Identification And Salt Tolerance Analysis Of F-box Genes In Vicia Faba L.

Vicia faba L.,belonging to the genus vetch of the Fabaceae,is the seventh largest edible legume crop in the world.It has very important commercial and nutritional value.Vicia faba is widely cultivated for its ultra-high nitrogen fixation capacity.However,it is often damaged by abiotic stresses such as drought,waterlogging and saline-alkali.Among them,salt stress caused great losses to agricultural production and reduced the yield.We can use the genetic engineering can effectively improve plant salt tolerance,and the key lies in mining salt tolerance related genes.Studies have shown that F-box genes have widely participated in plant growth and development as well as play an important role in responding to abiotic stress pathways.At present,researches on plant F-box gene mainly focus on a few plants such as Arabidopsis thaliana,Triticum aestivum L.and Glycine Max.There are few researches and reports on the F-box gene in Vicia faba L.In this study,F-box gene family members were identified at the transcriptome level under salt stress.By analyzing the transcriptome data of Vicia faba L under salt stress and using bioinformatics methods,F-box genes related to salt tolerance were screened,and the over-expressed F-box gene material was obtained by transforming Arabidopsis thaliana and tobacco.The salt tolerance and its mechanism were further analyzed in this study.The main research results are as follows:1.Genetic analysis of F-box gene family in Vicia faba L.: Based on salt-stressed transcriptome sequencing(RNA-seq)data,161 Vicia faba F-box genes were annotated and all contain F-box conserved domain.According to different C-terminal domains,they were divided into 11 subfamilies.The analysis of the conserved domain showed that the F-box conserved motif contained an extremely conserved tryptophan residue.By comparing and analyzing the evolutionary tree constructed by the F-box family of Vicia faba and the F-box family of Arabidopsis thaliana,it was found that most of the genes in the same C-terminal domain were clustered together and C-terminal domain is the main basis of subgroup classification.The results of subcellular localization prediction showed that: 124 F-box genes were located outside the cell,and 37 F-box genes were located in the nucleus.The analysis of gene structure showed that there were no introns in the DNA sequences of the F-box family genes of Vicia faba,and all of them were composed of UTR zone and CDS zone.2.Expression Analysis of F-box Family Gene in Vicia faba L.: The expression data of F-box family genes between two broad bean cultivars(Y134 and Y078)for 16 h and 24 h were compared and analyzed.The results showed that the expression of F-box gene in Vicia faba was different at two different treatment time points,the expression was more obvious at 16 hours after salt treatment.The results of q RT-PCR analysis showed that there were five different genes in the F-box family.The expressions of Vf056266.1,Vf062764.1 and Vf024236.1 were all up-regulated at 16 hours after salt treatment,Vf060904.1 and Vf045761.1 were both down-regulated at16 hours after salt treatment.3.To construct a transgenic Arabidopsis thaliana material with F-box overexpression : Two differentially expressed genes,Vf045761.1 and Vf062764.1,were cloned from Vicia faba L tissues.Arabidopsis thaliana overexpression vectors are constructed,introduced into wild-type Arabidopsis thaliana by floral dip,and the seeds were harvested to the homozygous T3 generation for functional verification.It is found that the germination rate and root length of the overexpressed Vf062764.1gene are higher than those of the empty vector under four salt concentrations(0 m M,100 m M,150 m M,and 200 m M),and the difference is significant at 150 m M and 200 m M.The difference is inconspicuous for overexpression of Vf045761.1 gene compared to the empty vector.Under high-salt stress,seedling phenotypes and growth physiological indicators of the Vf062764.1 overexpression gene are significantly improved compared with the empty vector.Seedling leaves of transgenic plants are more green,while empty vector plants are severely wilted;dry weight,fresh weight and root length are more than that of empty vector,malondialdehyde content is lower than the empty vector and the difference is significant;superoxide dismutase and proline content are significantly higher compared with the empty vector.In contrast,overexpression of the Vf045761.1 gene is not noticeable compared to the empty vector.4.Viral vector transformation of tobacco: Vf045761.1 and Vf062764.1 were overexpressed by virus expression vector in tobacco.It was observed that the over-expressed Vf062764.1 plants grew better under high salt stress than the empty vector and without injection plants.The leaf shrinkage is small and the root system is lush.Plants with overexpression of Vf045761.1 gene grew slightly better than those empty vector and without injection plants,and leaf shrinkage appeared,with no obvious difference.