The Mutant Lirbary Construction Of E3 Ubiqutin Ligase Involved In Cotton Fiber Development

Ubiquitination is a major form of post-translational modification,which determines the fate of proteins.Ubiquitinated proteins may change their activity or be degraded directly by the 26S proteasome,and E3 ubiquitin ligase plays a key role in the recognition of target proteins in the process.Protein ubiquitination systems are widely involved in biological developmental processes.Cotton is an important cash crop in China,and fiber is its main product.The development process of cotton fiber is regulated by several factors.At present,studies on the mechanism of cotton fiber development mainly focus on the transcriptional level.However,the effect of some factors of protein level on cotton fiber development has not been reported.Therefore,in this study the role of protein degradation system in cotton fiber development will be explored.By analysis of the transcriptomes of fiber through 4 development stages including initiation,elongation,transition,secondary wall thickening we found that E3 ubiquitin ligases were expressed,and some of them were preferentially expressed in some special stages.In this study,a library of E3 ubiquitin ligase mutants was developed by CRISPR/Cas9 gene editing technology,and the function of an E3,GhATL68,involved in fiber development were further analyzed.The main results were as follows:1、Genome-wide identification of E3 ubiquitin ligase genes:According to the RNA-Seq data in the public database,a total of 172 E3 ubiquitin ligases which were preferentially expressed in some special stages were screened out.2、Design of sgRNA:2 sgRNA per gene were designed,due to the restriction of target sequence screening criteria,there was only one sgRNA for 26 genes,and a total of318 sgRNA were designed in the end.The vectors with one sgRNA were constructed,then 318 vectors were divided into 8 sublibraries for cotton transformation,and each sublibrary contained 40 sgRNA involving about 20 genes.A total of 612 T₀ plants were obtained,and 582 transgenic plants were transformation positive.A total of 220 sgRNA were detected by sgRNA coverage analysis.It accounted for 69.18%of the total number of designed sgRNA.Most E3 ubiquitin ligase mutants contain one sgRNA.Only 17plants contained 2 sgRNA,and plants containing 2 sgRNA accounted for 2.74%of the total mutants.3、Identification of mutant library genes:A total of 133 E3 mutants were obtained,and the gene coverage was 77.33%.The proportion of missing editing type,insertion editing type and replacement editing type was 69.41%,24.91%and 5.67%,respectively.The longest deletion fragment was 41bp and the longest insertion fragment was 29bp.All the mutant genotypes could be detected in the progeny plants,and the progeny of the chimeric plants with complex editing types would lead to next generation separation.4、The fiber phenotype investigation of T1 mutants:The mature fiber phenotypes of31 mutant plants were analyzed.Compared with the wild type,the fiber length of Ghir＿A09G018170 and Ghir＿A04G009770 mutants did not change obviously,but the micronaire decreased.The fiber of Ghir＿D11G002970 mutant was shorter and the micronaire was lower.5、Fuction analysis of E3 ubiqutin ligases gene GhATL68:T₄GhATL68 mutant lines were obtained,and the fibers of atl68 mutant were shortened.The ubiquitination experiments in vitro showed that GhATL68 had E3 ubiquitin ligase activity,and GhATL68 could interact with GhUBC32（E2 ubiquitin-conjugating enzyme）.The results of IP-MS showed that GhATL68 interacted with sucrose synthase GhSS-A13.And GhATL68 may target and ubiquitinate GhSS-A13.