Isolation And Characterization Of PYL Genes In Cotton(Gossypium Hirsutum)

Cotton is a very important crop species and it is planted on large scales in many countries, including China. Cotton fiber is natural textile fiber and it’s very important for textile industry. However, adverse environment conditions, such as drought, drowning, cold and high salinity, do much harm to the cotton plant growth and cause the reduction in cotton fiber production. How to improve the resistance of cotton to these adverse conditions is one of the major problems which researchers have been focused on. It seems to be a very promising strategy to solve this problem by introducing new stress-tolerant genes into the cotton genome through the transgene technology.Abscisic acid (ABA) is a phytohormone and plays vital roles in response to abiotic stresses. The ABA receptor PYL proteins, together with clade A PP2Cs, kinase SnRK2s and the downstream responding factors, constitute the core ABA signaling pathway. Functional studies of these genes in cotton may help revealing the ABA signaling in cotton and the screen of stress-tolerant genes of cotton.In this study, we isolated 27 PYL genes from cotton genome and performed sequence and phylogenetic analysis, tissue expression and stress induced expression analysis and studied the interactions between PYL proteins and PP2C proteins and the phenotypes of transgenic Arabidopsis. The main results are as follows:1. Isolation and phylogenetic analysis of cotton PYL genesAccording to the sequence similarities with Arabidopsis PYL proteins, we isolated 27 candidate PYL genes from cotton genome, designating them as GhPYL1-27. These genes encode about 200 amino acids with an ORF of about 600 base pairs and they are highly conserved on the crucial secondary structures. According to the homology with Arabidopsis PYL proteins, GhPYLs can be categorized into three subgroups.2. Tissue expression and stress induced expression analysis of cotton PYL genesThe tissue expression analysis by semi-quantitative RT-PCR revealed that GhPYLs had different tissue expression patterns. For instance, GhPYLl had a relative higher expression in root, hypocotyl and cotyledon. GhPYL8 was strongly expressed in root and there were no significant differences in the expression of GhPYL26 among 8 kinds of tissue. The qRT-PCR assay of 5 GhPYLs after drought treatment showed expression of 5 tested GhPYLs were down-regulated after drought in the cotton cultivar Jinmian 13.3. The interactions between cotton PYL proteins and PP2C proteins in yeastYeast-2-hybrid assay showed that GhPYL8, GhPYL10, GhPYL12, GhPYL22, GhPYL25, GhPYL26 can selectively interact with GhPP2C proteins in yeast.4. Subcellular localization of GhPYL1We constructed 35S GFP:GhPYLl expression vector and transformed Arabidopsis through agrobacterium. Confocal images of root cells of transgenic seedlings showed localization of fluorescent signal was in cytosol and nucleus, indicating that GhPYLl is localized in cytosol and nucleus.5. Sensitivity of transgenic Arabidopsis to ABAWe constructed overexpressing vectors of GhPYL10, GhPYL12, GhPYL26 and transformed wild-type Arabidopsis. Statistical analysis of germination rate and green cotyledons percentage of WT and transgenic lines on MS plates containing ABA showed transgenic Arabidopsis overexpressing GhPYL10, GhPYL12 or GhPYL26 were more sensitive to the ABA-mediated inhibition of seed germination and early seedling growth.6. Sensitivity of transgenic Arabidopsis to saltStatistical analysis of germination rate and green cotyledons percentage of WT and transgenic lines on MS plates containing low concentrations of NaCl showed that transgenic Arabidopsis overexpressing GhPYL10 or GhPYL12 had slower germination rate and less green cotyledons than WT and that transgenic Arabidopsis overexpressing GhPYL26 germinated more slowly than WT.7. Sensitivity of transgenic Arabidopsis to osmotic stressStatistical analysis of germination rate and green cotyledons percentage of WT and transgenic lines overexpressing GhPYL10, GhPYL12 or GhPYL26 on MS plates containing mannitol showed that there were no significant differences between WT and transgenic lines.8. Root length analysis of transgenic Arabidopsis overexpressing GhPYLStatistical analysis of primary root length of 10-day-old WT and transgenic Arabidopsis on MS plates showed that primary root length of transgenic Arabidopsis overexpression GhPYL12 or GhPYL26 are much longer than WT. This result suggests that overexpression of GhPYL12 or GHPYL26 promotes the primary root growth of Arabidopsis during seedling stage.