Cloning And Transient Expression Analysis Of Sugarcane Bacilliform Virus Promoters

Sugarcane bacilliform viruses(SCBV;genus Badnavirus.family Caulimoviridae)that are one of the economically important pathogens present in most of the sugarcane-planting regions of the world,causing significant damage to the sugarcane production.The promoters derived from Badnavirus genome are widely used in crop genetic transformations.In this study,we try to clone the various promoters encoded by different SCBV genotypes which share highly genetic variation for supplying the suitable promoters for molecular breeding of transgenic crop.Various SCBV isolates collected from different geographic regions were cloned and sequenced and different SCBV genotypes were identified by phylogenetic analysis and sequence identity based on partial RT/RNase H seuqences at the amino acid(aa)and nucleotide(nt)levels.The activities and cis-acting elements of the SCBV promoters were also investigated.A 3.0 kb DNA fragment contained partial RT/RNase H and promoter region was cloned and sequenced from six SCBV isolates.Based on the RNase H sequences from the six SCBV isolates in this study and other ten reported isolates,sequence identity and pairwise sequence comparisons(PASC)revealed that the sixteen isolates shared 52?95%identity and 0.02?3.00 genetic distance at nt level and 50%?100%identity and 0?1.24 genetic distance at aa level.Phylogenetic analysis showed that six SCBV isolates in this studywere grouped into SCBV-H,SCBV-Q SCBV-P,SCBV-N,SCBV-Q and SCBV-R and two SCBV isolates from other countries were grouped into SCBV-E and SCBV-L.For investigating the differences of SCBV promoter strength driving the reported enhanced Yellow Fluorescent Protein(EYFP),eight plant expression plasmids contained individual SCBV promoters fused with EYFP were constructed.Transient expression levels of eight plasmids were verified in onion epidermis,and were then performed in sugarcane young leaf segments.Our results revealed significant variation of EYFP transient expression driven by the eight SCBV promoters in sugarcane leaf segments.EYFP expression level(Gray valuexpixels)ranged from 3.45×104 to 1.00×106 and EYFP-expressing foci counts ranged from 40?499.The highest EYFP expression level under SCBV-TX promoter was increased by 4.20 and 8.06-fold than that of Ubi and CaMV35S promoters,respectively.The EYFP expression level under SCBV-CZ70 promoter was increased by 2.10 and 4.02-fold than that of Ubi and CaMV35S promoters,respectively.EYFP-expressing foci under the SCBV-ROC27 and SCBMOV-MOR promoter were increased by 4.26-and 2.03-fold compared with the Ubi promoter,and wherein creased by 5.29-and 2.53-fold,compared with the CaMV35S promoter,respectively.Bioinformatic analyses revealed that a typical promoter structure TATA-box and various cis-acting elements,i.e.enhance the element of CAAT-box and CAT-box associated with the meristematic tissue-specific expression,were available within SCBV promoters.However,the credible functions of these cis-acting elements need to be verified in vivo.In conclusion,eight SCBV promoters were cloned from different viral genotypes and different promotional activities and cis-acting elements were observed here.The findings will supply with various alternative SCBV promoters in genetically modified crops.