黄麻内参基因筛选及次生细胞壁合成相关基因的表达分析

Reference genes screening for expression analysis of secondary cell wall synthesis related genes in jute(Corchorus capsularis)

选择合适的内参基因进行校正和标准化,既能提高实时定量荧光PCR的准确性,也为分析黄麻次生细胞壁合成相关基因的表达模式奠定基础。本研究通过常用的内参基因,结合课题组已有的基因组数据和转录组数据,初步筛选出8个内参基因(CcTUBα、CcACT、CcDnaJ、CcTUBβ、CcUBQ、CcEF1α、CcUBC和CcUBI)。为验证这些候选内参基因的稳定性,以优良品种‘黄麻179’为材料,对种子萌发后14 d的根、茎、叶进行qRT-PCR分析。经Ct值的变异系数、软件geNorm和NormFinder的综合分析,确定表达最稳定内参基因为CcDnaJ,最佳内参基因组合方式为CcDnaJ+CcUBQ+CcUBI。通过种子萌发后10 d下胚轴、60 d和90 d茎皮的转录组数据分析次生细胞壁合成相关基因的表达模式,发现木质素合成基因Cc4CL1、CcCCoAOMT1的表达量在种子萌发后60 d茎皮最高,而种子萌发后90 d茎皮表现为下降;纤维素合成酶基因CcCesA4、CcCesA7、CcesA8和木聚糖合成基因CcIRX8、CcIRX9、CcFRA8的表达量在种子萌发后10 d下胚轴最高,而种子萌发后60 d、90 d茎皮的表达量均有下降,表明这些基因参与了黄麻纤维发育。以CcDnaJ+CcUBQ+CcUBI作为内参基因组合,qRT-PCR分析5个次生细胞壁合成相关基因,即Cc4CL1、CcCCoAOMT1、CcCesA4、CcCesA7、CcesA8,在种子萌发后7 d下胚轴和14 d茎的表达模式,发现这5个基因在种子萌发期后14 d茎的表达量均高于种子萌发期后7 d下胚轴,暗示着黄麻纤维的形成开始于7~14 d之间,同时表明CcDnaJ+CcUBQ+CcUBI的内参基因组合具有实用性。

Selecting suitable reference genes for calibration and standardization can not only improve the accuracy of real-time quantitative PCR(qRT-PCR),but also lay the foundation for analyzing the expression pattern of genes related to secondary cell wall synthesis in jute.In the present work,8 candidate reference genes were screened on the homologs of common reference genes by using available genomic and transcriptomic data of the research group.To evaluate the stability of candidate genes,we used different tissues(roots,stems and leaves)of‘Huangma 179’at the 14 days after germination(DAG)as materials for qRT-PCR.Then qRT-PCR data were analyzed by coefficient of variation(CV)of Ct value,the softwares of geNorm and NormFinder,respectively.The results showed that the most stablest reference gene was CcDnaJ,and the best combination of reference genes was‘CcDnaJ+CcUBQ+CcUBI’.The expression patterns of secondary wall synthesis related genes were analyzed by transcriptomic data of hypocotyls at the 10 DAG,stem barks at the 60 DAG and 90 DAG.The electronic expression of lignin synthase genes,Cc4CL1 and CcCCoAOMT1,in stem barks at the 60 DAG were the highest,and then decreased at the 90 DAG.The expression of cellulose synthase genes,CcCesA4,CcCesA7,CcesA8,and xylan biosynthesis genes,CcIRX8,CcIRX9,CcFRA,in hypocotyls at the 10 DAG were the highest,and decreased in stem barks at the 60 DAG and 90 DAG.These findings indicate that these genes were involved in jute fiber development.Furthermore,the expression patterns of 5 secondary wall synthesis related genes,Cc4CL1,CcCCoAOMT1,CcCesA4,CcCesA7,and CcesA8,were analyzed by qRT-PCR using the reference gene combination of‘CcDnaJ+CcUBQ+CcUBI’at different stem developmental stages,i.e.hypocotyls at the 7 DAG and stems at the 14 DAG.The results revealed that relative expression of Cc4CL1,CcCesA4,CcCesA7,and CcesA8 genes in stems at the 14 DAG were higher than that in hypocotyles at the 7 DAG,suggesting that jute fiber formation starts at between 10 and 14 DAG,and the reference gene combination of‘CcDnaJ+CcUBQ+CcUBI’was available as well.