| Lotus corniculatus L. (L. corniculatus) is a perennial leguminous herb and belongs to the genus of Lotus. It is widely distributed in Europe, Asia, North America and Oceania. Owing to the fine quality, high biomass yield, resistance to adverse environmental conditions and easy storage performance, crowtoe has been used as one of the most excellent forage grass around the world. Flavonoids and carotenoids are two major categories of secondary metabolites influencing the quality of crowtoe. In recent years, a conserved microRNA family, miR156, has been shown to participate in regulation of the biosynthesis of flavonoids and carotenoids. Meanwhile, overexpression of miR156 in plants can enhance biomass yield, increase shoot branch number, delay flowering, etc. Although a lot of work has been carried out, the regulatory mechanism of miR156 in many plant species are still unclear.In the present work, a miR 156 precursor gene was cloned from Lotus corniculatus L. var. japonicus (L. japonicus) and was designated as pri-LjmiR156a. The authenticity and the functions of this gene were further investigated by constitutive expression in the same plant species. In addition, a number of putative genes targeted by LjmiR156 were identified from L. japonicus by bioinformatic analysis and the possible regulatory networks of miR156 in lotuso were preliminarily clarified. At the same time, the transcriptome and the digital gene expression profiles of L. corniculatus were analyzed with the help of next-generation high throughput sequencing method, and more than a hundred genes encoding the key enzymes of flavonoid biosynthesis were identified. These findings have provided new evidence to understanding of the regulatory networks formed by miR156 and its target genes, and can be used in promotion of the biomass and the contents of flavonoids and carotenoids by genetic manipulation.The detailed achievements are shown as below:1. By using the sequence of Arabidopsis Pre-AtmiR156 as template, a precursor gene of miR156, Pri-LjmiR156a, was isolated from L. japonicus genome. The miR156 overexressing plants were produced via genetic transformation mediated by Agrobacterium. Transgenic plants showed significant increase in biomass yield, delay in flowering, and disturbances in flavonoid biosynthesis and distribution. Late-flowering of multiple transgenic lines resulted in reduction of the fertility. Meanwhile, the symbiosis between the plants and the rhizobium was repressed by the overexpression of miR156. Compared to wild-type plants, miR156 overexpression transgenic plants exhibit 50% less nodules.2. The putative targets of miR156, including a number of SPL genes, were determined in L. japonicus by bioinformatic analysis. In miR156 overexpressing plants, the transcriptional levels of these SPL genes were selectively repressed. By employing a modified 5’-RACE protocol, the cleavage sites in mRNA of three LjmiR156 targets, including TC70253, AU089181 and TC57859, were confirmed. Further experiments on examination of the transcript abundances of the key genes in symbiosis revealed that the expression of multiple genes could be inhibited by enhanced level of miR156, such as ENOD40-1, ENOD40-2, POLLUX and CYCLOPS.3. To further examine the potential regulatory function of miR156 in forage crops, the transcriptome of a close relative species of L. japonicus, L. corniculatus, was profiled using high throughput sequencing method, and a large amount of genetic information was acquired. As a result,45,698 unigenes were obtained in total. Among them, approximately 150 unigenes were classified into key enzyme encoding genes involved in flavonoid biosynthesis. Meanwhile,2,998 unigenes were annotated to be transcription factor encoding genes by homologous search of the database of legume transcription factors. Four unigenes, including unigene322, C12952.Contigl-2, Unigene 19239 and Unigene2664, were characterized as the orthologs of Arabidopsis TTG1, PAP1, PAP2 and TT2, respectively, due to the high similarity between them.4. The expression patterns of the flavonoid biosynthesis related genes in different tissues, such as flowers, siliques, leaves and roots, were analyzed by using the digital gene expression libraries. The results indicated that these genes showed different expression profiles in different tissues. For instance, unigene322, a WD40 family gene, expressed in all the examined tissues, whereas a homologous gene of ANR was inclined to express in flowers, siliques and roots, the ANS family genes displayed a higher activity in flower, roots and leaves. |
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