Function Analysis Of A Cotton E2 Gene GhUBC2L And Establishment Of The CRISPR/Cas9 System In Cotton

Protein ubiquitination is a key biochemical process for cell normal growth.Proteins modified with different ubiquitination state can change its activity or gain death.E2 ubiquitin-conjugating enzyme is one of the key enzymes in this process.It decides the specificity of the target together with E3 ligase,transfers the ubiquitin molecule to the target,affects the processivity of ubiquitin chain formation,and determines the length and linkage specificity of ubiquitin chain.Plant E2 family has dozens of members.Most literature-reported gene function analysis comes from Arabidopsis.They have a wide regulation in plants,including photomorphogenesis,vegetative and reproductive growth,flowering time control and response to a variety of biotic or abiotic stresses.Relative gene function analysis is rare in cotton,which is one of the important economy crops that is worldwide cultivated.The CRISPR/Cas9 system has become the most popular genome editing tool because of its site-specific editing,higher efficiency and robustness.It has been widely applied to generate gene mutants.However,rare mutants were utilized to conduct gene function analysis in cotton research before.So construction of the CRISPR/Cas9 system in cotton can promote cotton gene function analysis to the genome wide level.There are two parts of research in this thesis.One is to conduct function analysis of a cotton E2 gene GhUBC2 L.The other is to construct the CRISPR/Cas9 system in cotton.Main results are as follows.A.GhUBC2 L regulates the development of cotton organs.1.We cloned a cotton gene GhUBC2 L that was annotated with E2 function.It is quite homologous to AtUBC1 and AtUBC2,both in nucleotide and protein sequences.In vitro assay verified that GhUBC2 L protein had ubiquitin conjugation activity.This protein accumulated almost anywhere in the cell.2.Overexpression and RNA interference of this gene were conducted in cotton.The RNAi transgenic cotton lines showed slower growth rate compared to the control,resulting in obviously decreased organ size of leaves,flowers and mature seeds.The fiber development was also inhibited.Conversely,leaves and flowers in GhUBC2 L overexpression lines were obviously enlarged.Detailed analysis of the organ cells illustrated that GhUBC2 L might affect organ size through regulating cell proliferation and cell expansion.3.Through yeast two hybrid screening,we found that GhUBC2 L interacted with a predicted E3 ligase GhUbox8.This interaction was verified with both in vitro and in vivo experiments.In vitro assay verified that GhUbox8 had autoubiquitination activity,a characteristic that almost all RING type E3 ligase have.GhUbox8 also can form dimer in yeast.We detected histone modification in different transgenic plants and found that H2 Bub and H3K4me3 were obviously decreased in RNAi lines,especially for H2 Bub.But we had not deciphered the relationship between GhUbox8 and these changes.4.The expression level of LOX1,a key gene for JA biosynthesis,and a cell proliferation related gene MADS1 were reduced in the RNAi lines.Finally we speculated that GhUBC2 L might interact with more than just GhUbox8 to regulate histone modification and affect expression level of some development related genes.B.Construction of the CRISPR/Cas9 system in cotton.1.We cloned a cotton specific U6 promoter that was homologous to that of AtU6-26 gene.It was used to modify the CRISPR/Cas9 vector that can generate more than one sgRNA.A series of sgRNA target sites were designed for the exogenous gene DsRED2 and an endogenous gene GhCLA1.Genetic transformation was conducted through agrobacterium mediated method in cotton.2.All designed 6 sgRNA target sites in DsRED2 were edited,with nucleotides deletion being the most obvious mutation type.Knockout of DsRED2 reversed the DsRED2 overexpressing cotton to wild type,resulting in diminished red fluorescence in all organs.The seeds also lost red color.Further analysis revealed that the edited gene mutation was inheritable from T0 to T1 generation.The wild type target sites gained new mutation in T1 plants.3.For GhCLA1,all designed 4 sgRNA target sites were edited,with nucleotides deletion being the most obvious mutation type.A pair of sgRNA in the vector can target two sites in the GhCLA1 gene simultaneously,resulting in a large fragment deletion.Complete knockout mutants of GhCLA1 had an albino phenotype all the seedlings,with a ratio of 75%.After analyzing the genotype at the target sites and phenotype of independent albino plants,we found that this obvious phenotype was resulted from mutation of all the GhCLA1 copies.4.To improve genotyping of the target gene in a lot of mutant plans,a barcode-based high throughput sequencing method was developed to check 30 plants in a run.Similarly,26 potential off-target sites were checked with high throughput sequencing method.There was no detectable mutation at those potential off-target sites.All these results proved that our CRISPR/Cas9 system has high efficiency and specificity for gene editing in cotton.This system will promote the development of cotton mutants library in the future.