Identification Of MtN3/saliva/SWEETs Gene Family And Functional Analysis Of BcNS In Brassica Campestris L.

The MtN3/Saliva/SWEETs-type genes are an evolutionally conserved group of genes that are prevalent in higher eukaryotes, and they are also found in protozoa, metazoa, fungi, bacteria, and archaea. The MtN3/Saliva/SWEETs domain is also named as the PQ loop repeat, and it comprises a pair of repeats, each spanning two transmembrane helices connected by a loop. Although a distinct type of genes based on MtN3 and saliva has been identified, the exact biological functions of MtN3 and saliva themselves remain to be unclear.In this study,29 MtN3/Saliva/SWEETs genes have been identified in the Chinese cabbage genome. The classification, protein properties, gene structure, chromosomal distribution, evolutionary relationship and expression pattern in different tissues and the different periods of flower development of MtN3/Saliva/SWEETs genes in Chinese cabbage have been analyzed. In addition, the expression and biogical function of Bra000116(BcNS) was described by sub-cellular localization method and transgenenic technology. The main results are as follows:(1) The full-length cDNA sequences of all 29 newly identified MtN3/Saliva/SWEETs were isolated through genomes on line database. The open reading frame (ORF) length ranged from 500 bp to 1000 bp, encoding polypeptides of 175 to 316 amino acid residues. By prediction techniches online, the molecular formula of these gene family numbers ranged from 20 to 35 kDs, and the isoelectric point ranged from 7.59 to 9.62. In addition, except for the chromosome 5, the other’s 9 chromosomes have been observed the distribution of the family genes. There are various cis-acting elements, most of which were related to the optical response. It seems to the MtN3/Saliva/SWEETs playing the essential roles in photosynthesis process. Subsquently, by transmembrane region prediction, all the members contain transmembrane regions, and 23 members contained seven transmembrane proteins, and these extramembrane regions are generally very short but highly conserved within the intramembrane area, and the transmembrane area is relatively conservative, moreover, there is one phosphorylated amino acid sites in the two pieces of highly conservative membrane. These results show that the intramembrane region is one of the important functional domain, the regulatory mechanism is likely to be reversible phosphorylation/phosphorylation, controlled by some kinase/phosphatase. This may be an important entry point in researching function of MtN3/Saliva/SWEET family gene. Evolution analysis shows that the gene existing function in the evolutionary tree are in a relatively remote branch, which means that they might play a separate role in the development process.(2) The expression patterns of MtN3/Saliva/SWEETs genes in different chinese cabbage tissue and floral developmental stages were analysed by qRT-PCR. Except for the six pairs of primers Bra021577\Bra016421,Bra026487,Bra025595,Bra009700,Bra035879 expression hardly, other 14 pairs amplifying product showed tissue specificity, and the difference of expressing quantity are more than 100 times. These results of MtN3/Saliva/SWEET gene expression analysis show that different MtN3 Saliva/SWEET gene expression level in different flower organ is very obvious in different flower development stages of Chinese cabbage. The MtN3/Saliva/SWEET gene families mainly participate in the development process of Chinese cabbage.(3) A pB7YWG2.0-BcNS-YFP over-expression vector has been successfully constructed. Recombinant plasmid was introduced into fresh onion epidermal cells by the particle bombardment method with a PDS10000/He. The results showed that obvious fluorescence signal can be detected in the cell membrane in the onion epidermal cells, this means the ORF fragment of BcNS can be instantaneous expression in the cell membrane. The membrane proteins are mainly carrier protein, cell activity related ion pump, channel protein and protein receptors, etc., this result suggests that the BcNS gene might be relevant with the stability of cytomembrane or membrane transporting protein.(4) Anti-sense plant expression vector pCAMBIA-BcNS has been constructed, and transferred into flowering Chinese cabbage by agrobacterium-mediated method. The molecular assay including PCR> Histochemical staining detection and qRT-PCR showed that pCAMBIA-BcNS constructs were integrated into the genomes of BcNS transgenic plants with homomycin resistance. Growth performance, floral organ development of transgenic plants were observed, these results showed that floral development was normal but lateral nectary division from the base by down-regulated expression of BcNS in transgenic plants. Due to technical limitations, we cannot determine the nectar composition of the transgenic plants.