Preliminary Functional Analysis Of A Cold-induced Seedling Chlorosis Gene Cisc(t) In Rice

PPR (pentatricopeptide repeats) gene family, one of the largest gene families discovered inplants, is characterized by tandem arrays of pentatricopeptide repeats, which play essential rolesin mitochondria or chloroplasts, probably via binding to organellar transcripts. Previous studiesof gene fine mapping conducted in our laboratory indicated that a PPR gene located on ricechromosome9is likely to be the candidate of gene CISC(t), which may cause the phenotype ofcold-induced seedling chlorosis when it is mutated. This study aimed to confirm the function ofthe CISC(t) via genetic complement test and overexpression test, to reveal the expression patternof CISC(t) by GUS staining of tissues and semi-quantitative RT-PCR, and to understand thetarget places of CISC(t) protein via subcellular localization experiment. The main results are asfollows.1. Bioinformatics analysis Indicated that the CISC(t) candidate gene encodes a putativepolypeptide of585amino acids, which contains many characteristic PPR motifs. Besides, thisprotein also contains three extra conserved motifs E, E+and DYW in the C terminal.2. To confirm its subcellular localization, a CISC(t) fused with GFP driven by the CaMV35Spromoter was introduced into the protoplasts of Arabidopsis thaliana by PEG method. Stabletransgenic protoplasts were then examined with confocal laser microscopy. The result showedthat CISC(t) protein is localized in the chloroplast.3. The full-length sequence of CISC(t) candidate gene from japonica variety Nipponbare wasintroduced into Dular, an indica variety showing the mutant phenotype of cold-induced seedlingchlorosis, by Agrobacterium tumefaciens-mediated transformation. The results showed that thetransgenic seedlings did not show albino phenotype under low temperature, suggesting that thePPR gene from Nipponbare is really the CISC(t) gene, which can complement Dular seedlingphenotype at19℃4. Semi-quantitative RT-PCR analysis using Nipponbar and Dular seedlings at27℃and19℃showed that the expression of CISC(t) gene is weak in leaf at27℃but significantly increased at19℃, suggesting that CISC(t) expression is related to temperature. GUS expression test drivenby CISC(t) promoter indicated that CISC(t) gene is expressed in phyllula, leaf segment andsheath base, but not in leaf, stem, root and lemma/palea. Interestingly, CISC(t) gene is temporallyand spatially expressed in the flower.5. Total RNA was extracted from14-day-old plants. A total of21published RNA editing siteswere amplified and sequenced by specific primers. The results suggested that CISC(t) geneprobably participates in the editing of chloroplast transcript RpoB RNA.