The Functional Characterizations Of Ubiquitin Ligase Gene GhATL68 In Cotton Fiber Development

Ubiquitin proteasome degradation system is the main way to degrade protein which requires ubiquitin activating enzyme,ubiquitin conjugating enzyme and ubiquitin ligase.Ubiquitin ligase E3 can specifically recognize and ubiquitinate proteins.Generally,the poly-ubiquitination proteins are degraded to peptides by 26 S proteasome.Ubiquitin ligase E3 plays an extremely important role in every process of plant development.Cotton fiber is derived from the outer integument,which is an excellent system for plant cell development.However,there are few reports about ubiquitin ligase in cotton,especially in cotton fiber development.Therefore,we found an ubiquitin ligase gene that was dominantly expressed in fiber rapid elongation period from gene expression profiles of upland cotton.According to its homologous genes in Arabidopsis,this gene was named Gh ATL68.The functional characterizations of Gh ATL68 in cotton fiber development were explored through Agrobacterium mediated transformation.The main experimental results are listed as follow.1.Sequence analysis of Gh ATL68Homologous proteins were found by using the BLASTP function of the NCBI website.The results showed that Gh ATL68 was more similar to At ATL68,Gr ATL67 and Tc ATL67.Conserved domain of Gh ATL68 was analyzed,which showed that Gh ATL68 had a C3H2C3 type RING structure that is characteristic structure of ubiquitin ligase family.2.Phenotypic identification of transgenic cottonOverexpression and RNAi cotton were gained.Finally we got two overexpression and RNAi lines for each,which had low copy number of T-DNA insertions and expected gene expression level.Then the fiber phenotype was observed in the T2 generation of transgenic lines.At 8 DPA,the development of bolls in RNAi lines was gradually suppressed.At 10 DPA,this suppression was more obvious.In addition,the fiber length in RNAi lines was significantly shorter than that of negative control lines and wild type,while the fiber and boll developments of the overexpression lines were not changedobviously.The mature fiber in RNAi lines was also significantly shorter than that of the negative control and wild type,while the length of the fiber in overexpression lines was not changed clearly.Moreover,The breaking strength and uniformity of fibers in RNAi lines were lower than that of the negative control and wild type,but the fiber quality of overexpression lines didn?t change noticeablely.These results suggested the decreased expression level of Gh ATL68 can retard the development of cotton bolls and worsen fiber quality;the elevated expression level of Gh ATL68 had no effect on fiber development.3.The possible mechanism of Gh ATL68 on cotton fiber developmentIn order to explore the mechanism that how Gh ATL68 might function in fiber development,we used the yeast two-hybrid system to screen the yeast library that express proteins in fiber elongation period.A protein Gh STP7 that might interact with the target protein was screened out.Gh STP7 codes a sugar transporter and its expression pattern was analyzed and it preferentially expressed at 5 DPA,10 DPA,15 DPA,which was consistent with the expression profile of Gh ATL68.The subcellular localization analysis had shown that Gh ATL68 played a role in the nucleus,cytoplasm and cell membrane,while the STP protein was reported to be located on the cell membrane.In summary,Gh ATL68 may affect the development of cotton fiber by affecting the transport of saccharide through Gh STP7 in the cells.