| Fasciclin-like arabinogalactan proteins(FLAs)are a subclass of arabinogalactan proteins(AGPs).Located at the cell surface,in the cell wall/plasma membrane,they are implicated in many developmental roles yet their function remains largely undefined.Fasciclin(FAS)domains are putative cell-adhesion domains found in extracellular matrix proteins of organisms from all kingdoms,but the juxtaposition of FAS domains with highly glycosylated AGP domains is unique to plants.Previous studies shown that FLAs play important roles in many developmental process.In this research,bioinformational analysis,semi quantitative PCR and CRISPR/Cas9 methods were used to identify FLAs gene in Populus,and to investigate the function of PtrFLA31 and PtrFLA34.Main results are as follows:According to amid acid sequence of AtFLAs,cooperated with keywords such as ’fasciclin’and ’fasciclin-like’,we have identified 46 Populus FLAs,the length of which ranged from 210aa to 446aa.For the 46 PtrFLAs,39 of which contained N-terminal signal peptide,38 genes contained 1 FAS domain,while 8 genes contained 2 FAS domains.27 C-terminal GPI anchor proteins were predicted using big-PI Predictor software.Multiple sequence alignment of fasciclin domains from Populus was conducted using Clustal X2.0 software,which contained highly conserved regions,H1 and H2(with the length of about 10 amino acids for each).[Tyr/Phe]-His([Y/F]H)motif was located between them according to Pfam database.Compared with any other species,Pro residue was absolutely or extremely conserved in these three regions.We conducted protein-based phylogenetic analysis and pair-wise sequence comparison of 21 AtFLAs and 46 PtrFLAs using MEGA6.Previous studies indicated that the 21 AtFLAs can be divided into four groups,named A-D.Here,we found that the 46 PtrFLAs can also be classified into four clades.Compared with Arabidopsis thaliana,the number of A group increased sharply,which implicated that genes belonged to A group play important roles in secondary growth.All of proteins belonged to A class contains a single fasciclin domain that was flanked by two AGP-like glycosylated regions.The semi quantitative PCR showed that all of the A group of PtrFLAs expressed highly in xylem.As for the expression in stem,they expressed tiny in the first two stems,and increased gradually from the third innode to the tenth.Except PtrFLA3 and PtrFLA20,which expressed highly in all tissues,all the PtrFLAs are specificately expressed in xylem.Both PtrFLA31and PtrFLA 34 contains a single fasciclin domain that was flanked by two AGP-like glycosylated regions,phylogenetic analysis showed that they belong to a same clade.PtrFLA31/34 double genes were knocked out via Cas9/gRNA,Phenotypic observation exhibit that compared to wild type,the mutant plants grow faster and the leaves are more narrow and longer. |
