Sequence Analysis And Function Exploration Of Tubulin And Spr1 Gene Family In Two Willows

Microtubules(MT) are hollow cylinders made up of α/β tubulin heterodimers by noncovalent bonding. They regulate their dynamics and arrays by microtubule-associated proteins(MAPs) to maintain cell shape and structure, particpate in cytoplasmic striaming, regulate mitosis, control cell polarity growth, cell wall establishment, cell differentiation and signal transduction in the physiological activities of the plant. Therefore tubulin and MAPs gene families have been widespread attention for a long time. In this study, S. matsudana and S. matsudana var. ‘Tortusoa’ were used for experimental material. The gene structure, phylogeny, and expression pattern of α and β tubulin gene families, and the expression pattern and potential function of SPR1 gene family were studied preliminary. The main results are as follows:(1) The α and β-tubulin genes from the two willows were cloned and identified. Sequence similarity, phylogeny, chromosomal localization and expression patterns were further analyzed. The results showed that each willow contained eight α and 20 β-tubulin genes.(2) Each α-tubulin gene shared more than 78.3% and 88.4% nucleotide and amino acid sequence similarity with one another in each willow species. And there was over 88.2% amino acid sequence similarity of the α-tubulin between the two willows. While the amino acid sequence similarity among Salix and other plants were larger than 85.1%. We also found that half of the eight Salix α-tubulin proteins contained a C-terminal methionine, leucine, glutamic acid or glutamine, instead of the more typical C-terminal tyrosine. In the phylogenetic analysis, α-tubulin proteins formed two distinct classes, which suggested these genes might originate from different ancestors. The C-terminal Y-type TUA1 genes were highly expressed in basal, middle, elongation and apical region of one year old branches of willows, therefore they were predicted to involve in the process of xylem development.(3) Each β-tubulin gene shared more than 74% and 86.6% nucleotide and amino acid sequence similarity with one another in each willow species. There was over 85.8% amino acid sequence similarity of the β-tubulin between the two willows. While the amino acid sequence similarity among Salix and other plants were larger than 81.5%. In the phylogenetic analysis, β-tubulin proteins formed four classes, and it was presumed that Salix β-tubulin gene families had undergone salicoid duplication and tandem duplication events combined with the study of chromosomal location of poplar β-tubulin. However, TUB11 and TUB12 were possibly derived from segmental duplication or replicative transposition. The expression pattern analysis showed a tissue specificity of β-tubulin genes in willows, and a section of duplicated-gene pairs showed different expression pattern in tested tissues. High degree of sequence similarity, evolutionary expansionand of members and expression pattern diversity of Salix α and β-tubulin gene families might confer flexibility in cell division and growth which is of important significance to the development and growth habit of perennial woody plants.(4) In addition, six SPR1 genes were also cloned from each of the two willows. Sequece similarity analysis showed that orthology genes of SPR1 from the two willows were consistent. While six homologs share sequence similarity only in the N- and C-terminal regions, whereas the internal sequence is highly variable. Phylogenetic analysis revealed that plant SPR1 genes were divided into two classes in which SmSPR1 genes distributed in both of them. In the six genes, only SP1L1 and SP1L2 expressed and the former was the main expressed gene. It is highly expressed in elongation and middle region of one year old branches of the willows, while expressed lower in basal and apex region. GUS histochemical assay of transgenetic tobaccos with PSPR1+GUS discovered that GUS gene can be drived to express in root elongation region, shoot xylem and cambium. Overexpression of SmSP1L1、SmSP1L4 and SmSP1L5 made the cotyledons of arabidopsis seedlings showing counterclockwise twist growth, and lead to short siliques and few seeds. Immunofluorescence labeled microtubules in hypocotyl cells of the etiolated seedlings of Arabidopsis revealed that the orientation of the microtubule array were altered in different degree. These results suggested that SPR1 genes from willows primarily expressed in rapid elongated cells, and may have the function that regulated the orientation of the microtubule array.