C2H2锌指转录因子FpCzf7参与假禾谷镰孢的生长和致病性

doi:10.13560/j.cnki.biotech.bull.1985.2021-1457

摘要/Abstract

摘要：

假禾谷镰孢引起的小麦茎基腐病已成为威胁我国小麦安全生产的重要病害,但是对假禾谷镰孢致病的机理还了解的比较少。C2H2锌指转录因子在人、植物和真菌中广泛分布,调控生长、发育和逆境胁迫等多种生理过程。本研究在假禾谷镰孢中鉴定到一个编码C2H2锌指转录因子的基因FpCzf7,利用PEG介导的原生质体转化,获得FpCzf7基因缺失的突变体（Δfpczf7）。生物学表型分析发现,与野生型（WT）和回补菌株（Δfpczf7-cp）相比,Δfpczf7在PDA培养基上的菌丝生长速率明显减慢、气生菌丝减少;在CMC液体中的分生孢子产量减少,但是分生孢子形态和萌发无差异。致病性测定结果显示,与WT和Δfpczf7-cp相比,Δfpczf7在大麦叶片、小麦胚芽鞘和小麦穂部的致病性均显著降低,DON毒素合成明显减少。综上所述,FpCzf7参与假禾谷镰孢的生长、产孢、致病性和毒素的产生。

关键词: 小麦茎基腐病, 假禾谷镰孢, C2H2型锌指蛋白, 致病性

Abstract:

Fusarium crown rot caused by Fusarium pseudograminearum has become one of the most destructive diseases of wheat in China. However,its molecular mechanism of pathogenicity is known little. C2H2 zinc finger transcription factors have been characterized from a wide variety of organisms,including animals,plants and fungi,and they are involved in the physiological processes such as growth,development and stress responses. In this study,a gene FpCzf7 encoding C2H2 zinc finger transcription factor was identified,and its deletion mutants（Δfpczf7）was obtained by PEG-mediated genetic transformation. Further analysis revealed that Δfpczf7 mutants grew significantly slowly on PDA plates and less aerial hyphae compared with that of wild type（WT）and the complement strains（Δfpczf7-cp）. Conidiation of the FpCzf7 deletion mutant in CMC liquid reduced as well,but their conidia morphology and germination were normal. Also,Compared to WT and Δfpczf7-cp,the pathogenicity caused by the FpCzf7 deletion mutant decreased on wheat coleoptile and spike and barley leaves,and DON toxin production reduced in the Δfpczf7 strain. Collectively,these results indicate that FpCzf7 is involved in growth,conidiation and pathogenicity and toxin production in F. pseudograminearum.

Key words: Fusarium crown rot, Fusarium pseudograminearum, C2H2 zinc finger protein, pathogenicity

赵静雅, 彭梦雅, 张时雨, 单艺轩, 邢小萍, 施艳, 李海洋, 杨雪, 李洪连, 陈琳琳. C2H2锌指转录因子FpCzf7参与假禾谷镰孢的生长和致病性[J]. 生物技术通报, 2022, 38(8): 216-224.

ZHAO Jing-ya, PENG Meng-ya, ZHANG Shi-yu, SHAN Yi-xuan, XING Xiao-ping, SHI Yan, LI Hai-yang, YANG Xue, LI Hong-lian, CHEN Lin-lin. Role of C2H2 Zinc Finger Transcription Factor FpCzf7 in the Growth and Pathogenicity of Fusarium pseudograminearum[J]. Biotechnology Bulletin, 2022, 38(8): 216-224.

图/表 8

表1 本研究用到的引物

Table 1 Primers used in this study

Primer	Sequence（5'-3'）
F1	CATCAAATGCAGGAACGGAGC
R1	CAATATCATCTTCTGTCGACGGTTTCATGGGCTGC- TAGAAATC
F2	ATAGAGTAGATGCCGACCGCGGGTTCCATCGTCT- TGCCGTGGTGAATTG
R2	CAATTGAACGAGGAAACACG
HYG/F^[16]	GTCGACAGAAGATGATATTG
HYG/R^[16]	GAACCCGCGGTCGGCATCTACTCTAT
YG/F^[16]	GATGTAGGAGGGCGTGGATATGTCCT
HY/R^[16]	GTATTGACCGATTCCTTGCGGTCCGAA
F3	GACCTACCCATGTTTACTTG
R3	CTTCGAAATCTGCATCGCATG
G1	CAACATCATATCGAACCCAC
G2	GAGAGTGAGAAGACATGG
H850F^[16]	TTCCTCCCTTTATTTCAGATTCAA
H852R^[16]	ATGTTGGCGACCTCGTATTGG
H855R^[16]	GCTGATCTGACCAGTTGC
H856F^[16]	GTCGATGCGACGCAATCGT
PKNTG-F	CTATAGGGCGAATTGGGTACCGACCTACCCATGTT- TACTTG
PKNTG-R	GCAGGCATGCAAGCTTATCGATGGAGCTTTTGCT- GCTCTTG
nei-F	GACCACGGTTTCCACTACTC
GFP-R	GATGCCCTTCAGCTCGATGCGGTTCA
RTF	TTCAACACCACCAGCATCA
RTR	TGTGGTGCAAGTTCTCGTT
TEF1a-RTF	TCACCACTGAAGTCAAGTCC
TEF1a-RTR	ACCAGCGACGTTACCACGTC

图1 FpCzf7的结构域及基因转录水平 A：FpCzf7的C2H2锌指基序;B：FpCzf7在侵染阶段的相对表达水平。MY和IF18h、IF30h、IF48h、IF3d、IF5d、IF7d分别表示假禾谷镰孢野生型菌丝阶段和侵染小麦胚芽鞘18 h、30 h、48 h、3 d、5 d、7 d

Fig. 1 Domains and expression levels of FpCzf7 in F. pseudograminearum A：The C2H2 zinc finger motifs in FpCzf7. B：The relative expression levels of FpCzf7 during infection stages. MY and IF18h,IF30h,IF48h,IF3d,IF5d,IF7d represent the wild-type mycelia and the F. pseudograminearum infected the wheat coleoptile at 18 h,30 h,48 h,3 d,5 d,and 7 d,respectively

图2 假禾谷镰孢FpCzf7基因的敲除 A：FpCzf7基因缺失策略示意图和PCR扩增结果（M：DNA分子量标准;A1：964 bp的FpCzf7上游片段;A2：997 bp的下游片段;HPH：1 350 bp的潮霉素片段;A1H1：1 762 bp的FpCzf7和HPH的上游融合片段;H2A2：1 928 bp的HPH和FpCzf7下游融合片段）;B：FpCzf7基因缺失突变体PCR检测（M：DNA分子量标准;H：750 bp的潮霉素（HPH）片段;G：751 bp的FpCzf7基因片段;F：1 673 bp的FpCzf7和HPH上游融合片段;R：1 568 bp的HPH和FpCzf7下游融合片段）

Fig. 2 FpCzf7 gene knockout in F. pseudograminearum A：Schematic representation of the FpCzf7 deletion strategy and PCR amplification（M：DNA size marker;A1：a 964 bp fragment of FpCzf7 upstream;A2：a 997 -bp fragment of FpCzf7 downstream;HPH：a 1 350 bp fragment of the hygromycin phosphotransferase gene;A1H1：a 1 762 bp fragment of FpCzf7 and HPH upstream cassette;H2A2：a 1 928 bp fragment of FpCzf7 and HPH downstream cassette）. B：PCR assay of the FpCzf7 deletion mutants（M：DNA size marker;H：a 750 bp fragment of the HPH;G：a 751 bp fragment of the FpCzf7 gene;F：a 1 673 bp fragment of FpCzf7 and HPH upstream cassette;R：a 1 568 bp fragment of FpCzf7 and HPH downstream cassette）

图3 野生型（WT）、Δfpczf7和Δfpczf7-cp在PDA平板上生长的菌落和菌丝测定 A：生长3 d的菌落形态;B：生长3d的菌落直径（**P < 0.01,t测验）C：生长24 h的菌丝形态（标尺=50 µm）

Fig. 3 Mycelia growth and morphology assays of WT,Δfpczf7 and Δfpczf7-cp on PDA plates A：Colonies morphology for 3 d. B：Colony diameters for 3 d（**P < 0.01,t-test）. C：Hyphal morphology for 24 h（Bar=50 µm）

图4 分生孢子产生和萌发测定 A：野生型、Δfpczf7和Δfpczf7-cp的分生孢子及萌发的芽管;B：WT、Δfpczf7和Δfpczf7-cp的产孢量（**P < 0.01,t测验）;C：WT、Δfpczf7和Δfpczf7-cp分生孢子的萌发

Fig. 4 Conidia production and germination assays A：Conidia and germinated tubes of WT,Δfpczf7 and Δfpczf7-cp. B：Production of conidia of WT,Δfpczf7 and Δfpczf7-cp（**P < 0.01,t-test）. C：Conidia germination rates of WT,Δfpczf7 and Δfpczf7-cp

图 5 大麦叶片和小麦胚芽鞘接种 A：野生型、Δfpczf7和Δfpczf7-cp菌丝块接种大麦叶片后3 d;B：大麦叶片病斑直径（**P < 0.01,t测验）;C：野生型、Δfpczf7和Δfpczf7-cp菌丝块接种小麦胚芽鞘后3 d;D：小麦胚芽鞘病斑长度（**P < 0.01,t测验）

Fig. 5 Inoculation on barley leaves and wheat coleoptiles A：Barley leaves inoculated with mycelial blocks of WT,Δfpczf7 and Δfpczf7-cp at 3 d. B：The lesion diameters on barley leaves（**P < 0.01,t-test）. C：Wheat coleoptiles were inoculated with mycelial blocks of WT,Δfpczf7 and Δfpczf7-cp at 3 d. D：Lesion lengths on wheat coleoptiles（**P < 0.01,t-test）

图 6 小麦盆栽和小麦穗接种实验 A：野生型、Δfpczf7和Δfpczf7-cp接种小麦10 d;B：小麦茎基腐病病情指数（**P < 0.01,t测验）;C：野生型、Δfpczf7和Δfpczf7-cp分生孢子悬浮液接种小麦穗部20 d

Fig. 6 Experiments of pot-culture and wheat heads inoculation A：Wheat seedlings inoculated by WT,Δfpczf7 and Δfpczf7-cp for 10 d. B：The disease severity index of Fusarium crown rot.（**P < 0.01,t-test）. C：Wheat heads were inoculated with conidial suspension of WT,Δfpczf7 and Δfpczf7-cp strains for 20 d

图 7 DON毒素的产生 **P < 0.01,t测验

Fig. 7 DON production **P < 0.01,t-test

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