Expression And Function Analysis Of Rice PPR Protein Gene CISC

Pentatricopeptide repeats(PPR)family is one of the largest gene families in plant,which belongs to nuclear genes.Previously our lab fine mapped the cold-induced seedling chlorosis(CISC)gene and determined the gene is a PPR gene by complement test.This study analyzed the expression of CISC and related plastid genes by quantitative RT-PCR.In this study we made subcellular localization of CISC protein,analysis of chloroplastic rpoB RNA editing site and sequence analysis of CISC gene.The main results are as follows;1.CISC protein is located in chloroplast.We first transferred pCAMBIA1302-CISC construct into Agrobacterium GV3101,which was then introduced into tobacco leaves.We found GFP fluorescence of pCAMBIA1302-CISC and the red fluorescence of chlorophyll overlapped almost completely in tobacco cells by a laser scanning confocal microscope.According to the results we concluded that CISC protein is located in the chloroplast.2.The expression of CISC.We performed quantitative RT-PCR using Dular and Nipponbare seedlings grown under the conditions of 26 ? and 19?.We compared the expression of Nipponbare CISC in the shoot and root at 26? and 19? and found both were reduced while that of Dular increased.Furthermore,at 26 ?,the expression of Dular cisc was lower than that of Nipponbare.At 19? the expression of Dular cisc compared to 26??increased instead of reducing.A quantitative RT-PCR was performed at booting stage using Dular and Nipponbare at 26?.The results indicated that in Nipponbare,the expression of CISC in flag leaf and mature leaf was higher than that in other parts.In flag leaf,flag sheath and mature leaf of Dular,the expression of cisc was higher than that in other parts.Moreover,in flag leaf,flag sheath and mature leaf the expression of Dular cisc was higher than that of Nipponbare,while in mature sheath the expression of Dular cisc was lower than that of Nipponbare.3.The expression of related plastid genes.We examined the transcription levels of other genes associated with either chlorophyll biosynthesis,chloroplast development,or photosynthesis at temperatures of 26? and 19? in Dular and Nipponbare.Eight genes were selected,including those encoding two reaction center polypeptides(psaA and psbA),Rubisco large subunit(rbcL),rRNA genes(rrn16S and rrn23S),PEP(plastid encoded RNA polymerase)genes(rpoB and rpoC1),and nuclear encoded gene(CAB1).For the expression of related plastid genes in Nipponbare at 26 ? and 19? as the background,the results indicated that,compared with 26 ?,at 19?,the expression of Dular class ? genes(rbcL,psaA and psbA)decreased(psaA and rbcL)or unchanged(psbA)?the expression of Dular class ? genes reduced(rrn16S)or essentially unchanged(rrn23S),the expression of Dular class ? genes(rpoB and rpoCl)went up.4.Dular is defective in RNA editing of rpoB transcript.Total RNA of leaf was extracted at seedling stage at the 26? and 19? in Dular and Nipponbare.It was then reversely transcripted to cDNA.We amplified the cDNA with specific designed primers.From the sequenced results,we observed that rpoB at 156,182 and 187 codons appeared editing of C to U which are not complete.For Nipponbare,the editing efficiency of 19? was higher than that of 26?.However at both 26? and 19?,the editing efficiency was almost the same in rpoB of Dular.At 26 ? the editing efficiency of Dular was higher than that of Nipponbare,while at 19? the editing efficiency of Dular was lower than that of Nipponbare.5.Sequence analysis of CISC gene 8 bp deletion.We first undertook the cold stress test,which was followed by the extraction of DNA at seedling stage.We designed a pair of specific primer to amplify the target segment.The results showed that some rice varieties seedling had chlorosis at 19? but no 8-bp deletion appeared.