Optimization of T-DNA insertion-mediated mutagenesis of Leptosphaeria biglobosa and mutant screening

doi:10.19802/j.issn.1007-9084.2020256

Abstract

Abstract:

Blackleg of oilseed rape (Brassica spp.) is caused by Leptosphaeria biglobosa, which has been found to occur widely in oilseed rape-producing areas in our country, resulting in a substantial seed yield loss in the industry of oilseed rape. However, the mechanisms for ecological adaptation and pathogenesis of L. biglobosa have not been elucidated. Therefore, it is necessary to conduct in-depth studies on molecular mechanisms for ecological adaptation and pathogenesis of L. biglobosa. In order to fulfill this objective, this study was done to mutate L. biglobosa by Agrobacterium tumeficens-mediated transformation (ATMT). The contents included optimization of the factors affecting the ATMT, evaluation of the quality of the transformants, and screening of mutants. The results showed that the optimum ATMT factors for L. biglobosa Lb731 were: hygomycin B concentration at 50 μg/mL, transformation recipients (conidia) from the 15-day-old cultures (20℃), conidial concentration at 2 × 10^7-8 conidia/mL, co-incubation of A. tumeficens-recipients at 25℃ for 72 h. The transformation efficiency reached 80 transformants per million conidia on average. The T-DNA insertion frequency reached up to 100% based on PCR assays with 72.7% single-copy insertion among the transformants based on Southern blottings. Results also showed that the hygromycin-resistance trait in the transformants inherited stably through nuclear mitosis. A total of 32 mutants were screened from 2136 transformants, including 11 mutants with suppressed mycelial growth, 7 mutants with deficiency in pigment biosynthesis, and 14 mutants with deficiency in conidial production. Meanwhile, 7 out of the 32 mutants were identified to completely lose pathogenicity (virulence) on oilseed rape. Using the hiTAIL-PCR technique, three DNA sequences flanking the T-DNA-insertion sites were obtained from three mutants. Overall, these results laid a solid foundation for further elucidation of the molecular mechanisms for ecological adaptation and pathogenesis in L. biglobosa.

Key words: Leptosphaeria biglobosa, Agrobacterium tumeficiens-mediated transformation (ATMT), T-DNA insertional mutagenesis, mutant screening

CLC Number:

Q785

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.

Figures/Tables 7

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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	检测潮霉素抗性基因，探针 Detection of the hph gene as probe
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）

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]	SONG Pei-ling, HUANGFU Hai-yan, SHI Zhi-dan, YAN Meng-jiao, HAO Li-fen, HUANGFU Jiu-ru, JIA Xiao-qing, GUO Chen, YANG Yong-qing, LI Zi-qin. Agrobacterium tumefaciens-mediated transformation of Leptosphaeria biglobosa [J]. CHINESE JOURNAL OF OIL CROP SCIENCES, 2021, 43(2): 293-.
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