盐穗木miR167d和预测靶基因ARF8在盐胁迫下的表达与相关性分析

Expression Pattern and Correlation Analysis of the Halophyte Halostachys Caspica miR167d with Predictive Target Gene ARF8 under Salt Stress

【目的】盐穗木(Halostachys caspica)是一种藜科多年生盐生灌木,对盐分适应性极强。miR167是作用于生长素信号通路上的内源非编码小RNA分子,它通过靶向生长素响应因子ARFs(auxin response factors)来调控生长素响应基因的表达。研究从高盐(600 mmol/L NaCl)处理48 h盐穗木根的小RNA文库中筛选到差异表达的miR167d,并从该物种的转录组数据中预测到其靶基因ARF8。该文开展了高盐胁迫下盐穗木miR167d和预测靶基因ARF8的表达模式和相关性分析。【方法】通过荧光定量PCR试验检测和分析高盐胁迫下盐穗木miR167d和预测靶基因的表达模式和相关性;利用烟草瞬时表达试验间接地鉴定盐穗木miR167d对预测靶基因ARF8的靶向关系;采用同源克隆方法结合RACE技术获得盐穗木miR167d的靶基因ARF8的全长序列,并进行生物信息学分析。【结果】(1) 600 mmol/L NaCl处理盐穗木,其同化枝中miR167d和ARF8的表达均受到诱导,胁迫48 h时,miR167d的表达最高并与靶基因的表达呈现显著的负相关性,HcARF8基因的表达随胁迫处理时间的延长呈现先增加后下降的趋势,推测盐穗木miR167d和ARF8两基因可能在响应盐胁迫过程中发挥作用。(2)构建融合GFP的含预测靶基因HcARF8的植物表达载体并转化农杆菌,烟草瞬时表达试验的结果表明,拟南芥miR167d对盐穗木ARF8基因具有剪切作用,间接证明盐穗木miR167d对预测的靶基因HcARF8具有靶向切割作用。(3)克隆获得的盐穗木ARF8基因全长2 861 bp,ORF 2 442 bp,编码813个氨基酸,在编码区与盐穗木miR167d高度互补匹配。生物信息学分析该基因编码的蛋白高度保守,与同科植物甜菜ARF8同源性达到87%,都具有能与生长素相关元件(B3 DNA绑定元件、生长素响应元件,生长素诱导转录IAA超家族的元件)结合的功能域。【结论】盐穗木miR167d和HcARF8基因具有靶向关系,高盐胁迫处理盐穗木,其同化枝中两基因的表达都受到诱导并呈现显著的负相关性。这些结果为后续阐明盐穗木miR167d与其作用的靶基因ARF8的生物学功能奠定基础。

【Objective】Halostachys caspica belonging to Chenopodiaceae is perennial halophyte with extremely strong resistance and strong adaptability to salinity.miR167 is an endogenous non-encoding RNA,which plays an important role in the auxin signaling pathway and it regulates the auxin genes expression by targeting ARFs.Differentially expressed miR167d was chosen from the species roots'small RNA libraries by the treatment of high salt stress(600 mmol/L NaCl)for 48 h and predictive target gene is ARF8 using the Halostachys caspica transcriptome data.In this paper,the expression pattern and correlation of this species'branches miR167d and predictive target gene HcARF8 were studied under high salt stress.【Method】qRT-PCR and tobacco transient expression experiments were carried to explore the expression pattern and correlation of the two genes miR167d and HcARF8 in the Halostachys caspica branches under salt stress.The full-length sequence of target gene HcARF8 was obtained by RACE and PCR technologies,and bioinformatics analysis was performed.【Result】(1)miR167d and HcARF8 of the Halostachys caspica branches were both induced under high salt stress by qRT-PCR,HcmiR167d was significantly up-regulated at 48 h and well negatively correlated with the predictive target gene,the expression of HcARF8 increased first and then decreased with the extension of salt-stressed treatment time.These two genes(Hc miR167d and HcARF8)might participate in the salt stress biological process.(2)Halostachys caspica ARF8 fusion GFP vector was transformed into agrobacterium and transformed tobacco,the result of tobacco transient expression showed that Arabidopsis thaliana miR167d had a shearing effect on HcARF8 gene,and then indirectly proved that HcmiR167d could cleave the predictive target gene HcARF8.(3)The obtained Halostachys caspica ARF8 gene was 2,861 bp,ORF 2,442 bp,coding 813 amino acid,which could be highly matched with Hc miR167d in the coding region.HcARF8 was highly conserved,and had 87%homology with the ARF8 of the species Beta vulgaris belonging to Chenopodiaceae,and had functional domains that could bind to auxin-related elements(B3 DNA binding element,auxin response element,auxin-induced transcription)by bioinformatics analysis.【Conclusion】miR167d and HcARF8 of the Halostachys caspica branches were both induced and had a good negative correlation under high salt stress.These results have laid some foundations for the further study on biological function and salt tolerant mechanism of the halophyte Halostachys caspica miR167d and its target gene ARF8.