| Arabidopsis thaliana, as a model plant, has been the focus of scientists’research in the past ten years. Based on its research, people have improved not only their cognitive level of function genes of plants, but also trying to establish platforms for crop and ecological research. Therefore, T-DNA insertion mutant library was constructed to study the large amount of function unknown genes.The purpose of this paper is to study a function unknown gene AtRRM1(At3g23900) in Arabidopsis thaliana, about its expression and its possible correlation functions. TAIR functional annotation of the gene is the RNA recognition motif gene. The full length CDS of AtRRM1gene is2964bp, encoding987amino acids, containing two conservative domains according to the results of CDD database analysis.After the screening of AtRRMl homozygous inserted lines and observation of its phenotypic traits, we found that at the same stage, the height of mutant lines are significantly higher than that of the wild type plants and even in early stage of development, the hypocotyl of the mutants are longer. In addition, leaf color is shallower, petioles are longer, and leaf area is larger. Then, we used pMDC83to construct the whole length expression vector of the gene, transformed into the mutant lines. As a result, we found the phenotypes of transformed lines are complemented by different degree, consistent with the expression level of AtRRM1gene.About2kb sequence upstream of the ATG was cloned into the pBI101GUS-expression vector to analysis its expression in WT background. For semi-quantitative PCR and quantitative PCR, we took samples from both the mutant lines and wild type plants. GUS staining and PCRs share similar results on gene’s expression location and expression level, showing gene expresses in various organs but at different degree. GUS activity was observed in the leaf vein, small anthers, flowers and tender siliques except for older siliques and roots. However, expression in stems is different in sections.Conducted the bioinformatics analysis of proposed promoter we found that, the cis-elements of light reaction are in the major, no matter from kinds or from numbers. Therefore, we designed a light experiment. Seedlings were grown under three different conditions:light, dark and light for2days then in dark. After a certain time, we detected the germination rate and the height of seedlings. Data suggests that hypocotyl of mutant lines are inhibited under dark environment, though light can promote its hypocotyl elongation and at the same time improve the germination rate. This indicates the gene is sensitive to light and may be involved in light process.Plant height and plant hormones are closely linked, especially for gibberellin, which have the most closely relationship. Sprayed GA and PAC to the wild type and mutant lines, then detected GA related genes. We discovered in mutant lines, AtRRMl gene expression decreased while the GA3oxl gene expression increased in vivo. GA3oxl rose means increased synthetic amount of GA. So, we conclude that the height of mutant lines is an outcome of interaction of GA and light.Finally, we searched homo logy sequences of AtRRMl gene and made an evolution analysis. The gene share high affinity with B. rapa and B. oleracea gene (Bo1037328, Bra014989). According to the high homology of B. rapa, we designed primers at the region of start and stop codon, cloned a copy from Brassica napus, named BnRRM1. Searched homology sequences in B.napus rape database and got high homologous sequences, combined with the known sequences of B. rapa and B. oleracea gene, designed primers in common conservative region to amplify target sequence in ZhongShuang11(Brassica napus) with high fidelity enzyme, classified these fragments according to the specific SNP loci after sequencing. In total, we got seven different copies, making a foundation for future research. |
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