Cloning,Expression And Anti-tobacco Mosaic Virus Function Analysis Of Tobacco Resistance-related Gene NtHin1

Tobacco mosaic virus belongs to the genus of Tobamovirus,it has the worldwide host ranges,including solanaceae,cruciferae,poaceae,chenopodiaceae,leguminosae and many other plants,also brought serious harm on the agricultural economy.Breeding of resistant varieties is one of the main measures to control the disease,but the isolation and identification of related disease-resistant genes and the application of functional excision still need to be strengthened.HIN1?harpin-induced gene 1?is one of the proteins induced by Harpin,it plays an important role in defense response,growth development,senescence and stress resistance of plants.To identification the function of this gene in anti-tobacco mosaic virus has important theoretical and practical significance.In this study,Hin1 gene was isolated from four species of solanaceae plants,Nicotiana benthamiana,pepper,potato and tomato by molecular cloning.The DNA sequence and its encoded protein sequence were analyzed by bioinformatics tools.Subcellular localization and tissue-specificity expression of the NtHin1 has been identified and the plant expression vector pFGC5941-NtHin1 has been successfully constructed.The Nicotiana benthamiana over-expressing NtHin1 was obtained by Agrobacterium-mediated transformation and genetic transformation.On the basis of this materials,we test the function of anti-tobacco mosaic virus and plan to explore its antiviral mechanism by transcriptome sequencing.In this study,Hin1 gene was cloned from four solanaceae plants:687 bp of NtHin1,684 bp of CaHin1,675 bp of potato StHin1 and 675 bp of SlHin1.W clustering analysis showed that the highest homology was NtHin1 and CaHin1 attach to 89.9%.The phylogenetic tree showed that the closest relationship was from other solanaceae plants and was far from rice and sorghum monocotyledons.The result of protein prediction of NtHIN1 indicated that it encoded 229 amino acids,the theoretical molecular weight of the protein was 26.2 kD,the theoretical isoelectric point was 9.39,the molecular formula was C₁₁₈₂H₁₈₄₀N₃₂₀O₃₂₇S₁₃ and has the conserved domain LEA-14.The RFP fusion expression vector pCV-NtHin1 was constructed and transiently expressed in Nicotiana benthamiana leaves by agrobacterium transformation.Subcellular localization was showed that NtHIN1 distributed on the plasma membrane of the leaf epidermis cell of Nicotiana benthamiana,which is consistent with the predicted results.The results of RT-qPCR showed that the NtHin1 was of tissue-specificity,whose expression decreased from tobacco roots,flowers,leaves to stems.The plant expression vector pFGC5941-NtHin1 was successfully constructed according to the NtHin1 sequence,and then transiently expressed in Nicotiana benthamiana before being inoculated with TMV-GFP.After 4 days of exposure,the naked tobacco leaves of the treated group did not appear the green fluorescence and the control group of leaves have shown clearly green fluorescence.Then the treatment group showed visible green fluorescent spots on day 5,while the control group's green fluorescence had been extended to lobus cardiacus.The sporadic fluorescence of the treated group only slightly expanded until day 7,whereas the control group's green fluorescence had spread to all the lobus cardiacus.The results of ELISA also demonstrated that the virus replicated was seven times in the control group after 7 days inoculation.Subsequently,using the way of agrobacterium tumefaciens mediation obtained positive genetic transformation of plants,T₀ generation of tobacco mosaic virus inhibition effect was the same with transient expression.After selection,the T₁generation transgenic transformation plants were obtained.There was no significant difference in morphological compared with wild type,and also no allergic necrosis spots were observed.After inoculation of TMV-GFP by friction,macroscopic green fluorescent spots appeared on the second day.Compared with the treated group,the control group has more spots.On the third day after inoculation,the spots began to become larger and began to diffuse.On the fourth day,the control group had expanded to the whole lobus cardiacus while the treatment group did not extend to.Also,the green fluorescent spots of the treatment group were also relatively small.The results of RT-qPCR of GFP also indirectly prove that the accumulation of viral RNA levels is indeed lower than the control group.In conclusion,NtHin1 overexpression could inhibit tobacco mosaic virus.By comparing the transcriptome data of wild-type and T₀ transgenic plants,102differentially expressed genes were found,of which 48 were up-regulated and 54 were down-regulated.They are involved in 23 variety of physiological and biochemical processes in plants,including plant-pathogen interactions,plant hormone signaling,endocytosis,and RNA degradation.After verified by real-time PCR,we found that tobacco PARN,CNGCs and Rab11 genes were upregulated between treatment and control groups.After treatment with jasmonic acid,Hin1 and Rab11 in Nicotiana benthamiana began to increase at 5h and 2h and the trend of the two genes was basically the same during 5-12h.The expression level of COI1,a receptor gene of jasmonic acid pathway,was higher than that in wild type,indicating that the jasmonic acid biosynthesis pathway maybe was induced.However,the protein interaction test proves that there is no direct interaction between NtRAB11 and NtHIN1,and the specific relationship between them needs further study.