Expression Analysis And Functional Verification Of ARF And GRF Family Genes Related To Tobacco Leaf Development

For tobacco,the leaves are harvesting part.Leaf development determines the final yield and quality of tobacco.In this study,46 auxin response factor(ARF)family members in tobacco were identified.According to the expression of NtARFs in different tissue or organ and NAA treatment,the key gene NtARF16 for leaf development was selected.According to the expression of GRF gene family in leaves of “HongHuaDaJinYuan” and its two mutants,NtGRF8 as a key gene for leaf development was selected.NtARF16 and NtGRF8 were isolated and cloned respectively,their functions were preliminarily verified.The main results are as follows: 1.The ARF family in Tobacco comprised of 46 members.According to the structure and phylogenetic analysis of the ARF genes in tobacco,tomato,and Arabidopsis,they were divided into six groups: ?~?.35 NtARFs had a typical ARF protein structure,including the N-terminal DBD domain,Aux_Resp domain and CTD domain(Aux_IAA).Seven different types of cis-acting components were identified in 46 NtARFs genes.These seven types of cis-acting components include light response elements,hormone response elements,development-related elements,environmental stress response elements,promoterrelated elements,Site binding elements,other elements.The NtARFs were also regulated by microRNAs,and the target site of nta-miR160 was predicted in NtARF30,NtARF31,NtARF32,NtARF33,NtARF34,NtARF36,NtARF40,NtARF41,and NtARF42 while the target sites of nta-miR167 were predicted in NtARF23,NtARF24,NtARF26,NtARF25,NtARF27,and NtARF22.2.qRT-PCR was used to analyze its expression pattern in different tissues after exogenous NAA application.NtARFs had tissue expression specificity,and most of the ARF genes were expressed in seeds.NtARF8,NtARF9,NtARF14,NtARF44,and NtARF45 showed similar expression patterns at different time points after NAA treatment.Generally,overall gene expression increased within 48 h after treatment.NtARF11,NtARF16,NtARF24,NtARF28,and NtARF34 showed similar expression patterns and the general trend of gene expression increased first and then decreased within 48 hours.The response of NtARF16 to NAA was the most significant.It was found that NtARF16 protein was located in the nucleus by the methods of Agrobacterium infection and DAPI nuclear staining.Compared with wild type plants,the leaf area of the transgenic Arabidopsis thaliana transformed with the NtARF16 gene increased by 58%,the number of cells increased by 43.1% and the size of cells increased by 22%.3.The leaf area of two leaf size mutants(mutant 1 and mutant 2)decreased by 89.88% and 16.08%,respectively.Among them,the leaf cells of mutant 1 became smaller and the total number of cells decreased by 89.07%,the size of mutant 2 cells increased and the total number of cells decreased by 51.88%.the qRT-PCR analysis showed that the expression of 17 GRF family members in mutant 1 and 2 was decreased.Among them,NtGRF5,NtGRF8,and NtGRF25 in mutants 1 and 2 were the most downregulated genes.The expression level of NtGRF5 in mutant 1 and mutant 2 decreased by 69.72% and 65.57%,respectively.The expression level of NtGRF8 in mutant 1 and mutant 2 decreased by 80.91% and 66.81%,respectively.Similarly,the expression level of NtGRF25 in mutant 1 and mutant 2 decreased by 81.59% and 68.76%,respectively.The expression levels of NtGRF8,NtGRF19,NtGRF7 and NtGRF25 were higher than those of other members of the GRF family in the wild type plants leaves.According to the expression of GRF gene family in leaves of “HongHuaDaJinYuan” and its two mutants,NtGRF8 as a key gene for leaf development was selected.It was found that NtARF16 protein was located in the nucleus by the methods of Agrobacterium infection and DAPI nuclear staining.Compared with wild type plants,the leaf area of transgenic NtARF16 Arabidopsis increased by 41%,the number of cells and the size of cells increased by 10.9% and 61%,respectively.The leaf growth of mutant 1 was inhibited more significantly,the morphological characteristics were more obvious,the cells in the leaves of mutant 1 were smaller,NtGRF8 was almost not expressed in mutant 1,but the leaf cells of Arabidopsis transformed with NtGRF8 gene were increased,which indicated that NtGRF8 could promote the cell growth in the leaves.