<i>TaRanGAP2</i>在小麦抵抗叶锈菌侵染中的作用

doi:10.7668/hbnxb.20192497

摘要/Abstract

摘要：

为研究TaRanGAP2在小麦抵抗叶锈菌侵染的HR诱发中的作用,阐明小麦抗叶锈病分子机制,为小麦抗叶锈病育种给予分子水平的指导。以小麦近等基因系TcLr26及其轮回亲本Tc分别与叶锈菌生理小种260组成不亲和组合(TcLr26×260)及亲和组合(Tc×260),生物信息学分析发现,TaRanGAP2 的CDS全长为1 665 bp,编码554个氨基酸,对其保守结构域进行分析发现TaRanGAP2基因编码的蛋白属于Ran GTPase家族;利用RT-qPCR技术检测TaRanGAP2在这2个组合中的表达情况,发现在不亲和组合中TaRanGAP2表达量出现显著上调;利用烟草瞬时转化系统进行亚细胞定位检测发现TaRanGAP2定位于细胞质;利用病毒诱导的基因沉默技术沉默TaRanGAP2基因表达,在接种叶锈菌后与未沉默植株相比,TaRanGAP2基因沉默植株叶片的单侵染点HR面积显著增大,HMC数量显著增多;结果说明TaRanGAP2基因在小麦抵抗叶锈菌侵染的寄主过敏性反应发生具有重要作用。

关键词: 小麦, 叶锈菌, TaRanGAP2, 过敏性反应

Abstract:

The aim to study the role of TaRanGAP2 in HR induction of wheat resistance to Puccinia triticina,so as to lay a foundation for elucidating the mechanism of Puccinia triticina resistance and breeding for disease resistance in wheat at the molecular level.The wheat near-isogenic line TcLr26 and its recurrent parent Tatcher(Tc)were used to form incompatabile(TcLr26×260)and compatabile combination(Tc×260)with Puccinia triticina race 260,respectively.Bioinformatics analysis showed that the total length of CDS of TaRanGAP2 was 1 665 bp,encoding 554 amino acids.Analysis of its conserved domains revealed that the protein encoded by TaRanGAP2 gene belonged to the Ran GTPase family;RT-qPCR was used to detect the expression of TaRanGAP2 in the two combinations.It was found that the expression of TaRanGAP2 was significantly up-regulated in the incompatible combination;the subcellular localization detection of TaRanGAP2 by tobacco transient transformation system showed that TaRanGAP2 was localized in the cytoplasm;Virus induced gene silencing(VIGS)technology were used to silence TaRanGAP2 gene in TcLr26.After inoculation with Puccinia triticina race 260,it was found that HR area of TaRanGAP2 gene-silencing plants was significantly increased,and the number of HMCs were also significantly increased compared with the unsilenced plants control plants.These results proved that TaRanGAP2 played a positive regulatory role in the induction of HR in wheat resistance to Puccinia triticina.

Key words: Wheat, Puccinia triticina, TaRanGAP2, Hypersentive reaction

宋姗姗, 张欣婷, 王琦, 芦远景, 侯春燕, 王冬梅. TaRanGAP2在小麦抵抗叶锈菌侵染中的作用[J]. 华北农学报, 2022, 37(1): 158-164. doi: 10.7668/hbnxb.20192497.

SONG Shanshan, ZHANG Xinting, WANG Qi, LU Yuanjing, HOU Chunyan, WANG Dongmei. The Role of TaRanGAP2 in Wheat Resistance to Puccinia triticina Infection[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(1): 158-164. doi: 10.7668/hbnxb.20192497.

http://www.hbnxb.net/CN/Y2022/V37/I1/158

图/表 8

表1 引物序列

Tab.1 Primer sequence

引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')	引物用途 Primer application
qRT-F	ACGCGAACTACATCCCAGAC	RT-qPCR
qRT-R	CTTCGTCCTCGTCATCAGGG
GAPDH-F	CTGCATCATACGATGACATC	RT-qPCR内参基因
GAPDH-R	TGTCACCGACAAAGTCAGTG
TaRanGAP2-GFP-F	CGTCGACATGGCTTCAGCGGCGCAAGATTTC	亚细胞定位试验
TaRanGAP2-GFP-R	CACTAGTGTCCTCCTCCGCCTCCACCTTCAG
VIGS-TaRanGAP2-F	GTGCGGCCGCTCCCGCAAGTATGGCCTTTTG	VIGS试验
VIGS-TaRanGAP2-R	CAGCGGCCGCGCAGTCGTCACTGGACCTTTTTTC

表2 VIGS试验体系

Tab.2 System of VIGS assay

试验设计 Experiment design	反应混合液Reaction mixture
试验组Test group	α、β、BSMV:TaRanGAP2各2 μL+19.5 μL FES
对照组Control group	α、β、BSMV:00各2 μL+19.5 μL FES
指示组Indicating group	α、β、BSMV:PDS各2 μL+19.5 μL FES

图1 TaRanGAP2保守结构域及物种进化树分析
A.保守结构域:绿色为WPP保守结构域;红色区域表示LRR亮氨酸重复序列。B.红色方框内为TaRanGAP2。

Fig.1 Analysis of the conserved domain and species phylogenetic tree of TaRanGAP2
A.Conserved domain:Green area is the conserved domain of WPP;Red area is the LRR leucine repeat sequence.B.TaRanGAP2 is in the red box.

图2 叶锈菌接种对TaRanGAP2基因表达的影响
RT-qPCR值是3个独立试验数据的平均值±标准误,利用SPSS软件对其进行差异显著性分析,由同一字母表示的平均值无显著差异(P>0.05)。图4,6同。

Fig.2 Effect of Puccinia triticina inoculation on TaRanGAP2 gene expression
RT-qPCR values are means±s of three independent experiments data,SPSS software is used to analyze the significance of the difference.The mean value indicated by the same letter has no significant difference(P>0.05).The same as Fig.4,6.

图3 TaRanGAP2亚细胞定位

Fig.3 Subcellular localization of TaRanGAP2

图4 叶片表型及沉默效率检测
A.转染病毒后13 d叶片表型观察;B.利用RT-qPCR技术检测TaRanGAP2基因沉默效率。

Fig.4 Leaf phenotype and silencing efficiency detection
A.Observation of leaf phenotype 13 days after virus transfection;B.The detection of TaRanGAP2 gene silencing efficiency by RT-qPCR.

图5 VIGS沉默叶片接种叶锈菌后的荧光染色
紫外光激发观察HMC个数;蓝光激发观察HR面积。

Fig.5 Fluorescence staining of VIGS silent leaves inoculated with Puccinia triticina
Number of HMC observed by ultraviolet excitation;HR area observed by blue-light excitation.

图6 单侵染点HMC数量、HR面积统计
A.接菌后单侵染点的HMC数量统计;B.接菌后单侵染点的HR面积统计。

Fig.6 The statistics of HMC number and HR area of single infection site
A.Statistics of the number of HMCs at a single infection site after inoculation; B.Statistics of HR area of single infection site after inoculation.

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