| Expansin is an essential component of plant cell wall, which has a wall-loosening function and plays important roles in plant growth and various developmental processes along with the resistance activities. The previous study in our lab identified a grass expansin gene called AsEXP1 from a heat tolerant rough bentgrass (Agrostis scabrd) cultivar’NTAS’, which could be induced by high temperature and expressed much stronger in heat tolerant grass genotypes than in heat sensitive grass genotypes. This study analyzed the DNA sequences of AsEXPl homologous genes in turfgrasses through gene cloning and bioinformatic methods. The heat tolerance related function of PpEXPl gene homologous to AsEXPl cloned from Poa pratensis cultivar’Diva’were identified by over-expression of PpEXPl in tobacco(Nicotiana tabacum) plants through Agrobacterium tumefaciens mediated transformation approach. Furthermore, we investigated the function of different expansin genes in turfgrass through analysis of expression levels of expansin genes in two tall fescue (Festuca arundinacea) genotypes (fast growing genotype’K-31’and dwarf genotype’Bonsai’) in different hormonal and abiotic treatments. The main results were as follow:(1) The cloning and alignment of DNA sequences of AsEXPl homologous genes among 12 turfgrass genotypes from 3 genera showed that AsEXPl homologous genes from the same genus shared several specific mutations in the gene length and sequence which were different from other genera, and the diversity degree of intron regions was much higher than exon regions. Based on the variation of different homologous genes, three PCR primers were designed to identify the different grass genera, and determined in 32 grass genotypes from 6 genera. The results indicated that the primer pair cAs3F/cAs3R and cAs4F/cAs4R were able to identify the frequently-used bentgrass (Agrostis) genotypes and bluegrass(Poa) genotypes, respectively and cAs5F/cAs5R was only amplified in fescues (Festuca) and ryegrasses (Lolium). The phylogenetic tree was drawn based on the deduced amino acid sequences of 12 AsEXP1 homologous genes, in which the cultivars in the same genus were collected in the same branch, while the cultivars belonging to different genera were in different branches. The results implied expansin genes contained clues about evolution pathway of plants and could supply the important gene data to the grass phylogenetic relationship studies.(2) The wild type tobaccos and transgenic tobaccos which over-expressed PpEXPl were treated with high temperature (42℃) for 6 d at the same time. The results showed that wild type tobaccos were severely wilted and the most of leaves turned yellow, while the transgenic tobaccos performed well with a few leaves on the base of plants wilted. The physiological parameters of leaf including relative water content, relative electrolyte leakage, chlorophyll content, net photosynthetic rate, hydrogen peroxide content, malonaldehyde content, and superoxide dismutase activity indicated that transgenic tobaccos had stronger heat tolerance than wild type tobaccos. The transverse sections of leaf tissues were also examined by microscope. After heat stress, the wild type plants displayed some cellular disruption and disorganization of the mesophyll cells, with large intercellular spaces, while transgenic plants still maintained well-defined mesophyll cells and the transversal structure of the leaf appeared the same as the leave exposed to the optimal growth temperature. The above results indicated that the over expression of PpEXPl had significantly enhanced the heat tolerance of transgenic tobaccos.(3) The expression levels of five expansin genes were analyzed in different tall fescues under different treatments. The results showed that EXPA1 and EXPA3 expressed higher in the fast growing genotype’K-31’than slow growing genotype’Bonsai’and EXPA3 expressed stronger in the fast growing region than non-elongating region along the ’K-31’leaves. Leaf elongation rates were elevated by exogenous GA3 with EXPA1 and EXPA3 upregulated in the leaves of both tall fescues. Drought and salt stresses inhibited the leaf growth of grasses while the expression of EXPA3 was down-regulated by both stresses. The results above indicated that the EXPA3 gene expression was correlated to the leaf elongation rate, which laid the foundation of molecular mechanisms of turfgrass leaf elongation. |
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