The Expression And Function Analysis Of Pollen-specific Gene BcSKS11 During Pollen Development And Pollination Process In Brassica Campestris L.

Pollen development and pollination are complicated processes that are regulated by thousands of genes. Researches had showed that the pollen-specific expression genes, especially the ones associated with the synthesis and metabolism of cell wall, continuously expressed until over and done with the pollen tube growth. Recently, the large-scale pollen transcriptomics in Arabidopsis and Rice had also indicated mRNAs which played a role in pollen germination and pollen tube growth were stored in mature pollens. Now, there is a question. According to the efficiency of the pollen metabolism, why are those mRNAs produced in advance and how do they exist and escape from being degraded during the maturation and hydration of pollen by a complete style and transferred to the tissues they work. In our previous study, using Microarray technology for genome-wide transcriptional differential expression between the unpollinated and pollinated siliques in Chinese cabbage (Brassica campestris L. ssp. chinensis Markino var. communis Tsen et Lee), we detected a series of cell wall related genes which specifically up-regulated in pollinated siliques. Among them, we detected a putative multicopper oxidase (MCO) gene (Transcript ID:At3g13390) which may participate in pollen development and pollination. But the exact roles were not fully delineated. In plant, MCO which mainly includes ascorbate oxidase (AAO) and laccase affects the plants vegetative and generative growth. But the researches on their functions are still in a small proportion. Here, we cloned the new gene though 3'RACE and homological amplification methods and analyzed its structure and speculated its biological function. In addition, we used RT-PCR and In Situ Hybridization established its temporal and spatial gene expression patterns. Moreover, By Observing and comparing the vegetative and generative growth between the mutant sks11-1, sks11-2 and the wild type in Arabidopsis, we fixed the tentative function. By those studies, we aim to provide foundation of mRNA storage and transportation. The results obtained are as follows.(1) Through the combination methods like homologous amplification and RACE, we determined the full-length DNA containing 1911 bp and an ORF of 1668 bp which encoded a 555 Amine Acid. Comparison of the cDNA and DNA sequence by Clustal X software showed that it composed of three extrons and two introns, whose linkages accorded with the GT-AG rule. The putative protein included a hydrophobic signal peptide, a transmembrane helix, six distinct biologically significant sites in the protein sequence as follow:6 N-glycosylation sites,1 Glycosaminoglycan attachment site,11 Protein kinase C phosphorylation sites,4 Casein kinaseⅡphosphorylation sites,9 N-myristoylation sites,1 ATP/GTP-binding site motif A. Based on the online tools, we found it was a GPI-anchored protein and contained 3 putative domain, Cu-oxidase 3, Cu-oxidase, Cu-oxidase 2, which showed the structural characterization of AAO。Compared with other AAO, the protein is similar with them except lacking the completely Histidine residues to bind copper. And the similar amine acid sequences characteristics had been found in kinds of plants, for instance, the SKS proteins in Arabidopsis and NTP303 in tobacco. So we called this gene BcSKS11.(2) Here, we used RT-PCR and in situ hybridization to determine the expression pattern of BcSKS11. We ascertained that BcSKSll expressed strongly in flower but not in other tissues through RT-PCR method firstly. Further expression analysis among Petal, Sepal, stamen, pistil, nectary showed that it just expressed in stamen. In addition, BcSKS11 mRNA was first detected in the flower buds at stage IV and a highest level at stage V, which correspond to the microspore uninucleate stage and the mature pollen stage respectively. After pollination, BcSKSll still expressed and increased along with the time gone after pollination until 8 h-12 h, the expression level was highest and than decreased subsequently. While through In Situ Hybridization, we also established that BcSKS11 expressed in the pollen of buds at stage V.(3) By using "Three primers" for SALK lines, we separated the Arabidopsis homozygous mutant from T3 generation. Then, through observing the plant characters which included vegetative growth and the flower structure and analyzing the mRNA expression level of homology gene of BcSKS11, SKS11 in Arabidopsis, compared the mutant with the wild-type, we found that the mutant had no apparent differences with the wild type on the vegetative growth, male sterility, the flower structure, pollen morphology and pollen nuclear and blossom and bear fruit normally. And the pollen tube growth length in vitro after 5 h is almost the same between them. Yet growth patterns of mutant pollen tubes in the wild-type female organ 1 h after pollination had less germination rate than that of wild pollen tubes which indicated the role in pollen tube growth in vivo. What's more, SKS11 expressed lower in the mutant and simultaneously cause other related genes up-regulated or down-regulated which implied a potential relationship among them.