| The tung oil tree (Vernicia fordii) is one of the most important industrial oil tree in the world, was widely used in industry, for example, in paints, plastics, synthetic rubber, ink and so on. Meanwhile, the characteristic of security, renewable and environmentally friendly let the tung tree considered to be an important raw material for biodiesel. However, the current study of tung tree was mainly focused on conventional breeding direction, as the high-yield planting, cultivation optimal selection, thus, its molecular biology reasearch was very weak. Therefore, with the molecular techniques can we clone the oil biosynthesis-related genes and by the functional verification can help us establish a foundation to conduct the molecular breeding. The NF-YB (Nuclear Transcription Factor-Y, Subunit B) genes encode a subunit of the CCAAT box-binding factor (CBF), a highly conserved trimeric activator that recognizes and binds specifically the ubiquitous CCAAT promoter element with high affinity. The functional importance of NF-YB family genes have been proved in a variety of diverse experimental systems, proved their roles in embryo and plant development, flowering time, plant growth, and plant-environment interactions. The transcription factor LEC1 (Leafy Cotyledonl) is a member of NF-YB family, studies have proved its important roles in fatty acid synthesis. The research here cloned 7 NF-YB family genes, and characterized the phylogenetic relationships, exon/intron structures, protein structures, expression patterns about its tissue-specificity and in plant-pathogen interactions. Besides, we selected VfNF-YB14 (VfLEC1) gene to do the preliminary functional verification, we successfully constructed the plant expression vector, then transferred VfLEC1 into Arabidopsis plants. The main results were as follows::(1) To investigate the structure of VfNF-YB genes, the specific primers were employed to amplify the sequences from tung genomic DNA. DNA sequence comparison revealed that VfNF-YB1,7,9,11,13,14 had no intron structure, however, VfNF-YB5 had three introns. Structure modeling of seven VfNF-YB demonstrated highly conserved domains among them and there were a disordered region in N terminus and two long helix structure in C terminus. Phylogenetic analyses show that VfNF-YB family genes had conserved B domian, and divided them into non-LEC1 type genes VfNF-YBl,5,7,9,11,13 and LEC1-type gene VfNF-YB14. Besides, DNAman was used to identify levels of similarity between members of the VfNF-YB family. VfNF-YB1/VfNF-YB5 and VfNF-YB7/VfNF-YB13 had high identity,VfNF-YB11with VfNF-YB7and VfNF-YB13 had close similarity, but VVfNF-YB9 had low similarity with them. Generally, the conserved domains of individual VfNF-Y members showed much higher levels of similarity than did the full-length amino acid, suggesting that fragments of the VfNF-YB outside of the conserved domain have low identities.(2) VfNF-YB1,5,11 shared a predominant expression in kernels while varied little in the other three tissues, VfNF-YB1,9 reached a peak of expression in roots, and showed low transcriptional level in the other three tissues. VfNF-YB13 is expressed approximately at the same level in the four tissues. VfNF-YB14 is mainly expressed in vascular tissues, including roots, stems and leaves.(3) VfLEC1 (VfNF-YB14) was expressed in all tissues, the expression performance in the different organizations as:Leaf> stem> root> seed> flower> ovary> fruit> stamen. The expression of VfLEC1 had time-specificity, because it had high expression in the early period of fatty acid synthesis (11th in August). The gene structure revealed VfLEC1 had no intron structure, and encoded 242 amino acids. The predicted isoelectric point was 6.91, and the molecular weight was 27.03kD. Amino acid comparison showed VfLEC1 had a conserved region B, and had 16 conserved amino acid residues (M, I, R, H, D, I, Y, N, D, R, Q, T, A, E, K, T). There were three conserved regions a-helix (a1-3) and two connecting areas (L1, L2). Phylogenetic analysis showed that the tung oil tree had the closest evolutionary relationship with castor (.Ricinus communis)(4) Successfully constructed the expression vector pB110-VfLEC1,which has a seed-expressed specific promoter. According to the Arabidopsis growth condition and the level of expression of AtLEC1, we selected the appropriate mutants, and transferred VfLEC1 into Arabidopsis mutant plants successfully. We obtianed eight positive transformants, the expression differences between them was significant, the highest expression of the transformant (LEC1-7) is about ten times higher than LEC1-4,which had the lowest expression. The phenotypes of the mutant wild-type and transgenic arabidopsis in the early stage of seedling is not obvious.In the middle growth stage, the transgenic arabidopsis boltted and flowered more early compared with the wild-type, but boltted and flowered latter than the mutant. In the late stage, the transgenic arabidopsis had more branches compared with the wild-type. The seeds appearance difference of the mutant、wild-type and transgenic arabidopsis varied little,but the transgenic seeds had heavier grains,and the length, width value was larger slightly.The fatty acid content and the component analysis need a subsequent measurement, in order to reveal the role of transcription factors VfLEC1 in controlling oil synthetic biology. |
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