Molecular cloning,characterization and promoter analysis of LbgCWIN1 and its expression profiles in response to exogenous sucrose during in vitro bulblet initiation in lily

Lily(Lilium spp.) is an important ornamental flower, which is mainly propagated by bulbs. Cell wall invertases(CWINs), which catalyze the irreversibly conversion of sucrose into glucose and fructose in the extracellular space, are key enzymes participating in sucrose allocation in higher plants. Previous studies have shown that CWINs play an essential role in bulblet initiation process in bulbous crops, but the underlying molecular mechanism remains unclear. Here, a CWIN gene of Lilium brownii var. giganteum(Lbg) was identified and amplified from genomic DNA. Quantitative RT-PCR assays revealed that the expression level of LbgCWIN1 was highly upregulated exactly when the endogenous starch degraded in non-sucrose medium during in vitro bulblet initiation in Lbg. Phylogenetic relationship, motif, and domain analysis of LbgCWIN1 protein and CWINs in other plant species showed that all sequences of these CWIN proteins were highly conserved. The promoter sequence of LbgCWIN1 possessed a number of alpha-amylase-, phytohormone-, light-and stress-responsive cis-elements. Meanwhile, β-glucuronidase(GUS) assay showed that the 459 bp upstream fragment from the translational start site displayed maximal promoter activity. These results revealed that LbgCWIN1 might function in the process of in vitro bulblet initiation and be in the response to degradation of endogenous starch.

Molecular cloning,characterization and expression analysis of three key starch synthesis-related genes from the bulb of a rare lily germplasm,Lilium brownii var.giganteum

Starch is the predominant compound in bulb scales,and previous studies have shown that bulblet development is closely associated with starch enrichment.However,how starch synthesis affects bulbification at the molecular level is unclear.In this study,we demonstrate that Lilium brownii var.giganteum,a wild lily with a giant bulb in nature,and L.brownii,the native species,have different starch levels and characteristics according to cytological and ultra-structural observations.We cloned the complete sequence of three key gene-encoding enzymes(LbgAGPS,LbgGBSS,and LbgSSⅢ)during starch synthesis by rapid amplification of 5’and 3’complementary DNA(cDNA)ends(RACE)technology.Bioinformatics analysis revealed that the proteins deduced by these genes contain the canonical conserved domains.Constructed phylogenetic trees confirmed the evolutionary relationships with proteins from other species,including monocotyledons and dicotyledons.The transcript levels of various tissues and time course samples obtained during bulblet development uncovered relatively high expression levels in bulblets and gradual increase expression accompanying bulblet growth.Moreover,a set of single nucleotide polymorphisms(SNPs)was discovered in the AGPS genes of four lily genotypes,and a purifying selection fashion was predicted according to the non-synonymous/synonymous(Ka/Ks)values.Taken together,our results suggested that key starch-synthesizing genes might play important roles in bulblet development and lead to distinctive phenotypes in bulblet size.