Cloning, Expression And Function Analysis Of BcNS In Brassica Campestris L.

Brassica is one of the most important genus of Cruciferae, and is one of the vegetable crops cultured most widely in Chinese cabbage pak-choi (Brassica campestris L. ssp. chinensis Makino), a subspecies of Brassica campestris L, is a typical allogamy plant with significant heteosis, and the popularization and application of its Fl hybrid seeds can improve the level of production in Chinese cabbage. Earlier study in our laboratory has found that At2g39060 was up-regulated in the fertile B line of the Chinese cabbage-pak-choi (Brassica campestris L. ssp. chinensis Makino) genic male sterile AB line (Bcajh97-01A/E) based on an ATH1 gene chip. The protein At2g39060 encoded belongs to MtN3/saliva family, and the function of At2g39060 is unknown. In order to realize the structure and expression partern of this gene, we cloned its full length cDNA using homologous gene amplification and rapid amplification of cDNA ends (RACE). And then, we analyzed its sequence characteristics and predicted protein structure. Real time quantitive polymerase chain reaction (qRT-PCR) and in situ hybrization were performed to inverstigate the expression parttern of the gene during different stages of plant development and forecast the gene's function. Then, the antisense RNA expression vector of BcNS were constructed, and transfomed into flowering Chinese cabbage (B. campestris ssp. chinensis var. parachinensis Tsen et Lee) by agrobacterium-mediated method to obtain their loss-of-function mutants for confirming its function. The major study results were as follows:(1)We cloned the homologous gene of At2g39060 in Chinese cabbage-pak-choi based on a ATH1 gene chip, using homologous gene amplification and RACE.Then we named it Brassica campestris Nectary Stamen (BcNS). The lengths of cDNA and DNA of BcNS are 1603bp and 1069bp. Its DNA includes six extrons and five introns, and the lengths of extons are 49 bp,37 bp,211 bp,162 bp,120 bp,234 bp; the lengths of introns are 188 bp,84 bp,92 bp,94 bp,76 bp, respectively. The open reading frame of its cDNA is of 813bp, encoding 270 amino acid residues. The molecular weight of BcNS is 30110.08 Daltons, and its isoelectric point is 9.117. BcNS includes two N-glycosylation sites, one cAMP- and cGMP-dependent protein kinase phosphorylation site, one Casein kinaseⅡphosphorylation site, two N-myristoylation sites, four Protein kinase C phosphorylation sites, one Big-1 (bacterial Ig-like domain 1) domain profile, two MtN3/saliva family sites.(2) Real time quantitive polymerase chain reaction (qRT-PCR) and in situ hybrization were performed to inverstigate the expression parttern of BcNS. we detected the expression of this gene in the sterile plants and fertile plants of 'Bajh97-01A/B' of the level five flower buds and opening flowers, and the expression in open flowers was significantly higher than level five buds, but at other stages of buds no expression was detected. In addition, the gene mainly express in the nectaries and stamens of the sterile plants and fertile plants, while the expression in the petals and sepals are very low, and no expression exsits in the pistil. Expressions of BcNS in stamens of the sterile plants and fertile plants are almost the same, but the expressions are not high. BcNS is highly expressed in the nectary, and the expression quantity is about 26～47 times higher than that in stamens.And the gene expression in the nectary of the fertile plants is about 1.7 times as that in sterile plants. The expression of BcNS in floral organs shows that the gene specifically expressed in the nectaries and stamens, but expressions between the sterile and fertile lines are not significant different. This indicates that the gene has no direct relationship with the fertility of Chinese cabbage, and it may play a role in other areas. However, expression of the gene detected only in the nectary by in situ hybridization, and this may be due to the limitations of this technology.(3) Anti-sense plant expression vector pBI35S-BcNS was constructed, and transformed into flowering Chinese cabbage by agrobacterium-mediated method. The molecular assay showed that pBI35S-BcNS constructs were integrated into the genomes of BcNS transgenic plants with Kanamycm resistance. Growth performance, floral organ development of transgenic plants were observed, these results showed that floral development was influenced by down-regulated expression of BcNS in transgenic plants. These results suggest that, BcNS is a new flower development-related gene in Chinese cabbage.