<i>Pg&#x003B2;Glu4</i>基因调控大麦条纹病菌致病性的功能分析

doi:10.7668/hbnxb.20195746

摘要/Abstract

摘要：

前期研究发现大麦条纹病菌β-葡萄糖苷酶基因PgβGlu4在侵染阶段高表达,为了深入研究该基因的功能,构建PCE2-EGFP-PgβGlu4的亚细胞定位载体转化水稻原生质体,观察PgβGlu4在水稻组织中的定位;同时构建PgβGlu4基因RNAi载体,利用CaCl₂-PEG4000介导法制备QWC原生质体进行遗传转化;通过检测突变株的营养生长和致病性对PgβGlu4基因功能进行了分析。PgβGlu4与不同病原菌同源蛋白序列的进化树分析显示,PgβGlu4与小麦黄斑叶枯病菌具有较近的亲缘关系;亚细胞定位结果显示,PgβGlu4主要在细胞核与细胞膜中表达;经潮霉素验证得到4株PgβGlu4基因突变体;qRT-PCR分析结果表明,4个RNAi突变株PgβGlu4基因表达量较野生株分别下降了66.31%,68.60%,54.37%,69.89%;突变株菌落直径显著小于野生株,侵染大麦后发病率较野生株侵染组分别降低了56.69,52.76,47.43,53.30百分点;干扰突变株侵染后大麦叶片叶绿素相对含量为30.3~35.0,3个干扰突变株系侵染组大麦显著高于野生组;PgβGlu4基因沉默前后侵染大麦对其株高影响显著。结果表明,PgβGlu4基因参与调控大麦条纹病菌生长发育及其致病性。

关键词: 大麦条纹病菌, β-葡萄糖苷酶, RNA干扰, 致病性

Abstract:

To investigate the function of the β-glucosidase(βGlu)gene PgβGlu4 from Pyrenophora graminea,which previous studies found to be highly expressed during the infection stage,we constructed a subcellular localization vector of PCE2-EGFP-PgβGlu4 and transformed rice protoplasts,observed the fluorescence distribution and analyzed its location of existence.Simultaneously,the PgβGlu4 gene RNAi vector was constructed,and QWC protoplasts were prepared by CaCl₂-PEG4000 mediated method for genetic transformation.The function of PgβGlu4 gene was studied by detecting the vegetative growth and pathogenicity of the RNAi mutants.Phylogenetic analysis of PgβGlu4 and other homologous proteins from different pathogens showed that PgβGlu4 had a closer evolutionary relationship with that from Pyrenophora tritici-repentis.The subcellular localization results showed that PgβGlu4 was mainly localized in the nucleus and cell membrane.Four PgβGlu4 gene RNAi mutants were verified by hygromycin.qRT-PCR analysis showed that the expression of PgβGlu4 gene in four RNAi mutants decreased by 66.31%,68.60%,54.37% and 69.89%,respectively,compared with the wild isolate.The colony diameter was smaller than that of the wild isolate,and their incidence rate was reduced by 56.69,52.76,47.43,and 53.30 percentage points.After infection with the mutant strain of RNAi-PgβGlu4,the relative chlorophyll content in barley leaves ranged from 30.3 to 35.0,which was significantly higher than that of the wild-type group.The effect of PgβGlu4 gene silencing on the height of barley plants before and after infection was significant compared with that of the wild-type.The results indicated that the PgβGlu4 gene was involved in the regulation of the growth,development,and pathogenicity of Pyronophora graminea.

Key words: Pyrenophora graminea, β-glucosidase, RNA interference(RNAi), Pathogenicity

中图分类号:

S435.123

杨文娟, 祁天涛, 王艳婷, 孟亚雄, 汪军成, 王化俊, 司二静. PgβGlu4基因调控大麦条纹病菌致病性的功能分析[J]. 华北农学报, 2025, 40(5): 188-197. doi: 10.7668/hbnxb.20195746.

YANG Wenjuan, QI Tiantao, WANG Yanting, MENG Yaxiong, WANG Juncheng, WANG Huajun, SI Erjing. Analysis of the Function of PgβGlu4 Gene to Regulate Pathogenicity in Pyrenophora graminea[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(5): 188-197. doi: 10.7668/hbnxb.20195746.

http://www.hbnxb.net/CN/Y2025/V40/I5/188

图/表 11

表1 本研究所用引物序列

Tab.1 Primers sequences used in this study

引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')	用途 Purpose
PgβGlu4-qRT-F	GAGCACCCCACACTTTACGA	实时荧光定量PCR
PgβGlu4-qRT-R	AAAGTACGGGCGGTTGTTGA
EGFP-PgβGlu4-F	TCATTTGGAGAGAACACGGGGGACTCTAGAATGCCACAAA	靶位点测序
	CACCACCAGCCACCTATCCTCGCCCCGACTTCCTTCG
EGFP-PgβGlu4-R	TTGCTCACCATAAGCTTGTCGACGGAGCTCTTATGCATCTG
	CGCATACGTTTCCATGGCATTTTCAACAACCTTCTTCACCTC
PgβGlu4-KpnⅠ-F	GG GGTACCCCACAAGGCCTGTAAACGAT	干扰片段克隆
PgβGlu4-BglⅡ-R	GA AGATCTTTTCCATGGCATTTTCAACA
PgβGlu4-XhoⅠ-F	CCG CTCGAGCCACAAGGCCTGTAAACGAT
PgβGlu-Hind Ⅲ-R	CCC AAGCTTTTTCCATGGCATTTTCAACA
HygB-F	ATGAAAAAGCCTGAACTCAC	干扰菌株筛选
HygB-R	CTATTCCTTTGCCCTCGGA
PgActin-F	TGTTGACATGGCTGGTCGTGATC	内参基因引物
PgActin-R	TCGGCGGTGGTGGAGAAGG

图1 大麦条纹病原菌PgβGlu4基因的干扰载体示意图

Fig.1 Schematic diagram of PgβGlu4 gene interference vector of Pyrenophora graminea

图2 大麦条纹病菌PgβGlu4及其他病原真菌中同源蛋白的系统发育树

Fig.2 Phylogenetic tree of PgβGlu4 protein from Pyrenophora graminea and homologous proteins in other phytopathogenic

图3 大麦条纹病原菌PgβGlu4蛋白亚细胞定位质粒和双酶切电泳图谱 1.PgβGlu4亚细胞定位载体质粒;2.双酶切片段;M.DL12000 DNA Marker。

Fig.3 Subcellular localization plasmid and double enzyme digestion electrophoresis map of PgβGlu4 protein of Pyrenophora graminea 1.Plasmid of subcellular localization vector of PgβGlu4; 2.Double enzyme digestion electrophoresis;M.DL12000 DNA Marker.

图4 大麦条纹病原菌PgβGlu4蛋白在水稻细胞中的亚细胞定位从左到右分别为空载体EGFP和目的基因PgβGlu4编码蛋白的绿色荧光蛋白(GFP)、明场(DIC)和3个通道的叠加照片(Merged)。

Fig.4 The subcellular localization of PgβGlu4 protein of Pyrenophora graminea in rice cells From left to right are the superimposed photos of green fluorescent protein(GFP),bright field(DIC)and three channels(Merged)of empty vector EGFP and target gene PgβGlu4,respectively.

图5 pSilent-1∶PgβGlu4干扰载体的酶切验证电泳图 A.大麦条纹病原菌PgβGlu4基因的干扰片段验证:1,2.阴性对照ddH₂O,3.干扰片段PgβGlu4-1,4.干扰片段PgβGlu4-2,M.DL2000 DNA Marker;B.干扰载体pSilent-1∶PgβGlu4双酶切验证:1.干扰载体pSilent-1∶PgβGlu4-1,2.干扰载体pSilent-1∶PgβGlu4-2,M.DL2000 DNA Marker。

Fig.5 Gel electrophoresis image of double-digestion validation of interference vector pSilent-1∶PgβGlu4 A.Verification of interference fragment of PgβGlu4 gene of Pyrenophora graminea:1,2.Negative control ddH₂O,3.Interference fragment PgβGlu4-1,4.Interference fragment PgβGlu4-2,M.DL2000 DNA Marker;B.Double-digestion validation of interference vector pSilent-1∶PgβGlu4:1.Interference carrier pSilent-1∶PgβGlu4-1,2.Interference carrier pSilent-1∶PgβGlu4-2,M.DL2000 DNA Marker.

图6 大麦条纹病原菌PgβGlu4基因干扰突变菌株的潮霉素抗性PCR鉴定 M.DL2000 DNA Marker;1.PgβGlu4-RNAi-1;2.PgβGlu4-RNAi-2;3.PgβGlu4-RNAi-3;4.PgβGlu4-RNAi-4;5.ddH₂O;6野生株;7.空载体(pSilent-1)。

Fig.6 PCR verification of hygromycin resistance of PgβGlu4 gene RNAi mutants of Pyrenophora graminea M.DL2000 DNA Marker;1.PgβGlu4-RNAi-1;2.PgβGlu4-RNAi-2;3.PgβGlu4-RNAi-3;4.PgβGlu4-RNAi-4;5.ddH₂O;6.Wild strain;7.Empty carrier(pSilent-1).

图7 大麦条纹病原菌干扰突变菌株PgβGlu4基因的相对表达量不同组之间的差异采用方差分析(ANOVA),不同小写字母代表差异显著(P<0.05)。图8—10同。

Fig.7 Relative expression of PgβGlu4 RNAi mutants of Pyrenophora graminea Analysis of variance(ANOVA)was used for the differences test between different groups,and different lower case letters represented significant differences(P<0.05).The same as Fig.8—10.

图8 大麦条纹病原菌野生株和PgβGlu4基因突变株在PDA培养基的菌落生长情况和生长速率 A.大麦条纹病原菌表观形态图;B.大麦条纹病原菌菌丝显微结构图;C.PDA培养基上接菌7 d的野生株QWC及4个干扰突变株的生长速率;D.PDA培养基上接菌第7天各菌株的菌落直径。

Fig.8 Growth morphology and rate of wild strain and PgβGlu4 RNAi mutants of Pyrenophora graminea in PDA medium A.The apparent morphological diagram of Pyrenophora graminea;B.The microscopic structure diagram of mycelium of Pyrenophora graminea;C.The growth rates of wild-type QWC,interfering mutant strains were tested in PDA medium for 7 days;D.The colony diameter of each strain on the seventh day of inoculation in PDA medium.

图9 大麦条纹病菌PgβGlu4基因干扰突变株对大麦的致病力分析 A.WT野生株、PgβGlu4-RNAi干扰突变株侵染20 d感病大麦的盆栽症状;B.WT野生株、PgβGlu4-RNAi干扰突变株侵染大麦后的发病率;C.WT野生株、PgβGlu4-RNAi干扰突变株侵染大麦后的病情指数;D.WT野生株、PgβGlu4-RNAi干扰突变株侵染20 d后大麦植株株高;E.WT野生株、PgβGlu4-RNAi干扰突变株侵染20 d后感病大麦叶片的叶绿素相对含量。

Fig.9 Analysis pathogenicity of Pyrenophora graminea PgβGlu4 RNAi mutant strains on barley A.The potted symptoms of diseased barley plants infected with wild-type strain,PgβGlu4-RNAi interfering mutant strains at 20 days post-inoculation;B.Incidence statistics of barley infected by Pyrenophora graminea wild-type strain and PgβGlu4-RNAi mutant strains;C.Disease index of barley infected by Pyrenophora graminea wild-type strain and PgβGlu4-RNAi mutant strains;D.Height of barley leaves infected with wild-type strain, PgβGlu4-RNAi mutant strains after 20 days of infection;E.Relative chlorophyll content of barley leaves infected with wild-type strain,PgβGlu4-RNAi mutant strains after 20 days of infection.

图10 大麦条纹病菌野生株和PgβGlu4基因干扰突变株侵染大麦后的发病叶片台盼蓝染色情况 A.WT、PgβGlu4-RNAi干扰突变株侵染20 d后对感病大麦发病叶片的台盼蓝染色;B.WT、PgβGlu4-RNAi干扰突变菌株侵染20 d后感病大麦叶片的病斑长度。

Fig.10 The trypan blue staining condition of symptoms of barley leaves infected by wild type strain and PgβGlu4 gene RNAi mutants of Pyrenophora graminea A.Trypan blue staining of diseased barley leaves after 20 days of infection with wild-type strain,PgβGlu4-RNAi mutant strains;B.Lesion length of barley leaves infected with wild-type strain,PgβGlu4-RNAi mutant strains after 20 days of infection.

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PgβGlu4基因调控大麦条纹病菌致病性的功能分析

Analysis of the Function of PgβGlu4 Gene to Regulate Pathogenicity in Pyrenophora graminea

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PgβGlu4基因调控大麦条纹病菌致病性的功能分析

Analysis of the Function of PgβGlu4 Gene to Regulate Pathogenicity in Pyrenophora graminea

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