Cloning And Functional Analysis Of α-Expansin Genes From Anthocephalus Chinensis

Anthocephalus chinensis with the synonym of Neolamarckia cadamba belonging to the family Rubiaceae, is an evergreen broadleaf arbor and a fast-growing tree species which grows in Tropical Asia and Southern Asia. It was universally accepted as "a miraculous tree" in the World Forestry Congress in1972because of its rapid growth. Meanwhile, it possesses significant development and utilization value in South China. However, genetic information on A. chinensis is still unclear, especially in molecular mechanism study on fast-growing of wood. Expansin is a class of important cell growth regulator, and closely related to the growth and development of plants. So, in the study, we fitstly cloned a-expansin gene sequences in A. chinensis, and inditified the function of the a-expansin gene abundantly expressed in the cambium region, a-expansin genes were isolated by amplifying conservative domain binding with genomic walking and3’RACE techniques from four differential growth tissues in A. chinensis, followed by bioinformatic analysis. Based on these, we selected a suitable reference gene and analysed the temporal and spatial expression pattern of a-expansin genes. AcEXPA8gene expressed highly in cambium region was selected to analyse its copy number in genome, and we also predicted the protein structure and function by homology modeling. Meanwhile, we constructed the plant expression vector and transformed it into Arabidopsis thaliana to further indentify AcEXPA8gene function. The main results and conclusions obtained in this study are shown as follows:1. AcEXPA1-16genes were cloned by a cloning strategy. Firstly, the conservative regions of A. chinensis a-expansin genes were amplified with the degenerated primers, then, the flank sequences were obtained by genomic walking and3’RACE. Their GenBank accession Nos. were FJ417847, JF922686-JF922700, with corresponding genomic DNA sequences of GenBank accession Nos. GQ228823, JF922701-JF922715.2. The intron-exon structures of AcEXPAl-16are similar between them, and they all contain intronl and intron2which are conservative in a-expansin genes. All AcEXPAl-16introns conforn with eukaryotic intron splicing pattern of GT-AG, except the intron2of AcEXPA4with TA-GA.3. All amino acid sequences encoded by AcEXPAI-16gene contain conservative sequences of EXPA protein, including the N-terminal signal peptide, eight C residues, HFD motif and four W residues near the C terminus. But, there is a leucine (L) instead of the first W in AcEXPA12. All mature proteins of AcEXPA1, AcEXPA5, AcEXPA8and AcEXPA9cotain RIPGV sequence at N-terminal and KNFRV sequence at C-terminal. And AcEXPAl-16genes were divided into four subfamilies in the phylogenetic analysis.4. The screened cyclophilin gene AcCyP was suitable in RT-qPCR analysis as reference gene. AcEXPA1-16genes showed obviously tissue-specific expression in A. chinensis. In the four young tissues in growth, AcEXPA8was expressed highestly in cambium region. AcEXPA4, AcEXPA6, AcEXPA15, AcEXPA14and AcEXPA10were abundantly expressed in the tender leaf. AcEXPA13was only expressed in leaf, whereas AcEXPA16was only expressed in root. The expression of AcEXPA8was significantly different between different growing seasons in cambium region, highest in July, but lowest in January. And the abundance of AcEXPA8expressed in second wall formation region was significantly lower than that in camium region.5. AcEXPA8gene is present in A. chinensis genomic DNA as multicopy. Protein homology modeling showed that the3D structure of mature protein AcEXPA8is similar to maize ZmEXPB1which is the only known crystal structure of EXP protein so far, and also contains domain1, domain2and connection area between them.6. Overexpression of AcEXPA8in Arabidopsis could not only accelerate leaf formation, induce leaf area, cause leaf length growth greater than width growth, leading to the leaf shape becoming prolate, and increase the radius of fully mature rosette, but also increase plant height and stem diameter.7. In transgenic plant stem, overexpression of AcEXPA8promoted secondary growth in Arabidopsis, mainly leading to increase fiber cell length and cell wall thickness, but without changing the content of cellulose in the cell wall. In addition, the area of pith and cortex parenchyma cells increased obviously, and the length of cortex parenchyma cells along the circumference direction increased particularly.In this study, the AcEXPA gene family member expressions were analyzed in different young tissures, and the temporal and spatial expression pattern of AcEXPA8gene expressed abundantly in cambium region was further analized, followed by gene function analysis. These findings will lay the foundation for studying the relationship between a-expansin genes and growth rate and wood quality of the xylem in A. chinensis, and finally provide potential candidate genes for tree molecular breeding.