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小麦条锈菌诱导的TaRRP4基因的克隆与表达
任惠文, 杨子璠, 郭双元,等
西北农林科技大学 生命科学学院,旱区作物逆境生物学国家重点实验室
摘要:
【目的】克隆小麦条锈菌诱导的小麦TaRRP4基因,研究其在小麦与小麦条锈菌互作、非生物胁迫、外源激素处理下的表达特征,为小麦与小麦条锈菌互作中该基因功能的验证及小麦抗病育种提供参考。【方法】以小麦品种水源11、小麦条锈菌生理小种条中23号(CYR23)和条中31号(CYR31)为材料,采用RT-PCR法,从小麦条锈菌侵染的水源11小麦cDNA中克隆得到1个外切体亚基TaRRP4基因,利用生物信息学对其序列进行分析。采用实时荧光定量PCR(qRT-PCR)分析不同处理下TaRRP4基因在各个时间点的相对表达量,其中小麦条锈菌处理体系包含非亲和体系(CYR23与水源11)和亲和体系(CYR31与水源11),非生物胁迫处理包含高盐、干旱、低温和机械损伤处理,外源植物激素处理包含脱落酸、茉莉酸甲酯、乙烯利和水杨酸处理,另外分析该基因在小麦不同组织(根、茎和叶)中的表达情况。【结果】克隆获得小麦TaRRP4基因序列,其开放阅读框为951 bp,编码316个氨基酸;同源氨基酸序列比对结果表明,TaRRP4与拟南芥AtRRP4p氨基酸相似度达62.96%,且均属于外切体亚基RRP4家族,包含类核糖体蛋白S1和KH 2个RNA结合结构域。qRT-PCR结果表明,与小麦条锈菌诱导0 h相比,小麦条锈菌CYR31诱导后,TaRRP4在诱导24,48和72 h极显著上调表达;而受小麦条锈菌CYR23诱导后,TaRRP4仅在诱导48 h显著上调,且上调倍数较小。与各个非生物胁迫小麦处理0 h相比,高盐胁迫下,TaRRP4在处理12和48 h分别极显著和显著上调;干旱胁迫下,TaRRP4在处理2和6 h均极显著上调表达;低温胁迫下,TaRRP4从处理2 h开始极显著或显著上调,直至48 h表达量降低;而机械损伤处理对TaRRP4的表达量没有显著影响。与各个外源植物激素处理小麦0 h相比,脱落酸可在处理12和24 h诱导TaRRP4下调表达,茉莉酸甲酯对TaRRP4的表达量没有显著影响,而乙烯利和水杨酸均可在处理2,6和12 h诱导TaRRP4上调表达。同时,TaRRP4基因在小麦根、茎、叶中均有表达,且在叶片中的表达量最高,显著高于其在小麦根、茎中的表达量。【结论】克隆得到小麦条锈菌诱导的小麦外切体RNA结合蛋白TaRRP4基因,该基因可能通过脱落酸、乙烯利和水杨酸信号交流,在小麦条锈病的防御反应中发挥负调节作用,同时在小麦对非生物胁迫的抗逆应答过程中发挥重要作用。
关键词:  小麦条锈菌  TaRRP4基因  基因克隆  基因表达
DOI:
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基金项目:陕西省自然科学基础研究计划项目(2018JM3031)
Cloning and expression of TaRRP4 gene induced by Puccinia striiformis f.sp. tritici in wheat
REN Huiwen, YANG Zifan, GUO Shuangyuan,et al
Abstract:
【Objective】The TaRRP4 gene of wheat induced by Puccinia striiformis f.sp.tritici was cloned to study its expression characteristics in the interaction of wheat and stripe rust,abiotic stress and exogenous hormone treatments,which can provide references for validating function of this gene and disease-resistant breeding.【Method】Wheat Suwon 11 and wheat stripe rust strains 23 (CYR23) and 31 (CYR31) were selected and RT-PCR was used to clone RNA-binding protein TaRRP4 from Suwon11 infected by stripe rust.Bioinformatics was used to analyze its sequence and qRT-PCR was used to analyzere lative expression of TaRRP4 gene in wheat leaves at different time points under different treatments.The wheat systems infected by strip rust included incompatible treatment systems (CYR23 and Suwon 11) and compatible treatment systems (CYR31 and Suwon 11).The treatments of abiotic stress included high salinity,drought,low temperature and wound treatments.Exogenous plant hormone treatments included abscisic acid(ABA),methyl jasmonate(MeJA),ethrel(ETH) and salicylic acid(SA) treatments.The expression of this gene in different tissues (roots,stems and leaves) were also analyzed by qRT-PCR.【Result】The wheat gene TaRRP4 was obtained and its ORF length was 951 bp,encoding 316 amino acids.It had 62.96% amino acid similarity with Arabidopsis thaliana AtRRP4p,and both of them belonged to the RRP4 family of exosome subunits,including ribosomal protein S1 and KH RNA-binding domains.The qRT-PCR showed that relative expression of TaRRP4 was significantly up-regulated at 24,48 and 72 h after CYR31 induction,while it was only slightly up-regulated at 48 h after CYR23 induction.Compared with abiotic stress treatments for 0 h,TaRRP4 was significantly up-regulated at 12 h and 48 h under high salt.Under drought,relative expression of TaRRP4 was significantly up-regulated at 2 and 6 h.Under low temperature,relative expression of TaRRP4 was significantly up regulated from 2 h to 48 h,and then it decreased.Wound treatment had no significant effect on relative expression of TaRRP4.Compared with wheat treated with various exogenous plant hormones for 0 h,ABA induced down regulation of TaRRP4 relative expression at 12 h and 24 h,MeJA had no significant effect,while ETH and SA induced up regulation of TaRRP4 expression at 2 h,6 h and 12 h.TaRRP4 expressed in roots,stems and leaves of wheat,and leaves had significantly higher expression than roots and stems.【Conclusion】A wheat RNA-binding protein TaRRP4 gene induced by stripe rust was cloned,which may play a negative regulatory role in the defense response of wheat to stripe rust by signal communications of ABA,ETH and SA.TaRRP4 also involved in the anti-stress response of wheat to abiotic stress.
Key words:  Puccinia striiformis f.sp.tritici  TaRRP4 gene  gene cloning  gene expression