| Pollen development and pollination are multi-step processes that regulated by thousands of genes. Recently, the large-scale pollen transcriptomic and proteomes in Arabidopsis and Rice has also shown that mRNAs which played a crucial role in pollen germination and pollen tube growth were stored in mature pollens. During the multi-step process, current researches only focus on the function of genes in one step. Although several speculations believed that pollen grain accumulation and storage of a variety of mRNA may be used for pollen germination and pollen tube growth. But there has little researches about the function of these continuously expressed genes in this process, and there is not clear that how they established the link between expression regulation and different developmental processes.In our previous study, we analyzed the expression pattern of pollen development and fertilization-related genes by AT HI microarray of ’Aijiaohuang’ genie male sterile AB line (Bajh97-01A/B) in Chinese cabbage-pak-choi (Brassica campestris L. ssp. chinensis Makino), and found several gene which specifically up regulated in pollen and pollinated pistil. Among them, one is a JmjC domain containing histone demethylase gene (Transcript ID:At3g20810) which may involved in pollen development and pollination (unpublished data), the other is a methyltransferase gene (Transcript ID:At1g58120) which may participate in pollen development and pollination. In plants, JmjC domain-containing histone demethylase play important roles in plant growth and development. But, the function of JmjC histone demethylase is not clear in plant reproductive. Therefore, in order to get the structure and expression characteristics of the homologous gene of At3g20810and Atlg58120in Chinese cabbage-pak-choi,we cloned the new gene through homological amplification and3’RACE methods and analysis its structure and speculated its biological function, we analyze the expression pattern of this gene using qRT-PCR and in situ hybridization. Through antisense RNA directional inhibition, the function of them during pollen and fertilization was identified.(1)A pollen and fertilization related gene was isolated from B.campeatris. We determined the full-length DNA containing1956bp and an ORF of1245bp. Comparison of the cDNA and DNA sequence by Clustal X software showed that it composed of seven extrons and six introns. The secondary structural composition predicted by the PredictProtein server shows that there are46.8%helixes,34.5%sheet, and16.9%loop in the deduced protein structure. One JmjC domain of the gene was predicted by SMART, within the414deduced amino acids, JmjC domain located between the amino acid257to414. Phylogenetic tree indicated that the gene is closely related to At3g20810(AtJMJ30) in Arabidopsis, so we named it as JMJ30(BcJM.130).(2) Here, we used qRT-PCR and in situ hybridization to determine the expression pattern of BcJMJ30. It was highly expressed in open flowers and scapes compared with germinal siliques, leaves and roots. It was continuously expressed during flower buds in fertile plants but had its strong expression in the first two stages of the ones of sterile plants. BcJMJ30mRNA mainly expressed in the stamens and petals compared with other flower parts such as sepals, pistils and nectarys. And there was a significant increased expression level in the pollinated pistils. (3) Functional analysis of BcJMJ30by anti-sense technique revealed that it can affect the size, shape and plumpness of pollen,25.21%of the pollen grains from anti-sense transgenitc plants exhibited abnormal, and reduce the pollen germination rate, leading to abnormal pollen tube. Statistics show that, the ratio of abnormal pollen tube of it is higher than the control. Moreover, in vivo pollen germination assay showed that in transgenic plantlets can reduced the pollen germinated on pistil, after4h pollination there still exist some observable pollen on stigma of the pistil compared with the control.(4) A pollen and fertilization related gene was isolated from B.campeatris. The cDNA sequence of the gene was1870bp and contained an open reading frame of1260bp which encoded419amino acids. ProtParam database analysis of the protein indicated it was aliphatic and unstable. Genomic sequence of it contained no introns. The secondary structural composition predicted by the PredictProtein server shows that there are29.6%helix,18.6%sheet, and51.8%%loop in the deduced protein structure. Sequence prediction indicated that the gene might encode a methyltransferase.(5) We used qRT-PCR and in situ hybridization to determine the expression pattern of the gene. We confirmed that it was expressed at higher levels in open flowers than in other organs (siliques, scopes and leaves). In different pollen developmental stages it was continuatively expressed in flower buds at five developmental stages in fertile plants, and expressed at higher transcript levels in the flower buds of fertile plants compared to the sterile plants except Stage Ⅰ, especially on a very high level in the Stage V. So we named it as Brassica campestris Male Fertility22(BcMF22). Further expression analysis among Petal, Sepal, stamen, pistil, and nectary showed that it just expressed in stamen. After pollination, BcMF22was expressed at higher levels in pistils at1,2and4HAP than their corresponding controls, especially on a very high level in the4HAP.(6) Functional analysis of BcMF22by anti-sense technique revealed that it can affect the pollen development, and even reduce the viability of pollen,25.76%of the pollen grains exhibited abnormal. Histochemical staining showed that transgenic plants could reduce the fluorescence signal of pollen. Transgenic plants leaded to decrease the germination ratio of pollen, once pollen germination, then the top of pollen tube was burst, abnormal pollen tube accounted for73%. |
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