Genome-Wide Analysis Of Xylogen-Like Arabinogalactan Protein Gene Family And Biological Function Studies Of OsAPG13in Rice

Arabinogalactan-proteins (AGPs) are a class of hydroxyproline-rich glycoproteins that widely participated in the plant growth and development processes. Xylogen is a type of chimeric AGPs contains non-specific lipid transfer protein (nsLTP) domain. We performed the genome-wide studies, chromosome location and expression analysesof OsXYLPs gene family, by using the methods and techniques of bioinformation, molecular biology, genetics and cell biology. We successfully identified two T-DNA inserted mutants of OsXYLP7and OsXYLP16, and found that the mutant xylp7have a shorter stem length compared to wild-type. OsAGP13is a Lys-rich AGP, there were varied phenotype in RNAi plants because of the down-regulated expression of OsAGP13gene, and then the biological functions of OsAGP13during the development processes of cell wall and vascular bundles were study by using multiple experimental approaches. The main results are described as follows:1.BLASTP analysis was performed across RGAP and RAP-DB databases by using the protein sequences of ZeXYP1, AtXYP1, and AtXYP2as queries; and we downloaded the Hidden Markov Model (HMM) profile of nsLTP domain from Pfam (http://pfam.sanger.ac.uk/)(PF00234) in fasta format for doing the BLASTP search with default settings to screen the proteins containing nsLTP domain in RGAP and RAP-DB databases. After removing redundant sequences of the two above results and confirming the presence of AGP-like regions and AG-type glycomodules, we obtained21OsXYLP proteins in rice. To study the evolutionary relationship between XYLP proteins, an unrooted phylogenetic tree of21OsXYLPs and13AtXYLPs was constructed according the alignments of their full-length protein sequences,34XYLP proteins were clustered into four clades. We achieved the exact coordinates and orientations of OsXYLP genes from the RGAP database, and found that21OsXYLPs are located on seven chromosomes and most of them are located on chromosomes3and7. The analysis of gene duplication events showed that there were13OsXYLPs (61.9%)expanded via gene duplication, including tandemly and segmental duplications, this means that gene duplicationhave important roles in expandedprocesses of OsXYLP gene family. By using the data of microarrays, ESTs (expressed sequence tags), MPSS (massively parallel signature sequencing) and the results of qRT-PCR (quantitive real-time reverse transcription polymerase chain reaction), we performed studies on the expression patterns of OsXYLP genes and their responses under treatments of abiotic stresses and hormones. The analysis indicated that most of OsXYLP genes are highly expressed in tissues with vascular system (such as roots, leaves, stems and panicles), however, most duplicated OsXYLP genes showed different expression patterns, hinting that the duplication events might have given rise to genetic functional diversity in the evolution procedure. The results showed that four genes were regulated under the treatments of abiotic stresses, especially drought and salt stresses,moreover, a majority of expression levels of OsXYLP genes were up-regulated by NAA and GA hormones. Two T-DNA inserted knockout mutants of OsXYLP7and OsXYLP16were identified; and mutant xylp7has a defect in stem height.2. OsAGP13is a Lys-rich arabinogalactan-protein. We analysis the expression pattern of OsAGP13gene at the RNA level by using in situ hybridization technique, the results showed OsAGP13are highly expressed in vascular bundles of young stems, primary and second branch primordia of panicle. And we found that OsAGP13-RNAi plants showed unusual dwarf, less first and second branches of panicle, significantly reduced mechanical intensity, and early flowering. We observed and compared the structures of panicle branches from OsAGP13-RNAi and wild-type (WT) plants, and found that OsAGP13-RNAi plants have thinner cell wall of sclerenchymal cell, decreased large and small vascular bundle number, and some abnormal vascular bundles. The results of mechanical properties tests showed the breaking forces of the stems and leaves are weaker in OsAGP13-RNAi plants than in wild-type. The cell wall composition assay indicated that the contents of cell wall composition in OsAGP13-RNAi plants are decreased remarkably compared to WT. The lignin staining tests displayed that the content of lignin was lower in OsAGP13-RNAi plants than in WT, especially in the cell wall of sclerenchymal and xylem cells. The results of CW staining showed the content of cellulose was decreased significantly compared to WT. Immunochemical experiment by using rat monoclonal antibodies (LM10, LM11) showed that the content of xylan was lower in OsAGP13-RNAi plants than in WT plants.3. We explored the transcriptomic changes of OsAGP13-RNAi plants by comparing to WT using results of high-throughput RNA-Seq, and found that there were1656differentially expressed genes, including1023up-regulated genes and633down-regulated genes. At the same time, we performed the analysis of annotation and functional enrichment of Gene Ontology (GO) function. According to the results of GO function enrichment analysis and previous studies, we found that many differentially expressed genes involved in the development of cell wall and vascular bundles. And these genes were divide into three categories:the first category contains genes involved in the synthesis of cell wall materials; the second category contains genes involved in hormone response processes, including three kinds of hormones, auxin, gibberellin and brassinosteroid, the three hormones play important roles in the development of cell wall and vascular bundles; the third category contains transcription factor genes, mostly belong to MYB transcription factor genes family, and many members in this gene family were proved to regulate the expression levels of genes in lignin precursor synthesis pathway. We choose some genes from these differentially expressed genes to validate their expression levels by using qRT-PCR. The results showed that theywere broadly consistent to the RNA-Seq consequence, indicating that the down-regulation of OsAGP13indeed affect the expression of genes involved vascular bundle and cell wall development.In summary, the genome-wide identification and expression analysis of rice XYLP gene family provide a solid foundation for the future research on the biological functions of rice XYLP gene family, and this study contribute19new genes encoded arabinogalactan proteins to AGP gene family in rice, make the number of AGP genes increased from98to117. And, in this study, we performed detailed researches on the biological functions of OsAGP13during the development of cell wall and vascular bundles, which will help us to understand the roles of AGPs in higher plant development.