Alhagi Bacilliform Virus And Functional Analysis Of Its ORF4 Promoter And Protein

Badnavirus,members of the plant virus family Caulimoviridae,possess an open-circular double-stranded DNA genome of ~7.2–9.2 kb with intergenic region(IR)and three typical ORFs(ORF1,ORF2 and ORF3).The ORF3 is the largest ORF of badnavirus,and encodes a ~216 kDa polyprotein containing conserved domains such as aspartic protease(AP),coat protein(CP),reverse transcriptase(RT)and ribonuclease H(RNaseH).Of the known badnaviruses to date,the badnaviruses have been found in tropical,subtropical and temperate plants,but have not yet been found in plants in the alpine desert region.Alhagi sparsifolia Shap.,Which belongs to the family Leguminosae,is a perennial subshrub.This plant species grows in desert climate and is naturally distributed in the Central Asian.Recently,using the A.sparsifolia primary roots,we constructed a high-resolution RNA sequencing(RNA-Seq)database,wherein two transcripts show homology with the sequences of the known badnaviruses.This suggests a putative badnavirus present within this species,and is therefore tentatively named Alhagi bacilliform virus(ABV).Herein,a series of studies were performed to determine the ABV genome as well as functional analysis of the ABV coding gene and regulatory sequence as follows.1)Cloning and sequence analysis of ABVUsing the total DNA prepared from the A.sparsifolia seedlings of Taklamakan as template,the ABV nucleotide sequence with length of 7068 nt was obtained by PCR cloning.Although sequence analysis did not find the complete IR and ORF1 sequences,the putative ORF2,ORF3 and ORF4 of the badnaviruses were identified,in particular,the typical conserved domains of badnavirus,including AP,RT-RNaseH and CP,were characterized in the ORF3-encoded protein.Comparison of the amino acid sequences of such conserved domains with the corresponding sequence of the known badnaviruses showed that ABV was highly homologous to badnaviruses.According to the phylogenetic analysis and the ICTV-defined badnaviruses criteria,we proposed that the putative ABV belongs to badnavirus and is a new species of this genus.Using southern blotting and inverse PCR,we demonstrated that the ABV-related sequences has been integrated into the genome of A.sparsifolia,and is present in the form of endogenous pararetroviruses(EPRV).The integrated ABV elements might undergo complex DNA recombination.A total of 12 A.sparsifolia samples collected from different locations(including Taklimakan)in the northwest China,The results showed that,of the tested samples,the RT-RNaseH conserved region was not amplified only from the samples of three neighboring areas,Wensu,Wushi and Alal,indicating that ABV is widely present in A.sparsifolia of the northwest China.2)Functional analysis of the ABV ORF4 promoter and its proteinUsing qRT-PCR,we showed that PEG treatment could induce m RNA expression of the ABV ORF4,thus indicating that the endogenous ABV element may be positively correlated with drought resistance of A.sparsifolia.The ABV ORF4 promoter(ORF4-P)was cloned to result in a plant expression vector pORF4-P-GUS.With the method of floral dip transformation,we generated the transgenic Arabidopsis thaliana plants harboring ORF4-P::gus.Through histochemical staining and GUS quantification,the transgenic plants were analyzed and the ORF4-P was identified as a drought-inducible promoter.The ABV ORF4 gene was cloned and transient expression analysis showed that the ORF4 protein was located in both cytoplasm and nucleus.The ORF4 gene was constructed in pCAMBIA1305 to result in a plant expression vector pCAM-ORF4,which was used to transform A.thaliana via floral dip transformation.Compared with wild-type A.thaliana,the resulting transgenic plants showed increased number of rosette leaf as well as delayed bolting.Under the treatment of 100 mM mannitol,the transgenic plants with ORF4 developed roots significantly longer than those of the wild type A.thaliana plants.In conclusion,a new badnavirus ABV was found in the typical alpine desert plant,A.sparsifolia,and present in the form of endogenous pararetroviruses.According to the preliminary functional analysis of the ABV ORF4 promoter and protein,A.sparsifolia may utilize endogenous ABV elements to regulate the gene expression or expression of proteins for enhancing their ability to resist abiotic stress such as drought.