The Expression Analysis Of BcAHA8and Functional Identification Of BcSKS11during Pollen Development And Pollination In Brassica Campestris L.

Pollen development and fertilization are complicated processes which regulated by thousands of genes. The complete of the Arabidopsis’genome sequencing and the fast development of bioinformatics contribute greatly to the increasingly genomic data available to us, which make it easy and more convenient for us to study the process of pollen development and fertilization. In our previous study, we used Microarray technology for genome-wide transcriptional differential expression analysis between the unpollinated and pollinated siliques in Chinese cabbage (Brassica campestris L. ssp. chinensis Markino var. communis Tsen et Lee), and detected a series of genes which were specifically up-regulated in pollinated siliques. Among them, we detected a putative H--ATPase gene (Transcript ID:At3g42640) which may participate in pollen development and fertilization. The proton-pump ATPase (H+-T-ATPase) of the plant plasma membrane acts as a primary transporter by pumping protons out of the cell, thereby creating pH and electrical potential differences across the plasmalemma. Researchers have found that membrane transporters played crucial roles in pollen development, fertilization and could help plants fight against environmental stress. However, there is little study on H--ATPase. BcSKS11is a putative multicopper oxidase (MCO) gene, and our previous study indicated that it was preferentially expressed in pollen grain and the pollinated pistils. Thus, we predict it may play important roles in pollen development and fertilization. Therefore, in order to get the structure and expression characteristics of the homologous gene of At3g42640in Chinese cabbage-pak-choi.we cloned the new gene through homological amplification, analyzed its structure features and expression patterns. Moreover, through antisense RNA directional inhibition, the function of BcSKSll during pollen and fertilization was studied. The results are listed as follows.(1) A pollen and fertilization related gene was isolated from B.campeatris. We determined the full-length DNA containing3199bp and an ORF of2847bp. The secondary structural composition predicted by the PredictProtein server shows that there are41.9%helixes.9.8%sheet, and48.3%loop in the deduced protein structure. Cation_ATPase_N and E1-E2_ATPaseOne domain were predicted by SMART, indicating it is a H--ATPase gene.(2) We used RT-PCR. qRT-PCR and in situ hybridization to determine the expression pattern of BcAHA8. The results showed that BcAHA8was specifically expressed in flower of the fertile plants. Meanwhile the mRNA of BcAHA8was detected at the flower buds at stage V and the pollinated pistils. The in situ hybridization demonstrated that BcAHA8was expressed in binucleate and mature pollen stages, and the stigma of pistils which pollinated after2h and12h was detected the hybridization signal.(3) We constructed the RNA antisense vector of BcSKS11, pB135S-BcSKS, and and transformed it into Brassica ssica campestris ssp. Chinensis via Agrobacterium tumefaciens. The PCR analysis confirmed the transgenic lines.