| Pollen development is the key process of plant sexual reproduction. It’s not only associated with many distinctive cytological processes, such as mitosis and meiosis, but is also involved in some unique biological processes, such as the degradation of tapetum and the formation of pollen wall. Besides, pollen development is also important to agricultural production, such as male sterility, crop yield and seed quality. Thus, it is valid for theory and practice to study on pollen development. Histone lysine methylation, which is mediated by SET domain group (SDG) family, the only histone lysine methyltransferases in plant, has been proved to be involved in this process. Based on the released genome data, the members, genome location and sequence characteristics of SDG genes in Brassica rapa were analyzed. By comparing with the orthologous genes in Arabidopsis thaliana, the evolutionary pattern and the origin of the four main group in SDG family were discussed. Further, the modification patterns of H3K4me3 and H3K27me3 in pollen development were detected and a series of candidate SDG genes which might be associated with this process were screened through transcriptome sequencing. Finally, the expression pattern of a SDG gene, ASHR1, was detected by PT-PCR and in situ hybridization, and functional characterized by over-expression and mutant in B. rapa and A. thaliana. The main results obtained were summarized as follows.(1) By screening the genome database,67 SDG genes in B. rapa were detected, and in addition to the formerly discovered SDG family in A. thanliana, two more SDG genes in A. thaliana were found. The 67 SDG genes in B. rapa were distributed in ten chromosomes, with most located in the same blocks as their orthologs in A. thaliana. SDG family of B. rapa could be classified into seven subfamilies, with obvious differences existed in gene length, gene structure and domain architecture among these subfamilies. Compared to other species, B. rapa had more SDG genes and a larger proportion in comparing SDG gene number to genome size. Significant differences were obtained in the expression pattern and the subcellular localization among a series of paralogous genes between B. rapa and A. thanliana.(2) Comparing to orthologs in A. thaliana, more than half SDG genes in B. rapa in the four main subfamilies were found to be different in gene structure, including intron gain/loss, exon gain/loss and intron phase changing. Considering the orthologs in other species, it was found that most of these differences came from B. rapa but not A. thaliana. Analysis on protein domain architecture also demonstrated that many SDG genes in B. rapa were different to their orthologs, with some domain existed only in B. rapa. Based on the transiently expression assays in tobacco leaf epidermal cells and onion epidermal cells, four pairs of SDG orthologous proteins between in B. rapa and A. thaliana were found to displaydifferent subcellular localization patterns. Analysis on molecular evolutionary rate indicated 1/3 SDG genes in B. rapa evolved faster than their orthologs in A. thaliana.(3) Analysis on the gene structure of SET domain of SDG family determined the evolution patter of the four main subfamilies, which indicated E(z) subfamily came from one ancestral gene, Ash subfamily came from three ones, Trx subfamily came from two ones and Suv subfamily came from three ones. The data of gene structure, domain architecture, subcellular localization and the rate of molecular evolution were integrated to estimate the group evolutionary rate of SDG genes among the four main subfamilies, which indicated Trx evolved fastest, followed by SUVR and E(z), with SUVH and Ash evolved at the slowest rates.(4) H3K4me3 and H3K27me3, the two main histone lysine methylation, were detected by immunofluorescence in the nucleus of pollen mother cell and unicellular microspore in B. rapa. So did the vegetative nucleus of binucleate pollen and trinucleate pollen and the sperm of trinucleate pollen. The signal for H3K27me3 was also detected in the generative nucleus of binucleate pollen. However, these two methylation modifications were not detected in tetrad.(5) Screening the transcriptome sequencing data from B. rapa indicated 44 SDG genes in B. rapa were expressed in flower buds at different stages. Most of them could be classified into five groups based on their expression pattern, which was corresponding to the developmental stages of flower buds. Among them,70% were differentially expressed in the different stages of flower buds, and 41% were differentially expressed in the flower buds of fertile line and sterile line. Through the comprehensive comparison with A. thaliana chip data, seven SDG genes which might function in the process of the pollen development were detected, with two of them having been proved to work in this process.(6) Through RT-PCR analysis, ASHR1 was proved to be higher expressed in the sterile line than the fertile line. Further investigation by in situ hybridization demonstrated that this gene were concentrated in unicellular microspore and binucleate pollen.(7) Five alternatively spliced transcripts were detected in BrASHR1 locus, with the expression level of BrASHR1.1 extremely higher than others. In At ASHR1 locus, two alternatively spliced transcripts were obtained, with similar expression level detected.(8) The over expression of ASHR1 in B. rapa and A. thaliana did not affect the pollen development but delayed the bolting time. The T-DNA insert mutant of ASHRI also produced normal pollen but bolting earlier than wild-type plants. RT-PCR analysis indicated the expression of FLC was inhibited in the ashr1mutant. |
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