Effect Of Auxin Response Factor GhARF6 On Growth And Development Of Gossypium Hirsutum

Cotton is an important material in the people's livelihood,and plays an important role in national economy.Auxin plays an important role in plant growth and physiological reactions,such as regulates cell elongation,division and differentiation,adventitious roots,fruit development and embryo formation.Auxin affects the growth and development of plants through regulating functional genes by it's signaling pathways.Therefore,the study of auxin signaling pathway and it's regulation mechanism of downstream gene is a research focus of plant biology in recent years.It is of great theoretical significance to study the biological function of auxin signaling transduction gene in cotton understand the role of auxin in cotton growth and morphogenesis.It will contribute to elucidate the complex molecular mechanism of auxin signaling in growth and development of cotton.ARFs(Auxin response factors)is the key factor in auxin signal transduction pathway,which can either activate or repress the expression of the primary auxin responsive genes.The discovery of ARFs family has led to a breakthrough in the understanding of auxin signaling pathway.As auxin response factors,ARF6 is associated the growth of plant apical meristem,the development of vascular tissues,the removal of dormancy and the development of stamen and gynoecium.In order to furtherly analyze the function of ARF6 in the development of Gossypium hirsutum,coding region and upstream of GhARF6 were firstly cloned in this study,the transcriptional level of GhARF6 in cotton tissus was examined and the expressional pattern of GhARF6 promoter in Arabidopsis and Gossypium hirsutum were analyzed.The effect of GhARF6 on the growth and development of cotton was performed by regulating the expression level of GhARF6 in Gossypium hirsutum through antisense,RNAi,and overexpression.The main results are as follows:1.Cloning and characterization analysis of GhARF6First,GhARF6,a homologous gene of ARF6,was cloned from Gossypium hirsutum,located on chromosome 5.Sequencing analysis indicated that GhARF6 contained a 2715 bp ORF.Putative protein encoded by the ORF contained 904 amino acid residues,the weight of molecular was 99.78 kDa,the isoelectric point was 6.33.The phylogenetic tree analysis of the amino acid sequences of 96 ARF in Gossypium hirsutum genome cultivars and 10 genes classified into five ARF subfamily in Arabidopsis thaliana genome were carried out.The result revealed that it contained 8 ARF subfamilies in Gossypium hirsutum genome,and GhARF6 shared highly indentity with Gossypium hirsutum D genome GhARF6-1(GhD05G3848)and A genome GhARF6-2(GhA05G1225).GhARF6-1,GhARF6-2,AtARF6 and AtARF8 belonged to same ARF subfamily.As transcription factors,amino acid sequence structure analysis showed that GhARF6 had typical ARF protein structure with a conserved N-terminal DNA-binding domain(DBD),a non-conserved middle region(MR),and a conserved C-terminal dimerization domain(CTD).To better understand the tissue-specific expression profiles of GhARF6 in Gossypium hirsutum,Real time quantitative PCR was employed to detect GhARF6 expression level in different tissues.The results showed that GhARF6 was expressed in all parts of cotton plants,and the expression level of GhARF6 was significantiy highest in stem and lowest in pistil and stamen among detected tissues.The downward trend of GhARF6 expression level in fibers and ovules from 0 DPA to 28 DPA was found.In fiber,the GhARF6 expression level from 0 DPA to 4 DPA was higher than other growth stage,and it maintained highly expression level in ovule from 0 DPA to 10 DPA,very low expression level at 26-28 DPA.These results suggested that GhARF6 played an important role at initial and elongation stage in the cotton ovule development process.To determine whether GhARF6 gene expression was also induced by auxin,the expression level in the wild type with and without auxin treatment was analyzed.Real time PCR results indicated that GhARF6 transcript in cotyledons and hypocotyles was induced,while not induced in roots by IAA treatment.The dose and time course experiment was also performed to determine the response of GhARF6 expression to aluminum treatment in roots following exposure to a range of concentrations of AlCl3(pH 4.2)or at different times to 50 ?M AlCl3.Wild type cotton seedlings were grown hydroponically for 12 hours after which they were exposed to either no Al or increasing levels of Al up to 200 ?M at pH 4.2.Our analysis revealed that GhARF6 was rapidly induced by Al,with the greatest expression found following treatment with 50 ?M Al3+ for 3 hours.2.Promoter of GhARF6 cloning and expression patternIn order to further understand the function of GhARF6,the promoter sequence(PGhARF6)of the 2100 bp was cloned.Bioinformation analysis of the promoter sequence revealed the presence of numerous plant cis-acting elements,including auxin response elements,light-responsive elements,tissue-related elements,salicylic acid response elements,defense and stress response elements,and specific parts to express related components.To directly investigate GhARF6 expression pattern in plants,the 2.1 kb upstream sequences of GhARF6 was fussed with the GUS reporter gene and introduced into wild type Gossypium hirsutum and Arabidopsis plants.Transgenic lines that were determined by kanamycin resistance and PCR amplification,were subsequently analyzed for pattern of GhARF6::GUS accumulation at different stages of plant growth and development.Histochemical analysis of all transgenic plants showed that the expression pattern of GhARF6 promoter driving GUS gene was same between in Gossypium hirsutum and Arabidopsis.GhARF6 was primarily expressed in all organs examined including roots,cotyledons,leaves,stems,and flowers.GUS activity was stronger at the roots and stems than other organs.GhARF6::GUS fusion protein mainly accumulated to the vascular of stems and roots detected by cross section.GUS analysis was also performed for roots that were either not treated or treated with 50?M AlCl3(pH 4.2)for 9 hours.As with Real time-quantitive PCR analysis for Al-inducibility,GUS analysis following Al treatment showed that Al exposure increased GhARF6 expression in roots.3.GhARF6 gene affected cotton growth and developmentIn order to further explore the biological function of GhARF6 in Gossypium hirsutum,we constructed overexpression(sense GhARF6)and suppression including antisense(antisense Gh ARF6)and RNA interference(GhARF6 RNAi)vectors under control of a cauliflower mosaic virus(CaMV)35S promoter and introduced them into Gossypium hirsutum genomic mediated by Agrobacterium.Forty plants of five independent transformants of antisense GhARF6,sixty plants of seven transformants of GhARF6 RNAi,and ten plants of two transformants of sense GhARF6,respectively,were eventually isolated on antibiotic plates and tested for the segregation of T0 seeds by GUS staining.Comparison to wild type cotton plants,small bracts,shorter petal and stamen filaments were found in antisense GhARF6 and GhARF6 RNAi transgenic plants.The arrested flower buds,undehisced anthers that didn't release pollen and immature gynoecia were also found in antisense GhARF6 and GhARF6 RNAi transgenic plants.At the same time,the phenomenon of high fruit shedding was observated in antisense GhARF6 and GhARF6 RNAi transgenic plants.The shedding of fruit was more serious of GhARF6 RNAi transgenic plants than antisense GhARF6,some plants even to 100%.However,sense GhARF6 transgenic cotton plants showed no significant differences in the stamen and gynoecium traits as compared with wild type cotton plants.Small cotton seeds and decreased seed number per boll were obviously found in GhARF6 suppression transgenic plants including antisense and RNA interference.Compared with the wild type,seed number per boll of the antisense GhARF6 lines 3-4-3,41-1-1 were reduced by 42%,33.5%,GhARF6 RNAi lines 2-2,2-4 were reduced 39.6%,29.7%.The length of mature fibers in transgenic cotton plants were measured.We found that fibers length of GhARF6 suppressor lines was shorter than wild type and sense GhARF6 lines,more significant difference was found in RNAi transgenic plants.Compared with the wild type,the antisense transgenic lines 3-4-3,41-1-1 and RNAi transgenic lines 2-2,2-4 were respectively decreased by 12%,14.5% and 15.4%,19.3%.However,it was not significant difference on seed number per boll and fibers length between the GhARF6 sense transgenic lines and wild type.These results indicate that the GhARF6 gene plays an important role in floral organ and ovule development of Gossypium hirustum in auxin signaling pathway,and more detail research such as downstream target regulated by GhARF6 to be performed.