeria maculans[J]. Fungal Genet Biol, 2007, 44(2): 123-138. DOI:10.1016/j.fgb.2006.07.006.
doi: 10.1016/j.fgb.2006.07.006 |
18 | 范雷. 灰葡萄孢T-DNA插入突变体库的构建及其致病相关基因研究[D]. 武汉: 华中农业大学, 2012. |
19 |
Möller E M, Bahnweg G, Sandermann H, et al. A simple and efficient protocol for isolation of high molecular weight DNA from filamentous fungi, fruit bodies, and infected plant tissues[J]. Nucleic Acids Res, 1992, 20(22): 6115-6116. DOI:10.1093/nar/20.22.6115.
doi: 10.1093/nar/20.22.6115 |
20 | M.R.格林, J.萨姆布鲁克, 贺福初主译. 分子克隆实验指南[M]. 北京: 科学出版社, 2017. |
21 |
Liu Y G, Chen Y L. High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences[J]. BioTechniques, 2007, 43(5): 649-656. DOI:10.2144/000112601.
doi: 10.2144/000112601 |
22 |
Fitt B D L, Huang Y J, van den Bosch F, et al. Coexistence of related pathogen species on arable crops in space and time[J]. Annu Rev Phytopathol, 2006, 44: 163-182. DOI:10.1146/annurev.phyto.44.070505.143417.
doi: 10.1146/annurev.phyto.44.070505.143417 |
23 |
杨龙, 吴明德, 张静, 等. 油菜黑胫病研究进展[J]. 中国油料作物学报, 2018, 40(5): 730-736. DOI:10.7505/j.issn.1007-9084.2018.05.016.
doi: 10.7505/j.issn.1007-9084.2018.05.016 |
24 |
Lowe R G T, Cassin A, Grandaubert J, et al. Genomes and transcriptomes of partners in plant-fungal- interactions between canola (Brassica napus) and two Leptosphaeria species[J]. PLoS One, 2014, 9(7): e103098. DOI:10.1371/journal.pone.0103098.
doi: 10.1371/journal.pone.0103098 |
25 |
Bolton M D, Thomma B P H J, Nelson B D. Sclerotinia sclerotiorum (Lib.) de Bary: biology and molecular traits of a cosmopolitan pathogen[J]. Mol Plant Pathol, 2006, 7(1): 1-16. DOI:10.1111/j.1364-3703.2005.00316.x.
doi: 10.1111/j.1364-3703.2005.00316.x |
26 |
Williamson B, Tudzynski B, Tudzynski P, et al. Botrytis cinerea: the cause of grey mould disease[J]. Mol Plant Pathol, 2007, 8(5): 561-580. DOI:10.1111/j.1364-3703.2007.00417.x.
doi: 10.1111/j.1364-3703.2007.00417.x |
27 |
Choquer M, Fournier E, Kunz C, et al. Botrytis cinerea virulence factors: new insights into a necrotrophic and polyphageous pathogen[J]. FEMS Microbiol Lett, 2007, 277(1): 1-10. DOI:10.1111/j.1574-6968.2007.00930.x.
doi: 10.1111/j.1574-6968.2007.00930.x |
28 |
Godoy G, Steadman J R, Dickman M B, et al. Use of mutants to demonstrate the role of oxalic acid in pathogenicity of Sclerotinia sclerotiorum on Phaseolus vulgaris[J]. Physiol Mol Plant Pathol, 1990, 37(3): 179-191. DOI:10.1016/0885-5765(90)90010-U.
doi: 10.1016/0885-5765(90)90010-U |
29 |
Badawy H M A, Hoppe H H, Koch E. Differential reactions between the genus Brassica and aggressive single spore isolates of Leptosphaeria maculans[J]. J Phytopathol, 1991, 131(2): 109-119. DOI:10.1111/j.1439-0434.1991.tb04737.x.
doi: 10.1111/j.1439-0434.1991.tb04737.x |
30 |
Keller N P. Fungal secondary metabolism: regulation, function and drug discovery[J]. Nat Rev Microbiol, 2019, 17(3): 167-180. DOI:10.1038/s41579-018-0121-1.
doi: 10.1038/s41579-018-0121-1 |
31 |
王梅菊, 刘晨, 吴明德, 等. 油菜内生细菌多样性分析及菌株CanL-30生防潜力评估[J]. 中国油料作物学报, 2018, 40(2): 258-268. DOI:10.7505/j.issn.1007-9084.2018.02.012.
doi: 10.7505/j.issn.1007-9084.2018.02.012 |
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