The Cloning And Functional Characterization Of A Cotton Negative Regulater GhJAZ2 In Jasmonic Acid Pathway

Among fiber crops,the cotton fiber is the most important raw material for the textile industry.The cotton fiber is a single cell that differentiates from the epidermal cells of the ovule.The number of fiber initiation and the length of mature fiber are important part of the yield.A lot of hormones and transcription factors effect cotton fiber development.Thus,it seems to be great potential to improve the fiber yield by regulating the number of epidermal cells that initiate the development process of fiber.The previous reports indicated that GA3,IAA and Eth could promote cotton fiber elongation.But high concentration of JA inhibit fiber initiation and an optimal concentration of JA promote elongation in ovule culture.Therefor we carried the research and obtained the following results.1.Optimal concentration of JA promote cotton fiber initiationIn order to find JA whether effect cotton fiber development,we detected the JA content in cotton ovules from-3 DPA(Days Post Anthesis)to 8 DPA in G.hirsutum YZ1.The JA content was highest in the-1 DPA ovules and sharply declined on the day of flowering.The JA content of the 0 DPA ovules showed great differences between the wild type(Xu142 and TM-1),the fuzzless mutants(GZnn,GZNn and n2)and the lintless-fuzzless mutants(Xin WX and Xu142 fl).The endogenous JA contents were higher in the wild type compared to the mutants,which suggested that a low level of endogenous JA might also be unsuitable for fiber initiation.To determine which JA level was suitable for fiber initiation,-2 DPA ovules were cultured in liquid BT medium with different concentrations of JA.And found that the ovules exhibited increased initiation in medium supplemented with 0.001 μM JA.These results suggested that an optimal JA concentration in vivo was very important for fiber development.2.Sequence analysis and cloning of Gh JAZ2 gene in cottonWe cloned a JAZ gene(Gh JAZ2)based on sequence information(ES802380)from NCBI.Gh JAZ2 was 1056 bp in length with an open reading frame of 792 bp,and the Gh JAZ2 protein contained 263 amino acids.Gh JAZ2 contained ZIM and Jas domains based on an alignment with Arabidopsis JAZ protein sequences.Phylogenetic analysis showed that Gh JAZ2 had homology with At JAZ1 and At JAZ2.Gh JAZ2 showed 33.21% identity with At JAZ1 and 43.28% identity with At JAZ2;therefore,we named this protein Gh JAZ2.Then,the expression pattern of Gh JAZ2 in different tissues was analyzed by q RT-PCR.Gh JAZ2 had high expression levels in the root,hypocotyl,flower and-1 DPA ovule but low expression levels in ovules attached with fibers and fibers at different developmental stages.3.Overexpression of GhJAZ2 resulted in less fuzz and shorter fibersTo test the function of Gh JAZ2,Gh JAZ2 over-expression(Ca MV 35S::Gh JAZ2)and down-regulation(Ca MV 35S::i Gh JAZ2)constructs were developed and transformed into cotton.We found that the fuzz was obviously reduced when Gh JAZ2 was overexpressed in the transgenic lines compared with the wild type.The seed indices were much smaller than the wild type and the RNAi lines.The length of overexpression lines were much shorter than RNAi and controls.To summarize,overexpressing Gh JAZ2 in cotton resulted in less fuzz fiber,shorter lint fibers and smaller seeds,but suppressing Gh JAZ2 had no significant effect.4.Gh JAZ2 regulates cotton fiber initiation through interacting with Gh MYB25-like,Gh GL1,Gh MYC2,Gh WD40,Gh JI1.To evaluate the mechanism underlying the effect of JAZ on fiber initiation,we used the yeast two-hybrid system(Y2H)to screen its interacting proteins.Gh JAZ2 as a bait was used to screen the-2 to 4 DPA ovule/fiber c DNA library to identify Gh JAZ2-interacting proteins.Approximately 80 positive clones were identified.Among the interacting factors,we focused on transcription factors such as the R2R3-MYB transcription factors Gh MYB25-like and Gh GL1,the b HLH factor Gh MYC2,the WD40-repeat protein Gh WD40 and an unknown protein Gh JI1.Next,bimolecular fluorescence complementation(Bi FC)assays confirmed the interactions between Gh JAZ2 and Gh MYB25-like,Gh GL1,Gh MYC2,Gh WD40 and Gh JI1.5.Gh JAZ2 represses transcriptional activation activity of Gh MYB25-likeTo date,only Gh MYB25-like was reported amongst these transcription factors that impacted fiber development.In order to characterize the function of Gh MYB25-like in vivo,the Gh MYB25-like gene driven by 35 S promoter was overexpressed in cotton.The Gh JAZ2 overexpressing lines were crossed by Gh MYB25-like overexpressing lines to develop their F1(Gh JAZ2×Gh MYB25-like)generation.The number of initial lint fibers in Gh MYB25-like overexpression lines was significantly increased,but the initials of the F1 plants and the Gh JAZ2 overexpression lines had no statistical differences.Gh MYB25-like was a transcription activator,and Gh JAZ2 repressed the transcription activity of Gh MYB25-like.Gh JAZ2 could interact with Gh MYB25-like and then inhibited the transcriptional activation function of GhMYB25-like.6.The redundancy of JAZ might explain why the RNAi lines did not show changes in fiber initiationThe number of initials and the length of fiber in RNAi lines showed no difference with OE lines.Thus we analyzed thirty cotton JAZ members based on the G.hirsutum TM-1((AD)1 genome)sequence database.A phylogenetic tree and heat map of the JAZ members were constructed,and found that three JAZ genes preferentially expressed at the fiber initiation,two other JAZ proteins had high sequence similarity with Gh JAZ2 and another JAZ protein had an obvious peak in the-1 DPA ovule during the fiber development stages.Only Gh_D01G1406 had a positive interaction with Gh MYB25-like,Gh GL1,Gh MYC2,Gh WD40 and Gh JI1.The expression of Gh_D01G1406 was increased in two RNAi lines but did not change in the OE lines.So there was other cotton JAZ genes with functional redundancy during cotton lint and fuzz initiation.