| Loropetalum chinense var.rubrum is one of the main color-leafed flowers for landscaping utility in the lower-middle reaches of Yangtze River basin and its adjacent south area. The lush greening of L. chinense var. rubrum is attained in summer and therefore its leaf phenotypic stability is an important aspect for quality evaluation. In order to investigate the molecular mechanism of temperature effect on leaf colour,’ChangyeMeihong’,’Huayejimu2,’DayeHong’ and’MeihongXiyeqing’ four materials were used to study Molecular analysis of gene expression and Choice reference genes against Loropetalum chinense var.rubrum.The main results are as follows:1. Screened Differentially Expressed Gene fragments:The RNA of4L. chinense var.rubrum materials in different month was extracted and amplified with the combination of both RT-PCR and cDNA-SRAP. Four pairs of high polymorphism primers:MelEml, Me1Em2, Me2Em1and Me2Em2in combination with Agarose Gel Electrophoresis and Polyacrylamide gel electrophoresis were selected in the amplification system. The differential expression of MelEml primer was screened from these4pairs. By testing the comparison expression of three pairs of specific genes (CHS, DFR, and ANS) in four flowers species, an ideal gene-CHS was screened for further reclaiming and sequencing.2.Analysis of differential gene expression:The sequence of specific gene expression was used to search available NCBI sequencing with the BLAST algorithm. The Data showed that CHS gene of four L. chinense var.rubrumis species share high homology with Juglans regia L. Hydrangea, tea plant, Camellia sinensis, Vitis vinifera and Casuarina equisetifolia. But the nucleotide sequence homology of Ml E1is still needed to confirm in further studies.3.The choice of internal reference genes:The targeted materials in this study were the buds, leaves, immature stem, petals and callus of L. chinense var.rubrum ’DayeHong’. The expressions of four common internal reference genes (a-tubulin, β-actin,18S rRNA and GAPDH) were studied by applying qRT-PCR against defined organs and tissues of L. chinense var.rubrum. The qRT-PCR analysis with Genorm and Norm Finder algorithms revealed that GAPDH could be employed as a reference gene to study the gene expression in different organs and tissues while β-actin in different mature leaves and callus. |
