Genome-wide Identification And Characterization Of TCP Transcription Factors And Study On The Role Of EIN3 In Upland Cotton(Gossypium Hirsutum)

Cotton(Gossypium hirsutum L.)is one of the important economic crops in China,is the primary fiber crop and important oil crops.Cotton fiber is a branchless epidermis(trichome)or seed hairs that are differentiated from epidermal cells outside the ovules of the ovary.TCP transcription factor,as a plant-specific transcription factor,participates in multiple processes of plant development,for example,TCP transcription factors are involved in the formation of plant meristem and somatic differentiation However,there are few studies on the regulation of fibroblast development in tetraploid cotton.In this study,74 non-redundant cotton TCP genes were isolated and identified.We have studied their expression patterns and interaction patterns.GhTCP22,which was preferentially expressed in fiber elongation,was chosen as a candidator to study its function in cotton.In addition,we also constructed vectors of GhEIN3(an ethylene signal key factor)for RNAi,overexpression and CRISPR-Cas9,and transformed these vectors into cotton,obtained embryos of these vectors Callus and transgenic seedlings.Overexpression of GhEIN3 in Arabidopsis thaliana could enhance the tolerance of Arabidopsis to salt,suggesting that cotton EIN3 may positively control plant responses to salt stress.The results obtained in this paper are as follows:1.Identification and Structural Analysis of Cotton TCP Family Gene and Its Evolutionary RelationshipAccording to the genomic database of tetraploid terrestrial cotton,74 non-redundant GhTCP genes were isolated and identified.We designated the 74 putative TCP genes as GhTCP1-A/D to GhTCP25-A/D.The members of GhTCP family were divided into 11 evolutionary subgroups(A to K),and members on the same evolutionary subunit shared high conserved motif and similar intron and exon distributions.According to the TCP protein classification of Arabidopsis thaliana,GhTCP could be divided into two subfamilies of class ?(TCP-P class)and class ?(TCP-C class),while class ? was further divided into two clusters,CIN cluster and CYC/TB1 cluster.2.The expression pattern of TCP gene family in cottonThe expression pattern of GhTCP family gene was analyzed by quantitative RT-PCR.The results showed that different GhTCP genes display different expression patterns in cotton tissues.Furthermore,some of these TCP genes were predominantly expressed in cotton fiber elongation phase,suggesting that these TCPs may be involved in regulating cotton fiber elongation.At the same time,we selected six GhTCP genes which specific or predominant expression in cotton fiber,and analyzed its expression patterns in cotton cultivar wild type(Xuzhou 142)and its fiberless mutant(fl).It was found that those gene showed significant difference in early ovules of WT and mutant,which suggested that these genes might play a role in cotton fiber initiation.3.Analysis of the interaction between Cotton TCP Protein and fibrous initiation related proteinIn order to explore the functional form of GhTCP in plants,we analyzed the interaction patterns among GhTCP family members by yeast two-hybrid assay.The results showed that most of the family members could form homodimers or heterodimers,especially Class I members.While family ? members could only interact with some of the GhTCP proteins.In order to investigate the role of GhTCP proteins in cotton fiber development,we have examined the interaction pattern between GhTCP 14a/GhTCP22 and some potential fiber initiation and elongation factors GhMYB2,GhMYB23,GhMYB25,GhMYB25-like,GhDEL65,GhSLR1,GhPIF3,GhARF6,GhBZR1,GhEIN3 and GhMYC2.The results showed that TCP14a and TCP22 could interact with GhSLR1,GhARF6,GhGL3,MYB23,GhMYB25 and TTG1.In addition,TCP14a could also interact with GhBZR1,GhEIN3 and GhEIN3.This suggested that GhTCP14a and GhTCP22 may be involved in the regulation network of cotton fiber development.4.Construction of GhEIN3 Expression Vector and Its Cotton TransformationWe identified the cotton EIN3 gene in the early work.The results of qRT-PCR showed that GhEIN3 might play an important role in the early development of cotton fiber.In order to study the function of the GhEIN3 in cotton fiber initiation,we constructed the overexpression vector,RNAi vector and CRISPR-Cas9 vector of GhEIN3,and transferred them into cotton by Agrobacterium tume faciens-mediated transformation.Each vector obtained transgenic cotton embryo genic calluses and some transgenic cotton seedlings,which provid the transgenic cotton material for next work,and the related work is in progress.5.GhEIN3 plays a positive role in transgenic Arabidopsis response to salt stressTranslate GhEIN3 into Arabidopsis thaliana,we obtained some homozygous transgenic GhEIN3 lines in Arabidopsis thaliana.With ACC treatment,GhEIN3 overexpressing plants showed shorter hypocotyls than wild-type and ein3eill double mutant under dark conditions,which indicated that overexpression of GhEIN3 was able to respond to ethylene signal.NaCl could induce the expression of GUS signal in pGhEEIN3::GUS transgenic Arabidopsis thaliana,which indicated that NaCl could enhance the activity of GhEIN3 promoter.Treated by NaCl,overexpression GhEIN3 transgenic plants showed significant higher germination rate and green plantlet rate than that of wild type.The results showed GhEIN3 can enhance the salt tolerance of transgenic Arabidopsis plant.