| Waterlogging stress is a major hidden danger in agricultural production.Pepper roots are shallow and not tolerant to waterlogging,resulting in a significant decline in the yield.Improving the waterlogging tolerance of peppers has become an urgent issue.With the rapid development of molecular biology,the mining and functional analysis of waterlogging-tolerant genes have become hot research topics.Abscisic acid insensitive 3/viviparous 1（ABI3/VP1）protein is crucial in regulating plant growth and responding to stress.There are few reports on the ABI3/VP1 gene in pepper.‘ZHC2’is a highly waterlogging-tolerant pod pepper cultivar.In this experiment,a specific ABI3/VP1 gene was identified from pepper‘ZHC2’through transcriptome analysis.The transcription factor gene is highly sensitive expression to waterlogging stress,and may be a key factor in response to waterlogging stress.We successfully cloned the gene and named it CaABI3/VP1-1.In order to further verify the function of the gene,peppers‘ZHC1’,‘ZHC2’,‘DFZJ’,and‘Nicotiana benthamiana’were used as tested materials to study the regulatory effect of the genethrough gene cloning,sequence analysis,homologous expression,heterologous expression,subcellular localization,physiological testing,and T3 transcriptome sequencing.The main research results were as follows:1.The expression of CaABI3/VP1 was analyzed by q RT-PCR.The expression trend of CaABI3/VP1 was the same in roots,stems and leaves,and the expression of CaABI3/VP1 decreased after flooding.At the T2 stage,the expression level in the roots was decreased by 62.79%,and that in the stems and leaves decreased by 43.81%and 55.36%,respectively.This was consistent with the changes in transcriptome,indicating that CaABI3/VP1 may be expressed throughout the plant.2.Successfully cloned the CaABI3/VP1-1 gene from pepper and constructed the p EGOEPubi-H-pepper-CaABI3/VP1-1-GFP vector.CaABI3/VP1-1 contains a 1,017bp open reading frame encoding 338 amino acids.Protein sequence analysis showed that CaABI3/VP1-1 contains two conserved B3 structural functional domains,lysine and glutamic acid are the main amino acid components,and the protein is a hydrophilic non-secretory protein with phosphorylation sites.The main component of protein secondary structure is irregular curling.CaABI3/VP1-1 and capsicum XP016577237.2 protein clustered in the same cladicle,the closest relative.3.Homologous modeling revealed that this protein had aα-spiral at the N-end and aβ-folding extension chain at the C-end.The confidence level of this model was set as 100.0%.The Ramachandran diagram of the protein shows that only 1%of the amino acid residues remained in the prohibited regions,verifying the accuracy of the model.A further 4 ns molecular dynamics simulation of the protein was performed,and it was found that the protein reached equilibrium after 2,300 ps,reaching a maximum value at 3,350 ps,with a fluctuation range of 1.174 to 1.414 nm,indicating that the protein structure was stable.4.The cis-acting elements of the gene promoter included ABRE related to abscisic acid reaction,plant hormones like TGA-element,CGTCA-motif,TATC-box,and TGACG,development like GCN4＿motif,adversity like LTR and ARE,metabolism like O2-site.In addition,a large number of conservative light regulatory elements had been found,such as G-Box,I-box,Gap box,GT1-motif,and Box 4.5.A CaABI3/VP1-1-GFP vector was constructed,and Agrobacterium tumefaciens was used to introduce CaABI3/VP1-1 into tobacco leaves for transient expression at 2-4 weeks.Fluorescence signals were detected by laser copolymerization microscopy,and subcellular localization showed that the protein was located in the nucleus.It had been confirmed that transcription factor must function in the nucleus to regulate expression.6.After homologous transformation of CaABI3/VP1-1 to peppers‘DFZJ’and‘ZHC1’,the overexpression lines were successfully identified by GFP green fluorescence screening and PCR detection.The CaABI3/VP1-1 overexpression lines had stronger roots and stems and lodging-resistant ability.At the same time,it increased the contents of soluble protein（SP）and proline（Pro）in the plants,stomatal density and pore size,reduced the accumulation of malondialdehyde（MDA）and reactive oxygen species（ROS）,and had higher catalase（CAT）activity than the wild type plants.Further,CaABI3/VP1-1 was transformed into N.benthamiana by A.tumefaciens mediated leaf disc transformation.Overexpression of CaABI3/VP1-1reduced the degree of root rot,also increased the content of SP,Pro,stomatal density,and CAT activity,reducing the accumulation of MDA in the plants.7.Transcriptome analysis of T3 lines revealed that LOC107844566,LOC107843480,LOC107879909,LOC107851756,and LOC107843452 may be downstream genes affected by CaABI3/VP1-1,which mainly affect MAPK signaling pathways-plant and plant hormone signal transduction.CaABI3/VP1-1 enhanced the waterlogging tolerance of transgenic peppers,which may be an important gene to improve the waterlogging tolerance.In summary,this experiment cloned the gene CaABI3/VP1-1 from pepper‘ZHC2’,and waterlogging stress induced its downregulated expression.CaABI3/VP1-1 subcellular localization was in the nucleus.The gene enhanced the waterlogging tolerance of plants through homologous expression in pepper and heterologous expression in tobacco.Therefore,CaABI3/VP1-1 was involved in the response of pepper to waterlogging stress and enhanced the waterlogging tolerance of plants. |
