Functional Analysis Of GhEIN3 And GhMYC2 In Cotton (Gossypium Hirsutum) Fiber Development

Cotton(Gossypium hirsutum),an important textile material,plays vital roles in economic development in the world.As the development of economy is getting faster and faster,the supply of cotton can't meet our needs.How to improve cotton production and quality has become a hot issue in cotton biology research.Cotton transgenic technology has provided favorable conditions for people to get cotton with high production and quality.But the molecular mechanism of cotton fiber development is still elusive and needs to be further studied.The growth and development of plants and plants hormones are inseparable.Ethylene and jasmonic acid can regulate different physiological processes including seed germination,pathogens defense,blossoming,fruit,senescence,biotic and abiotic stress response.It has been reported that ethylene and jasmonic acid have important roles in the process of cotton fiber development,but the mechanism is not very clear.EIN3 and MYC2 are two key regulators of ethylene and jasmonic acid signaling pathway respectively.To study their function in cotton fiber development,especially in initiating period help to elucidate the regulating mechanism of ethylene and jasmonic acid in the development of cotton fiber.In this study,two cotton genes which are homologous of Arabidopsis EIN3 and MYC2 are cloned and designated as GhEIN3 and GhMYC2.We mainly focused on the expression pattern of them in various tissues and different developmental stages of cotton fiber,subcelluar localization and transactivation activity,analysis of their function in overexpression transgenic Arabidopsis and transgenic cotton.The main results are as follows:1?The isolation of GhEIN3 and GhMYC2 and phylogenetic analysisGhEIN3 has an open reading frame(ORF)of 1842bp in length,encoding a protein of 613 amino acids.The molecular weight is 69.3KD and isoelectric point is 5.53.While GhMYC2 has an ORF of 2028bp in length,encoding a protein of 675 amino acids.The molecular weight is 73.6KD and isoelectric point is 5.44.Phylogenetic analysis showed that the revolution relationship of GhEIN3 and GhMYC2 were far from the homologous protein of Arabidopsis EIN3 or MYC2 but close to the homologous protein of cocoa respectively.2.Analysis of the expression of GhEIN3 and GhMYC2 gene in various tissues and different developmental stages of cotton fiberWe analysis the expression pattern of GhEIN3 and GhMYC2 through qRT-PCR,the results revealed that the expression of GhEIN3 could be detected in varied cotton tissues with different levels.It got a high expression in stem and it also expressed in ovule and fiber.The expression of GhEIN3 reached the top in 3d ovules after blossoming,suggesting that it may play a role in the early development of cotton fiber.GhMYC2,compared to GhEIN3,was expressed much lower in stem,nevertheless it had a higher expression in root and hypocotyl,further the expression of GhMYC2 in 1d ovule before blossoming,Od ovule and 6DPA fiber was also much higher.3.The subcelluar localization of GhEIN3 and GhMYC2To study the subcelluar localization of GhEIN3 and GhMYC2,we constructed the GhEIN3 and GhMYC2 with GFP fusion expression vector driven by CaMV 35S promoter.The vector then was transformed into agrobacterium.With the method of injecting agrobacterium which harbored the vector into tobacco leaf epidermal cells and observing the GFP green fluorescence under confocal microscope,we found that GhEIN3 and GhMYC2 were localized in the nucleus.4.GhEIN3 and GhMYC2 transactivation activity analysisThe yeast system experiments showed that GhEIN3 and GhMYC2 had transactivation activity.Further we found that 263-477 amino acid in the acidic region of GhMYC2 played an important role on its transactivation activity.5.Effects of hormones on the expression of GhEIN3 and GhMYC2The results suggested that expression of GhEIN3 could be rapidly induced by ACC,and reached maximum in 10h.Expression of GhMYC1 increased along with the extension of treatment time,but the rising amplitude was small.It indicated that GhEIN3 and GhMYC2 may be involved in ethylene and jasmonic acid signaling.6.Phenotypic analysis of overexpression GhEIN3 in transgenic ArabidopsisWe constructed GhEIN3 overexpression vector,then transformed the Arabidopsis and obtained the transgenic Arabidopsis.In dark condition,when GhEIN3 overexpression Arabidopsis was treated with ACC,the results showed that the hypocotyl and root length of GhEIN3 overexpression transgenic Arabidopsis was significantly shorter than that of wild type and ein3eill mutant.It indicated that GhEIN3 could response to ethylene.When treated with ABA,H2O2,NaCl,the germination rate of the seeds of transgenic plants was lower than that of the wild type.The root length of GhEIN3 overexpression Arabidopsis was much shorter than that of the wild type after ABA and NaCl treatment.At the same time,the chlorophyll content and relative fresh weight were less than the wild type.Continuing watering adult Arabidopsis with NaCl led to higher survival rate of wild type than the transgenic plants.To figure out why the transgenic Arabidopsis plants was sensitive to salt,we examined the content of H2O2 in Arabidopsis after treatment with NaCl by DAB staining.The staining results showed that when treated with 100mM and 150mM NaCl,the transgenic plants got deeper staining than the wild type indicating that the higher accumulated levels of H2O2 in GhEIN3 overexpression Arabidopsis than the wild type.7.Phenotypic analysis of GhEIN3 overexpression and RNAi transgenic cottonTo explore the function of GhEIN3 in cotton,we constructed GhEIN3 over expression and RNA interference vector.Then the vector was transformed into agrobacterium stain LBA4404,followed by the transformation of genetic cotton using the agrobacterium mediated DNA transfer.A Large number of transgenic cotton was obtained.The T0 generation transgenic cotton was used for preliminary analysis.The results showed that the long staple fiber of GhEIN3 overexpression transgenic cotton was much shorter than that of the wild type,but the short staple fiber and seeds did not change significantly.And the GhEIN3 RNAi transgenic cotton got thinner short staple fiber and bigger seeds but the long staple fiber remained the same as the wild type.8.Preliminary analysis of GhMYC2 transgenic ArabidopsisThe root elongation of GhMYC2 overexpression Arabidopsis was significantly repressed when it was cultivated on MS supplemented with 5?M MeJA indicating that GhMYC2 could response to JA signaling.Further studies are in progress currently.