Transcriptome Elucidation Of Tung Oil Tree Seeds And Cloning Of Key Oil Synthesis Genes

Tung oil tree (Vernicia fordii) originated in China is a famous woody oil plant equally celebrated with sasanqua (Camellia oleifera)、walnut (Juglans regia) and chinese tallow tree (Sapium sebiferum). Tung oil extracted from tung oil tree’s seeds has a high economic value and can be used to make industrial materials such as high-grade printing ink, biodiesel and new composite functional materials. Researches on oil synthesis of tung oil tree seeds will shed light on elucidation of the molecular mechanism of oil accumulation in woody plants. Furthermore, appling the results of this research in practice will increase the yield per unit area of tung oil and improve the tolerance of tung oil tree to adverse environment. However, researches on tung oil tree so far have focused on cultivation and breeding and there are no in-depth molecular mechanism studies. Related molecular biology researches on tung oil tree were restricted in homologous gene’s clone and have yet to involve identification of gene’s function in signal network. The current situation does not satisfy the requirements of genetis, molecular biology and breeding science of tung oil tree. This disadvantage is caused by the lack of useful referrible genic information. This research performed transcriptome sequencing of developing tung oil tree’s seeds using Illumina technology at June, August and October, respectively. Based on transcriptome sequencing, we pairwise compared 3 transcriptome to reveal oil synthesis-related pathways and cloned two key enzyme genes, FATA and KAS Ⅲ, to study their functions in oil synthesis. The main results are as follows:1. Sequence assembly, gene functional annotation and classification. This research performed transcriptome sequencing of tung oil tree seeds at 3 developmental stages (Ⅰ, Ⅱ and Ⅲ) using Illumina sequencing technology. Sequence assembly of 52,039,198 clean reads of transcriptome sequencing data at I stage obtained 98,909 Contigs and 61,001 Unigenes which involved 54,918 Contigs with a length range of 75-200 nt. Sequence assembly of 51,886,702 clean reads of transcriptome sequencing data at Ⅱ stage obtained 87,848 Contigs and 54,679 Unigenes which involved 47,735 Contigs with a length range of 75-200 nt. Sequence assembly of 52,734,436 clean reads of transcriptome sequencing data at III stage obtained 69,253 Contigs and 44,495 Unigenes which involved 34,274 Contigs with a length range of 75-200 nt. Accounting for 70.3 percentage, a total of 41,059 Unigenes could be matched to gene sequences already known in public database. Statistics on 3 transcriptome sequencing data, a total of 26,625 Unigenes had homologous genes in COG database and could be classified into 25 categories according to their predicted functions. Accounting for 51.8 percentages, a total of 30,280 non-redundant Unigenes could be classified into 61 categories. Pathway enrichment analysis classified all non-redundant Unigenes into 128 metabolic pathways.2. Expression profile elucidation of genes involved in oil synthesis pathways of tung oil tree seeds. Differentially expressed Unigenes obtained from pairwise comparisons of 3 transcriptome data could be classified into 57 (II vs I),59 (III vs II) and 61 (Ⅲ vs Ⅰ) categories using GO analysis, respectively. Meanwhile, these differentially expressed Unigenes also could be classified into 126 (Ⅱ vs Ⅰ),127 (III vs II) and 127 (III vs I) metabolic pathways using KEGG pathway enrichment analysis, respectively. Accounting for 0.24 percentages of all non-redundant Unigenes,54 Unigenes were involved in fatty acid biosynthesis pathway. After alignment of these Unigenes against KEGG database, we identified 14 homologous genes related to fatty acid biosynthesis and explored their expression profiles during oil synthesis stage of rung oil tree’s seeds. Accounting for 1.93 percentages of all non-redundant Unigenes, 428 Unigenes were involved in glycerophospholipid metabolism pathway. After alignment of these Unigenes against KEGG database, we identified 31 homologous genes related to glycerophospholipid metabolism and explored their expression profiles during oil synthesis stage of tung oil tree’s seeds.3. Cloning and characterization of tung oil tree’s FATA gene. We cloned full-length cDNA sequence of tung oil tree’s FATA gene with 1480 bp length. Based on full-length cDNA sequence clone, CDS and genomic sequences of FATA gene with 1113 bp and 3801 bp length, respectively, were cloned using PCR. Through sequence alignment, FATA protein sequence was found to possess high homology against other plants’ counterparts. This result demonstrated that the gene we cloned was FATA gene of tung oil tree. Phylogenetic analysis identified that tung oil tree’s FATA had closest evolutional relationship with Jatropha curcas FATA, followed by Ricinus communis, and had farthest genetic relationship with Medicago truncatula. Southern blotting analysis found that FATA gene only had one copy in tung oil tree’s genome. The expression of FATA gene of tung oil tree was detected in roots, leaves and seeds. Tung oil tree’s leaves cotyledons and radicles showed almost the same expression level of FATA gene whereas the seeds possessed higher expression level of FATA gene than these organs and the gene’s expression level was induced along with the seed development. In order to further study the regulation of gene expression, the promoter sequence of FATA gene was cloned using TAIL-PCR technology.4. Cloning and characterization of tung oil tree’s KAS Ⅲ gene. We also cloned full-length cDNA sequence of tung oil tree’s KAS Ⅲ gene with 1,651 bp length. Through sequence alignment, KAS III protein sequence was found to possess high homology against other plants’ counterparts. This result demonstrated that the gene we cloned was exactly KAS Ⅲ gene of tung oil tree. Phylogenetic analysis identified that tung oil tree’s KAS Ⅲ had closest evolutional relationship with Jatropha curcas and Ricinus communis KAS Ⅲ. Southern blotting analysis found that KAS Ⅲ gene only had one copy in tung oil tree’s genome. The expression of KAS Ⅲ gene of tung oil tree was detected in roots, leaves and seeds. Tung oil tree’s leaves cotyledons and radicles showed almost the same expression level of KAS Ⅲ gene whereas the seeds possessed higher expression level of KAS Ⅲ gene than these organs and the gene’s expression level was induced along with the seed development.In a word, our research based on RNA-Seq technology will not only shed light on elucidation of molecular mechanism of oil biosynthesis in tung oil tree’s seeds but also lay material and technical foundations for genetic improvement of tung oil tree.