عنوان مقاله [English]
Introduction: Dwarfism is one of the important traits in breeding of crops and horticulture plants. A dwarfing rootstock will produce trees with 15-50% of standard trees size. In modern intensive fruit tree orchards, dwarfing rootstocks are commonly used to reduce trees size, enabling high-density planting and easy management, thus achieving higher yield. Trees on dwarfing rootstocks can also exhibit other economically important traits, such as precocious flowering, increased yield and increased disease resistance. Dwarf rootstocks have been extensively studied and released in stone and pome fruits, because of presence of genetic materials and the simplicity of budding methods. Control of tree size using genetically dwarf rootstocks for achievement to higher density and mechanized orchard systems is now very important for walnut production in the world especially in Iran. Many different genes can be involved in appear of this. Mutations in GAI and PIP2 genes cause dwarf trait by two different mechanisms in some plant species. In this case, we study in silico analysis of GAI and PIP2 genes consist of conserved sequences and domains, exon and intron number, function of their proteins, targeting, secondary and tertiary structure, and post translational modification.
Materials and methods: The GAI and PIP2 mRNA and protein sequences (FASTA format) belonging to 17 monocotyledon and dicotyledon were downloaded from NCBI (http://www.ncbi.nlm.nih.gov) accessed, on September 2014. Several online web services and software were used for analysis of GAI and PIP2 mRNA and Proteins in plants. Comparative and bioinformatics analyses of PIP2 and GAI proteins were performed online at two websites NCBI (http://www.ncbi.nih.gov) and EXPASY (http://expasy.org/tools). Molecular Evolutionary Genetics Analysis (MEGA; version 4) program and CLUSTAL-W with default parameters were used for multiple alignments of sequences. The phylogenetic analysis of GAI and PIP2 protein was done with MEGA from aligned sequences. The motifs of protein sequences were found using the program of T-COFEE at website (http://www.ebi.ac.uk/Tools/msa/tcoffee/). The Neighbor-Joining (NJ) method was used to designing the phylogenetic tree. The predicted exons and introns in mRNA sequences were done by http://genes.mit.edu/GENSCAN.html website. The secondary structure of proteins was predicted by PSIORED online on http://bioinf.cs.ucl.ac.uk/psipred/. Prediction of 3D model of protein was performed using the 3D alignment of protein structure by BLASTp and PDB database as source. Also, targeting prediction of proteins was done online by TargetP at (http://www.cbs.dtu.dk/services/TargetP/) website.
Results and discussion: In phylogenetic investigation among 17 different species, Walnut species evolutionary stand in dicotyledonous and woody plants by both of GAI and PIP2 genes and protein sequence clustering. By multiple alignments and investigation in conserved sequence of these genes in plant revealed that despite differences in cDNA length, there were very similarities in conserved region, secondary and tertiary structure. Protein analysis in the GAI gene family showed that the following domains including DELLA, TVHYNP, VHIID, RKVATYFGEALARR, AVNSVFELH, RVER, and SAW were conserved in this proteins. In secondary structure of protein, β-sheets and α-helixes specified by PSIPRED software for both of GAI and PIP2 proteins. GAI protein had 9 β-sheets and 15 α-helixes in its structure, also PIP2 protein had2 β-sheet (at 180-188 and 248-253) and 8 α-helixes. In comparison of 3D structure, walnut PIP2 protein was very similar to chain A of PIP2 protein of spinach (Spinacia oleracea) and GAI protein of walnut was similar to B-subunit of Arabidopsis GAI protein with 48% similarity. The length of GAI protein was varied from 636 aa in Malus baccata var. xiaojinensis to 336 aa in Physcomitrella patens among species. In walnut, the length of GAI and PIP2 protein was 613 aa and 287 aa, respectively. PIP2 protein length was similar in different species among 257 aa in Triticum aestivum to 290 aa in Zea mays. By exon-intron and targeting analysis of sequence, it was found that GAI gene target was in nuclear and had just one exon without intron, and PIP2 gene in walnut had 4 exons and 3 introns with cell membrane targeting. In results, Tcoffee analysis revealed that PIP2 gene was very conserved across the evolution between plant species in compared with GAI gene.
Conclusion: Our results provide new insights into the evolutionary relationships of GAI and PIP2 proteins. The results of the sequence alignment showed that GAI and PIP2 in walnut and other species have high homology with each other. After this analysis, we can have a good perspective about molecular situation of walnut GAI and PIP2 genes. Result of this study can be used for make relationship between growth, flowering, and water uptake characteristics of these plants and their protein sequences. Also this research gives good information for us if we want to clone these genes from Iranian genotypes.