Cloning And Functional Analysis Of Fra A 1 Gene In Fragaria × Ananassa ’Benihoppe’

Cultivated strawberry(Fragaria ×ananassa Duch.)contains allergen protein Fra a 1,a certain group of people may have an allergic reaction;Fra a 1 belongs to the PR-10 family,may play a certain biological function during plant growth.’Benihoppe’ strawberry has the characteristics of high yield,good flavor,large fruit and good commerciality.It is currently one of the main domestic varieties.In this paper,the main three genes Fra a 1.01,Fra a 1.02 and Fra a 1.03 in the Fra a 1 family were cloned from ’Benihoppe’ strawberry,and their expression characteristics were analyzed.Fra a 1-GFP-pCAMBIA1302 was constructed.The fusion expression vector and the pCAMBIA1301-35SN-Fra a 1 plant overexpression vector were used to transform tobacco,and the function of the gene was explored.The main results are as follows:1.Design specific primers based on the published Fra a 1.01,Fra a 1.02 and Fra a 1.03 gene sequences in the NCBI database,and cloning Fra a 1.01,Fra a 1.02 and Fra a 1.03 gene from the ’Benihoppe’ strawberry leaf DNA and cDNA.The full-length DNA sequence of Fra a 1.01 gene is 594bp,591bp,584bp.The full coding region of cDNA is 483bp and 480bp,encoding 160 and 159aa,and contains an intron of 101 or 111bp in length;the Fra a 1.02 gene has no intron,the sequence of DNA and cDNA is 483bp,encoding 160aa;the full length of Fra a 1.03 DNA sequence is 599bp,the full coding region of cDNA is 480 bp,and the middle contains a 119bp intron,encoding 159aa.The molecular weights of the three predicted genes were 17766.13Da,17484.82Da and 17448.85Da,and the isoelectric points were 6.13,5.24 and 5.64,respectively.There were no transmembrane domains and signal peptides.2.The tissue specificity of Fra a 1 gene and its expression under the treatment of salicylic acid(SA),2-chloroethyl phosphoric acid(CEPA),methyl jasmonate(MeJA)and strawberry anthracnose(Colletotrichum fragariae Brooks)were analyzed by reverse transcreiption quantitative real-time PCR(RT-qPCR).The results showed that Fra a 1.01 was mainly expressed in roots,stems and leaves,and the highest expression in roots;Fra a 1.02 was highly expressed in roots,flowers,receptacles and achenes,and the highest expression in receptacles;Fra a 1.03 is mainly expressed in roots and achenes and has the highest expression in roots.SA induced the expression of Fra a 1.01 in leaves,and failed to induce its expression in roots and stems;CEPA induced Fra a 1.01 expression only in leaves,and induced in roots and stems;MeJA induced Fra a in roots The expression of Fra a1.01 was not significantly induced in stems and leaves.SA induced the expression of Fra a 1.02 in roots and leaves,and did not induce its expression in stems;CEPA and MeJA induced the expression of Fra a 1.02 in roots,stems and leaves.SA induced the expression of Fra a 1.03 in roots and leaves,and failed to induce its expression in stems;CEPA induced the expression of Fra a 1.03 in stems and leaves,and slightly induced its expression in roots;MeJA in roots,stems and leaves The expression of Fra a 1.03 was slightly induced in the middle.Strawberry anthracnose induced the expression of Fra a1.01 and Fra a 1.03 and failed to induce the expression of Fra a 1.02.3.In order to investigate the sub-cellular localization of Fra a 1,the Fra a 1 gene encoding frame lack of termination codon was connected with N terminal of green fluorescent protein(GFP),we generated 35S:Fra a 1-GFP-pCAMBIA1302 fusion construct and expressed the contruct in Nicotiana benrhamiana leaves by agroinfiltration.The subcellular localization of Fra a 1-GFP and GFP control were visualized with laser scanning confocal microscopy.The results revealed that Fra a 1-GFP was localized in both the plasma membrane and the nucleus,whereas the GFP control was localized in multiple subcellular compartments including the cytoplasm and the nucleus.4.The plant expression vector of Fra a 1.01,Fra a 1.02 and Fra a 1.03 gene in the Fragaria× ananassa Benihoppe’ was constructed and transformed into K326 tobacco(Nicotiana tabacum L.cv.’K326’)by Agrobacterium-mediated method.The transgenic events were confirmed by GUS,PCR and RT-PCR,and the transgenic lines were analyzed for salt tolerance and osmotic stress.RT-qPCR was used to analyze the pathogenesis-related proteins and anti-osmotic stress in transgenic tobacco lines.And the expression of antioxidant-related genes.Experimental results showed:the expression levels of NtPR1,NtPR3,NtPR3 and NtPR5 genes in the T0 generation of transgenic tobacco lines were up-regulated.The expression levels of antioxidant stress-related genes(NtSOD,NtPPO,NtAPX,NtPAL,NtRbohD and NtCAT)and osmotic stress-related genes NtSPS and NtSAM1 were up-regulated in T0 generation of transgenic tobacco plants.The T1 plants of transgenic tobacco lines showed salt tolerance and resistance to osmotic stress during seed germination.The trans-Fra a1.02 gene line was more resistant than Fra a 1.01 and Fra a 1.03.