The Gene Cloning And Function Exploration Of Maize Glossy6

The plants epidermal wax layer is the most important barrier between the environment and the plant itself, which plays important role on protecting the plants from drought, pest/disease stress, also it is very important on revolution from aquatic plant to terrestrial plant. So far, the studies related to waxes are mostly focused on the wax related pathway and the wax composition study, such as the reports in sorghum, maize, barley, rice and some of the model plants like Arabidopsis, moss. While the mechanisms of the waxes related stress resistance was still unclear. There are lots of genes participate in the waxes biosynthesis pathway, to understanding the waxes biosynthesis pathway and the resistance, the key is to understanding the different roles in the waxes biosynthesis. Cloning genes and learning their function has been promoted with the development of the Next-Generation Sequencing technology. Here we described a maize epidermal wax biosynthesis related mutant glossy6, the BSR-Seq technology was used for glossy6 fine mapping and the gene was cloned via Seq-Walking technology, in the 113.5-146.7 Mb BSR-Seq mapping interval, gene GRMZM2G139786 ?Chr3: 127,513,712-127,514,561 bp? was been proved in at least 6 glossy6 alleles which have different Mu insertions in the gene of interest. glossy6 candidate GRMZM2G139786, encode an unknown function protein DUF538, which contains 224 amino acids. Based on the study above, we explored the potential function of the GLOSS Y6, the mainly study as below:1. The phenotype of glossy6 is similar with most of the maize glossy mutants that the surface of the leaves show the wax deficiency during the seedling stage, and glossy6 mutant shows sever phenotypes than most of the other glossy mutants because of the significant bright green colored leaves which different from the normal wild-types with grey leaves. The water loss of detached leaves in glossy6 mutant shows faster than the wild-types which indicate the higher permeability of the glossy6 mutants.2. glossy6 mutant accumulate less wax crystal on the surface when observed with SEM, and the paraffin section shows that the adaxial cells are disordered in the glossy6 mutant, instead, in the wild-types, the surface cells are well organized. Furthermore, the TEM observation of the inner cells of leaf surface shows the lipid inclusion in the cells of glossy6 mutant ?Former work?. Which may explains the reason that the glossy6 mutant has less wax accumulation.3. We mapped and cloned glossy6 via BSR-Seq and Seq-Walking technology, GRMZM2G139786, encoding a DUF538 ?domain of unknown function? protein. glossy6 has a paralogs in maize, Ac203262.3_FG002/DAA47018.1/113842, the homologs in grass family like rice ?Oryza sativa Japonica Group?, Setaria italic and Sorghum bicolor have higher similarity than the ones in dicotyledon, like XP₀02889742.1_Arabidopsis lyrata subsp. Lyrata in Arabidopsis.4. Based on the observation above, we detected the lipid/wax content/composition of epidermal waxes ?waxes covered on the leave surface?, cutin ?waxes that embedded in the cutin layer?, the microsomes waxes ?the organ that produce wax precursor? as well as the total wax-free leaves with GC-MS. In the glossy6 mutant, the total wax deposit was reduced and the fatty acids, Alkanes, especially the primary alcohol and the Aldehydes with 32 carbon chain, were significantly deficient on the epidermal surface. While the total wax detected in the Microsomes was slightly increased in glossy6 but compared to the reduction on surface, it's not worth to mention. The overall lipids deposition in cutin layer do not have much difference between glossy6 mutant and wild-types. And also, not very much interesting conclusions observed in wax-free leaves.5. RNA-Seq analysis on glossy6 and the corresponding wild-type show that 24,426 genes expressed in glossy6 and wild-types, compared with the wild-types,53 genes were significantly down-regulated?log2FC<=-2; P value<=0.05? and 324 genes were significantly up-regulated in glossy6 mutant ?log2FC>=2; P-value<=0.05?. Except Maize CER3 gene was significantly high expressed in glossy 6 mutant, none of the other cloned maize wax genes were differential expressed among those DEGs ?differential expressed genes?. Combine with the wax/lipid related genes in Mapman pathway and the homologs/paralogs of Arabidopsis/Maize, we obtained more than 500 possible wax related genes, which most of the up-regulated DEGs were likely involved in wax/lipid biosynthesis pathway while few of them involved in the wax/lipid transporting.6. We performed sub-cellular localization of glossy6 in maize protoplast with PEG method. The transient expression of glossy6 in maize seedling leaves shows that glossy6 was located in both cell cytoplasm and plasma membrane except nucleus. The co-sub cellular of the glossy6 with the marker genes show that glossy6 was expressed in endoplasmic reticulum ?ER?, cytomembrane, vesicle as well as the pre-vacuolar compartment ?PVC?.