Gene Expression Profiling And Cloning Of Lycoris Aurea Leaves

Lycoris aurea, being a representative species of Lycoris Herb, is a fine decorative plant and medicinal plant. It has been researched in many aspects, mainly includes the cultivation breeding technology, the chromosome nuclear type analysis, the importance for medicinal purposes ingredient withdraws and so on, but the research being about its heredity growth aspect has not yet been reported. In this paper, we compaired the gene expression profiling between a mutant and the wild type Lycoris aurea leaves by DNA microarray. The results provide some clues for further discussion of its leaf growth mechanism.Lycoris aurea is parallel venation, the main vein with many lateral veins longitudinal parallel arrangement. There are secondary lateral veins (SLV) between each longitudinal veins. In generally, SLVs are not remarkable. In this paper, the material is one kind of Lycoris aurea mutant being called Raised Secondary Lateral Veins mutant (RSLV), because it has many Raised Secondary Lateral Veins in abaxial surface of its leaves. Its growing potential is weaker than wild type ones, and its blades are very thin. Moreover, the stamens of RSLV degenerate completely.Two cDNA libraries were constructed from RSLV mutant and wild type (WT) leaves respectively. A total of 3738 randomly selected clones, in phagemid form, were single-pass sequenced from the 5' end, resulting in the characterization of 3122 ESTs that were longer than 100 bp after elimination of vector sequence. They were grouped into 1749 contigs/singlets, which were then used to design an oligonucleotide DNA microarray. Following a multistep selection, 512 70-mer oligo-DNA probes were selected for attachment on the microarray slide. The gene expression profile of RSLV mutant and WT leaves was compared through the microarray on transcriptional level. The microarray experiment results were further confirmed by Quantitative Real-Time PCR (QRT-PCR).With the results of microarray and QRT-PCR, we identified 5 genes whose expressions changed more than 2-fold between RSLV mutant and WT leaves. They encode phloem protein 2 (PP2), ferritin, pectin methyl esterase (PME), chlorophyll a/b binding protein (CAB protein) and pyruvate decarboxylase (PDC) respectively.Furthermore, the full-length cDNA sequences of the 5 genes were acquired from RSLV and WT by RACE respectively. We compared the difference of the full-length cDNA sequences between RSLV and WT. The differential expressions of the genes are related to the formation of the RSLV mutant phenotype.