Development Of Transgenic Rice Lines With Resistance To Sheath Blight Using Dual Gene Expression Cassette

Rice sheath blight(SB),caused by Rhizoctonia solani,is one of the three most serious diseases in rice.To date,no rice germplasm has been found with complete and high resistance to this disease,which leads to the difficulty on developing resistant variety through conventional breeding strategy.In our previous studies,we found that overexpression of either GAFP2(Gastrodia Antifungal Protein 2)or OsPGIPl(polygalacturonases inhibiting protein 1)gene could significantly enhance rice resistance to SB disease.This allowed us to speculate that pyramiding the two genes and optimizing their expression pattern are able to obtain transgenic rice lines with better resistance as well as gene expression specificity.Consequently,in the present study,we adopted two rice green-tissue expression promotors PrbcS(from rice ribulose-1,5-bisphosphate carboxylase small subunit gene)and PcyFBpase(from rice cytosolic fiuctose-1,6-bisphosphatase gene),respectively,to direct the above two genes express for developing a dual-gene expression cassete ’PrbcS::OsPGIP1-PcyFBpase::GAFP2’.In addition,double T-DNA vector system was used in rice transformation for generating marker-free transgenic rice lines.Mainly results were as follows:1,total of 131 PCR-positive transgenic rice plants were obtained in the background of rice varieties WYJ24 and WLJ1.Based on the preliminary evaluation of agronomical traits,we selected 53 WLJ1 transgenic lines and 61 WYJ24 lines for further analysis.2,through Southern blot analysis,we found that most of transgenic lines contain more than three copies of dual-gene,and lines possess single copy and two copies were 15 and 9,respectively.We obtained 4 marker-free lines with single copy of dual-gene,and 4 lines with both single copy of dual-gene and marker gene.In most transgenic lines,the expression levels of target gene were significantly higher than that in the control,and showed no correlation with the copy number of target gene.The dual gene was highly expressed in leaf,young panicle,leaf sheath and stem,but weak or no expression in root and seed.The GAFP2 protein was detected highly expressed in single copy transgenic lines by Western blotting.These results indicate that the dual gene has been successfully integrated into rice genome and mainly expressed in rice green tissue.3,the resistance performance of 21 transgenic lines with single and dual copy of target gene were evaluated with artificial inoculation of SB fungus.In field,we found most transgenic lines showed significantly lower disease scores than that of the control.The disease scores differed between the transgenic lines and control are ranged from 0.3 to 2.8 with an average of 1.6(0-9 disease rating standard).Resistance of some lines were also evaluated using detached-stem method in growth chamber,which showed that most transgenic lines had significantly shorter lesion length than that of the control.All these results indicate that introduction of dual gene is able to significantly improve rice resistance to SB disease.4,some agronomical traits plus heading data were measured between 21 transgenic lines and controls,which showed that most lines displayed difference on one or more traits compared to the control but without consistent variation among different transgenic lines.This indicates that the trait variation of most lines are from tissue culture but not from the dual gene.5,the flanking sequences of dual gene of 13 single-copy transgenic lines were analyzed by reverse PCR strategy.Flanking sequences of the dual gene in 5 transgenic lines were amplified,which located on chromosome 1,3,5,9 and 11,respectively.By using specific PCR primers,two flanking sequences from rice line Y1-3 and Y10-1 were confirmed that located on 10.62Mb of chromosome 1 and 4.27 Mb of chromosome 3,respectively.Y1-3 is a marker-free and single-copy transgenic line,which showed over 2 disease scores lower than that of the control in field.This line has a high potential in rice breeding against SB disease,which has been applied for environmental release experiment of transgenic crops.