| In nature,protein is the basis of all life activities,regulating the physiological functions of life.The ubiquitin-proteasome involved in protein degradation pathway is the focus of many scholars.It regulates almost all life activities in higher eukaryotic cells,such as cell proliferation,differentiation and DNA repair,mainly through specific degradation of most proteins or partial processing of transcription factors.The regulation of cell cycle is a basic factor affecting the growth of plant organs.As an important cash crop,tobacco is the main raw material source of tobacco industry.The research of its organs has important biological significance and economic value.Studies have shown that DA1 protein,as a kind of ubiquitin-activated peptidase,plays an important role in the whole growth and development of plants such as Arabidopsis thaliana,affecting the size and morphology of various organs.However,the function of DA1 gene in tobacco is still not perfect.In this study,two AtDA1 homologous genes of Arabidopsis thaliana,namely NtDA1-1 and NtDA1-2,were identified and obtained from cultivated tobaccoHonghua Dajinyuan variety.Further,CRISPR/Cas9 gene editing technology and overexpression means were used to create transgenic materials of NtDA1 gene.The effect of tobacco NtDA1 gene on the growth and development of axillary buds,corolla and seeds of tobacco was studied.The main results obtained were as follows:1.Identification,cloning and expression analysis of the NtDA1 gene in tobaccoBy comparing the reported Arabidopsis AtDA1 protein sequences in tobacco database,two homologous genes with highly similar sequences were identified and named as NtDA1-1 and NtDA1-2,respectively.The ORF frames of NtDA1-1 and NtDA1-2 genes were 1644 bp and 1506 bp,encoding 548 and 502 amino acids respectively,with molecular weights of 62.08 k Da and 56.88 k Da.The similarity of CDS sequence and amino acid sequence was 96.74% and 95.38%,suggesting that their functions may be highly similar.Gene structure analysis showed that both of them contained 11 exons and10 introns.Multiple sequence alignment analysis showed that DA1 of multiple species contained the typical ubiquitin receptor interaction motif UIM and the single zinc finger binding domain LIM.Phylogenetic tree analysis showed that tobacco NtDA1-1 and NtDA1-2 are closely related to capsicum,potato and tomato,which are also Solanaceae,but are far related to brassica napus and Arabidopsis thaliana,which are cruciferae.The analysis of tissue expression profiles showed that NtDA1-1 and NtDA1-2 were expressed in nine tissues of tobacco,and were highly expressed in corolla,stamen and axillary bud,and their expression patterns showed high similarity.Subcellular localization indicated that NtDA1-1 was located in the cytoplasm and NtDA1-2 was located in the cell membrane.2.Production of the NtDA1 gene knockout and overexpression materialsIn order to explore the function of tobacco NtDA1 gene,we created a knockout line of tobacco NtDA1 gene.Three sg RNAs targeting NtDA1-1 and NtDA1-2 were designed based on CRISPR/Cas9 gene editing technology.After agrobacterium-mediated genetic transformation,T-cloning and Sanger sequencing,NtDA1 knockout transgenic plants were obtained.The editing form test showed that sg RNA2 could efficiently edit two NtDA1-1/2 genes at the same time,in the form of deletion,insertion or replacement of bases,resulting in codon replacement or frameshift mutation behind the mutation site.Through further screening of T1 generation plants,double knockout(T1 # 20)and single knockout mutants(T1 # 33 and 46)of NtDA1 gene were obtained,which were further cultivated and screened to obtain homozygous knockout plants of T2 generation in NtDA1-1/2 gene.In order to explore the function of tobacco NtDA1 gene from another aspect,overexpressing plants of tobacco NtDA1 were created.Overexpression vectors containing NtDA1-1 and NtDA1-2 were constructed by enzyme digestion and ligand,respectively,and overexpression positive plants were obtained by genetic transformation and Sanger sequencing.The results of q RT-PCR showed that the expression of NtDA1-1 in T0 # 23,24 and 43 plants was higher than wild type plants,and the expression of NtDA1-2 in T0# 6,14 and 20 plants was up-regulated.Due to the longer formation time of regenerated plants for genetic transformation,we only obtained the T2 generation of NtDA1 gene double-knockout homozygous mutant,single gene NtDA1-1 and NtDA1-2 knockout homozygous mutant,and the T0 generation of NtDA1-1 and NtDA1-2 overexpressed plants for subsequent studies.3.Functional study of tobacco the NtDA1 geneIn order to explore the function of tobacco NtDA1 gene,wild type,NtDA1 gene double-knockout plants and overexpressed plants were planted to observe whether they had an effect on tobacco phenotype.In order to study the effect of NtDA1 gene on branch development in tobacco,experiments were conducted on knocked out plants.The results showed that the deletion of NtDA1 gene did not affect the growth rate and length of axillary buds after topping,but reduced the number of axillary buds.These results indicated that the growth and development of axillary buds were regulated by multiple genes,and NtDA1 gene was only a part of the regulatory network.The corolla observation experiment showed that the corolla of overexpressed plants had larger petal notches(pits),and smaller petal radians which were more rounded.However,the corolla of double-gene knockout plants was longer and narrower than that of wild type,and the corolla phenotype of single-gene knockout plants was not obvious,indicating that NtDA1 gene was involved in the formation of the final form and size of the corolla in tobacco.Furthermore,the corolla paraffin section experiment showed that mesophyll parenchyma and vascular bundle cells of double-gene knockout plants were larger,while the single gene knockout mutant had no significant change,and the mesophyll parenchyma of overexpressed plants was smaller,indicating that NtDA1 gene was involved in affecting the size of parenchyma cells that make up palisary tissue.Seed phenotype observation and weight statistics showed that the seeds of the overexpressed plants became smaller and weighed significantly less,while the seeds of the double knockout mutant became larger and the seeds of the knockout plants weighed significantly more,indicating that NtDA1 gene affected the size and development of tobacco seeds.Referring to the expression of downstream transcription factors in AtDA1 gene mutant,we detected the expression of downstream homologous transcription factors of NtDA1 in tobacco.The results showed that NtDA1 peptidase in tobacco could inhibit the expression of transcription factors related to cell proliferation,and thus participate in the regulation of the growth and development of tobacco corolla and seeds.In conclusion,two ubiquitin-activated peptidases,NtDA1-1 and NtDA1-2 were identified and cloned in tobacco,demonstrating that they play an important role in the growth and development of tobacco corolla and seeds.This study provides a useful reference for exploring the development of tobacco organs and improving the metabolic pathway of DA1 gene. |
