响应白粉菌的小麦NBS基因筛选

doi:10.7668/hbnxb.20192379

摘要/Abstract

摘要： 核苷酸结合位点（NBS）基因是植物中广泛存在的一类抗病基因，通过分析小麦NBS基因受白粉菌侵染后的表达水平变化情况，可为小麦育种筛选抗白粉病相关基因。试验采用生物信息学方法，对小麦抗病品种受白粉菌侵染0，24，48，72 h的转录组原始数据进行组装，结果筛选出1 283条具有表达数据的TaNBS，其在21个染色体均有分布。表达差异分析结果表明，有395条TaNBS受白粉菌侵染后的表达水平变化显著；根据3个时间段（0~24 h，24~48 h，48~72 h）内的变化趋势将其分为降-升-升、升-升-降、升-降-升、降-降-升和升-升-升共5类。利用qRT-PCR技术，从升-升-升类中选择10个变幅最大的TaNBS进行验证，结果表明，有3个基因Ta7dlLoc004854、Ta7dlLoc000139和Ta3asLoc007663在实验室自育小麦抗病种质CH7124中受白粉菌侵染后保持显著上调；利用病毒诱导基因沉默技术，分别下调了上述3个基因在CH7124中的表达水平，获得沉默植株；接种鉴定试验结果表明，只有Ta7dlLoc000139的沉默植株对白粉菌的感染型由免疫变为高抗或中抗，说明Ta7dlLoc000139表达水平的降低引起了植株抗性减弱。序列分析结果表明，Ta7dlLoc000139全长4 042 bp，包含3个外显子和2个内含子，在其起始密码子前2 000 bp区域内包含大量的CGCG-Box、GATA-Box和CAAT-Box等调控元件。可为小麦抗病分子育种提供理论依据。

关键词: 小麦, NBS基因, 白粉菌响应, 表达水平, 基因沉默

Abstract: Nucleotide binding site(NBS) genes are a type of disease resistance genes widely present in plants.In order to screen the powdery mildew-resistant genes for wheat breeding,the changes in the expression level of wheat NBS genes after infection with Blumeria graminis f.sp.tritici(Bgt) were analyzed.Bioinformatics methods were used to assemble the original transcriptome data of a disease-resistant wheat variety infected by Bgt at 0,24,48,72 h,and 1 283 TaNBS with expression data were screened out,which were distributed on 21 chromosomes.Results of expression difference analysis showed that the expression levels of 395 TaNBS were significantly changed after being infected by Bgt,and they were divided into five types,including down-up-up,up-up-down,up-down-up,down-down-up as well as up-up-up,according to the change trend in three time periods(0-24 h,24-48 h,48-72 h).Using qRT-PCR technology,ten TaNBS with the most significant differences were selected from the up-up-up type for verification.The results showed that three genes Ta7dlLoc004854, Ta7dlLoc000139 and Ta3asLoc007663 remained significantly up-regulated in a self-breeding disease-resistant wheat germplasm CH7124 after being infected by Bgt. Using virus-induced gene silencing technology,the expression levels of the above three genes in CH7124 were down-regulated to obtain the silent plants.The results of the inoculation test showed that only the Bgt-infection type of Ta7dlLoc000139-silent plants changed from immune to high resistance or medium resistance,indicating that the decrease in the expression level of Ta7dlLoc000139 caused the plant resistance to weaken.Sequence analysis results showed that Ta7dlLoc000139 had a total length of 4 042 bp,contained three exons and two introns,and contained a large number of regulatory elements such as CGCG-Box,GATA-Box and CAAT-Box in the 2 000 bp region before its start codon.The research results could provide a theoretical basis for wheat disease resistance molecular breeding.

Key words: Wheat, NBS genes, Bgt response, Expression level, Gene silencing

中图分类号:

田志刚, 张树伟, 陈芳, 常利芳, 贾举庆, 张晓军, 李欣. 响应白粉菌的小麦NBS基因筛选[J]. 华北农学报, 2021, 36(6): 189-194. doi: 10.7668/hbnxb.20192379.

TIAN Zhigang, ZHANG Shuwei, CHEN Fang, CHANG Lifang, JIA Juqing, ZHANG Xiaojun, LI Xin. Screening of Wheat NBS Genes in Response to Powdery Mildew[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2021, 36(6): 189-194. doi: 10.7668/hbnxb.20192379.

http://www.hbnxb.net/CN/Y2021/V36/I6/189

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