| Reversible protein phosphorylation is an important mechanism of signal transduction that is mediated by protein kinases and protein phosphatase s which is adjusting various physiological mechanisms.It transfers signals through dephosphorylating or phosphorylating its substrates.At present,many kinase genes had been annotated and investigated to response to adverse stresses as a positive regulation factors,but the family of protein phosphatases haven't well investigated in contrast to kinase.PP2C-type protein phosphatases is a kind of serine/threonine protein phosphatases which regulating many important physiological events including mediating abiotic and biotic stresses triggered signaling pathways in yeast,plant and animals.Meanwhile,it is associating with the process for plant growth,regulating cell cycle,signal transduction and responsing environmental stresses.However,researches of PP2 C are mainly focused on well-characterized group A and B which playing an important role in ABA signal transduction pathways or several stresses signaling pathways,but the genes expression patterns and functions of other distinct groups have been poorly understood.It is worth mentioning that cotton is one of the important economic crops in China,which plays an important role in cotton economy through the world.But in practical production,adverse environment can cause serious influence on cotton.Therefore,reseaching the mechanism of stress tolerance by molecular biology could provide theoretical basis for breeding work.The functional studies of PP2 C in cotton can help cotton adapt to adversity stresses and obtained bring more economic benefit.In this study,we cloned the isolation of a cotton gene GhPP2C60 from G.hirsutum and demonstrated its expression patterns and potential functions.(1)The positive clone(A complete open reading frame)was 1086 bp which encoded 362 amino acid residues with the relative molecular mass and an isoelectric point of were 39.8KDa and 4.68,respectively.Sequence comparison of GhPP2C60 with that in other species showed that the gene sequence is highly conserved and also labelled that the predicted protein consisted of 11 conserved motifs.The phylogenetic analysis showed that GhPP2C60 has been defined to fall under the group I PP2 C family.(2)Using Agrobacterium-mediated transient transformation technology revealed that GhPP2C60 is cytoplasm localized.(3)qRT-PCR and Western blotting revealed that the GhPP2C60 expression could be induced by heat,cold and Pst DC3000,or inhibited under ABA and MeJA treatments which illustrated GhPP2C60 is involved in many signaling pathways and playing important role in defense response.(4)We obtained GhPP2C60-overexpressing plants using Agrobacterium-mediated transformation method and test its function.GhPP2C60-overexpression enhanced sensitivity to ABA in comparison to wild-type plants in seed germination,and influenced stomatal behavior in opening and closing.So we suspected that GhPP2C60 involved in ABA signaling pathway.(5)Transgenic plants overexpression of GhPP2C60 exhibited increased heat and Pst DC3000 stresses sensitivity that accelerated the accumulation of ROS,some genes of antioxidative enzymes were repressed consistently compared to wild-type plants,and the expression of defense-related genes were changed with or without treatments.Next,we explored functions in heat and Pst DC3000 treatments using GhPP2C60-silencing cotton which were obtained by virus-induced gene silencing technique and then tested that silencing GhPP2C60 enhanced heat and Pst DC3000 tolerance in cotton,which revealed that GhPP2C60 is a negative regulator in fighting with heat and Pst DC3000 stresses.(6)Genes related to JA/ABA are tested before and after pathogen and heat treatments,there are significant differences in the expression of JA-related and ABA-related genes after treatments which preliminary conclude that GhPP2C60 involve in ABA and JA signaling pathways thus performing functions in against biotic and abiotic stresses. |
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