Cloning And Functional Confirmation Of A Novel Pollen-Specific Gene BcMF20 Encoding C2H2 Zinc Finger Protein In Brassica Campestris L.

Brassica crops is a kind of important crops in agricultural production. The male sterile line was widely utilized in practice on produce F1 hybrid seeds. Much attention was paid to research on the plant breeding of the male sterile line and the basis for application in Brassica crops. The research on male sterility mechanism of Brassica contributed to the understanding of microspore and pollen development process and provided theory direction to create man-made male sterile line. In the former research of our lab, we established three different male sterile types,'Aijiaohuang'genic male sterility AB line (aihGMS'Bcajh97-01A/B'),'Poloma'genic-cytoplasmic male sterility (polG-CMS 'Bcpol97-05A') and'Ogura'cytolasmic male sterility (oguCMS'Bcogu97-06A') in Brassica campestris L. ssp. chinensis Makino, syn. B. rapa ssp. chinensis. These male sterile lines share a common maintainer line. Then, we conducted profiling comparisons between flower buds of these male sterile lines and their maintainer line using ATH1 genome array analysis. By analyzing the changes in expression pattern of genes acting downstream due to the gene mutation in different male sterile plant, 29 genes were identified. In this study, one C2H2 transcription factor in the 29 genes was selected for further characterization. The corresponding full-length cDNA and DNA was subsequently amplified by homologous gene amplification method. The gene sequence was analyzed and proteins functions were predicted. Spatial and temporal expression patterns were analyzed by reverse transcriptase PCR (RT-PCR), real-time quantitative PCR (RT-qPCR) and in situ hybridization. On this base the plant anti-sense RNA expression vector was constructed regulated by the constitutive promoter CaMV35S. The transgenic plants were obtained successfully using the floral dip method to transform the vector to the Arabidopsis (Columbia). Finally, molecular, morphological and cytological characteristics of the pollen were studied in transgenic plants, in order to elucidate the relation of the gene and pollen development, and understand the systematic regulation and molecular mechanism of pollen development in Brassica. The results obtained are as follows.(1) We cloned an cDNA of the gene differentially expressed in pollen using homologous expansion named Brassica Campestris Male Fertility 20 (BcMF20). Based on the cDNA of BcMF20, we designed primers to amplify DNA of BcMF20. A comparison of cDNA and DNA sequence showed that BcMF20 was composed of a 1359bp open reading frame, encoding an 50.874 kDa protein of 452 amino acids.The amino acid sequence of BcMF20 possesses the basic feature of C2H2 zinc finger protein, containing three finger, its finger type is MMB.(2) We isolated the homologue of BcMF20 from 18 cultivars from genera Brassica and Raphanus of family Cruciferae by homology cloning, and then compared these sequences with BcMF20 by multiple alignment. The similarity of DNA from BcMF20 homologous gene is from 86.9%-100%, which indicated that homologues of BcMF20 from family Cruciferae were highly conserved. All the genes are without introns, and totally identical in the CDs region. The encoded proteins are all C2H2 transcription factor contain three Zinc-finger, and the type of the Zinc-finger are MMB style as BcMF20. In the zinc-finger domain, the base sequences are conservation. When Blast BcMF20 according to NCBI database, found 12 Zinc-finger transcription factor genes have high identity to BcMF20, and all these sequences were analyzed by NJ to construct a phylogenetic tree. From the tree, BcMF20 is neighbored with three Arabidopsis thaliana C2H2 Zinc-finger transcription factors, and then become a tuft with four Zinc-fingers C2H2 transcription factor from Petunia.(3) Spatial and temporal expression patterns were analysed by RT-PCR and real-time quantitative PCR during the different stages of sterile and fertile plant development. The expression signal of BcMF20 was detected from fertile plant flower buds of stage 4 and stage5 by RT-PCR and real-time quantitative PCR. In contrast, little expression signal of BcMF20 was detected in same tissues of the sterile mutant by real-time quantitative PCR, and undetected from germinal siliques, leaves and scapes from sterile and fertile plant. The result indicated BcMF20 was an important gene in pollen development. BcMF20 was a'late'gene according to the classification of pollen expressed gene by Mascarenhas (Mascarenhas,1990). In situ hybridization indicated expression signal of BcMF20 was detected in uninucleate microspore and the tapetum cells, and expression signal was also detected in the maturing pollen and degradating tapetum. These results indicated that BcMF20 may has an effect on the tapetum and microspore development in uninucleate microspore stage.(4) Construct the anti-sense RNA vector with CaMV35S promoter, and introduced the vector into Arabidopsis thaliana by Floral Dip. Contrast with the empty vector, transgenic plant is normal during vegetable growth and the female organs, but have less pollen grain, and can not form turgid pods. The pollen from transgenic plant has low germination rate,13% and 15% in the T1 and T2 generations, respectively. 82% pollen from transgenic plant shows malformation under SEM. Contrast with normal pollen grain, the transgenic pollen display inanition, hollow and shrunken. Under TEM, we found two reasons to explain the low germination rate from transgenic pollen, one is the less of pollen grain in the pollen sac, and the other is the vacuole in the pollen. The abnormal pollen is likely due to the abnormal callose and the extine formation. The similarity in the expression pattern and the phenotypes resulting from the lack of their respective expression suggest that TAZ1, MS1 and BcMF20 could serve as components of the regulatory mechanism that controls the postmeiotic phase of tapetum development.