橡胶树树皮miRNA定量表达分析的内参筛选

Selection of miRNA Reference for Normalization of Quantitative Real-time PCR Analysis in the Bark of Rubber Tree(Hevea brasilien-sis Muell.Arg.)

世界所需天然橡胶主要来自橡胶树,橡胶树的乳管是合成和储存天然橡胶的组织,树干树皮中的次生乳管与天然橡胶生产密切相关,由维管形成层分化而来。次生乳管数量与天然橡胶产量呈显著正相关。因此,乳管分化是天然橡胶生产面临的一个重大理论课题。植物miRNAs是一类长约20~24个核苷酸的非编码小RNA分子,通过介导基因沉默在植物生长发育、细胞分化及逆境适应中起着非常重要的调节作用。橡胶树乳管分化过程中差异miRNAs的鉴定对于进一步认识乳管分化的分子机理具有重要的作用。实时荧光定量PCR(qPCR)已广泛用于miRNA的定量表达分析,选择合适的miRNA内参对于准确进行miRNA的表达定量至关重要。本研究以冠菌素(coronatine, COR)诱导橡胶树萌条分化次生乳管的实验系统,采用小RNA Poly A加尾的qPCR技术分析COR处理对形成层区3个非编码RNA(U6 snRNA、5S rRNA、18S rRNA)和6个miRNA(hbr-miR159b、hbr-miR396b、miR169-x、miR172-y、miR535-x、miR894-x)候选内参的表达稳定性的影响。geNorm和NormFinder软件的联合分析结果显示,表达稳定性最高的是hbr-miR396b和miR894-x,稳定性较差的是U6 snRNA。hbr-miR396b和miR894-x可作为合适的内参基因,用于分析COR影响下的miRNA相对定量表达,为鉴定橡胶树次生乳管分化相关的差异表达miRNAs奠定良好基础。

Rubber tree is the main source of natural rubber worldwide.Natural rubber is synthesized and stored in lati-cifer,a tissue composed of laticifer cells.The laticifer cells in the trunk bark are directly associated with natural rubber production and differentiated from the fusiform initials of vascular cambia.As the number of laticifer rings is positively correlated with rubber yield,the differentiation of laticifer from vascular cambia is a major theoretical subject faced to natural rubber industry.Plant miRNAs are a class of small noncoding RNAs about 20‒24 nucleotides in length and play a pivotal regulatory role in development,cell differentiation and adversity stress by mediating the the gene silencing.Quantitative Real-time PCR(qPCR)is widely used in the quantitative analysis of the miRNA expression levels,and the selection of appropriate internal reference of miRNA is crucial for accurating the miRNA expression levels.In the pre-sent study,the expression stability of three non-coding RNAs(U6 snRNA,5S rRNA,18S rRNA)and 6 mature miRNAs (hbr-miR159b, hbr-miR396b, miR169-x, miR172-y, miR535-x, miR894-x) were evaluated on the basis of coro-natine-induced secondary laticifer differentiation in the bark of rubber trees using poly (A)-tailed qPCR. According to the analysis of geNorm and NormFinder, hbr-mi396b and miR894-x were the top two stable miRNAs and U6 snRNA was the least stable gene in response to coronatine in the cambium tissue of rubber trees. The results showed that hbr-mi396b and miR894-x could serve as qPCR reference miRNA to analyze the miRNA expression pattern in COR-induced secon-dary laticifer differentiation. This study will provide a good basis for identification of the differentially expressed miRNAs related to secondary laticifer differentiation.