Isolation And Analysis Of Flanking Sequences From A Rice T-DNA Insertion Mutant Population And Cloning And Functional Analysis Of OsaTRZ2

Rice(Oryza sativa) is one of the most consumed staple food crops. With the completion of rice genome sequencing, functional genomics aiming at decipherment of function for every rice gene rises and develops rapidly. Large-scale mutant libraries constitute an important technique platform for functional genomic research in rice, and insertion mutant populations are one of the major parts of the platform. Because the mutant phenotype can be bridged to the genotype in insertion mutants conveniently through isolation of flanking sequence tags (FSTs), FST isolation becomes a key step to take advantages of insertion mutant populations. Functional tRNA molecules play essential roles in protein synthesis and other biological processes in all organisms. They are firstly transcribed as precursors, and then, become matured after undergoing a serial of processions. Removal of 3’trailer from tRNA precursors is an early step of the processions; however, the molecular mechanism beneath the step is not yet well understood in plants.In this study, large amounts of FSTs and mutants were isolated and screened out, respectively, using T-DNA insertion lines from Rice Mutant Database (RMD) as materials. Then, in combination with the chip data from Nipponbare callus at the stage of 6 hours after infection, we collected 72,090 FSTs to characterize the features of distribution and sequence constitution of T-DNA and Tos17 insertion sites and to speculate the factors effecting insertion site recoveries. In addition, OsaTRZ2, which encodes a tRNase Z that is responsible for tRNA 3’processing in chloroplasts, was also cloned and identified. The detailed results of this study are listed below:1.2,037 To transformants were checked by PCR, and 85.7% of the plants were T-DNA positive.2.5,711 T-DNA and Tos17 FSTs which could be well mapped to rice genome (E-value <10-5), were isolated from 7,548 transgenic lines with TAIL-PCR, adapter-ligated PCR and inverse PCR.3. Analysis of the distribution of T-DNA and Tosl 7 insertion sites at various levels of the genome hierarchy showed that the T-DNA and Tos17 insertion sites respectively displayed similar distributions in Zhonghua 11 and Nipponbare, suggesting that different japonica cultivars share the same factors effecting insertion site recoveries.4. Through comparing distributions of transcriptional activity levels and the insertion sites, our data revealed that distribution of the T-DNA insertion sites was highly positively correlated with transcriptional activity levels within rice genome, and especially within genic regions. However, a half of the main hot spots of the Tos17 insertion sites fell into chromatin regions with relatively low transcriptional activity levels, and the distribution of the Tos17 insertion sites was highly negatively correlated with transcriptional activity levels within genic regions. These data suggest that the transcriptional activity is an important factor for insertion site recoveries.5. Analysis of sequence constitution of the insertion-site nearby sequences showed that AT contents increased in the region 3’downstream of both the T-DNA and Tos17 insertion sites, and the increased extents correlated negatively to the numbers of insertion sites. The result was putatively caused by the preference of PCR technique to AT-rich sequences.6. Analysis of sequence constitution of the insertion sites revealed that G frequency declined about 2% consecutively within 5 base pairs (bp) 5’upstream of the T-DNA left-end insertion sites, whereas it increased about 2%-7% at the positions from 3 bp 5’upstream to 1 bp 3’downstream of the T-DNA right-end insertion sites. Such distinct but weak base biases appearing at T-DNA left-and right-end insertion sites agree with the microhomologous recombination model of T-DNA integration. Strong base biases appeared at the Tos17 right-end insertion sites, from which a consensus sequence, ANGTTGNNNCAACNT, could be deduced, reflecting the mechanism of Tos17 integration.7. Through phenotypic screening in fields,105 lines with abnormal panicle and 149 lines with abnormal leaf color, including 4 lines in which T-DNA cosegregated with the mutant phenotype, were obtained from 11,469 T1 lines from RMD.8. One of the cosegregated lines, OsaTRZ2, was an albino line, and the osatrz2 mutants survived less than 4 weeks after germination. Pigment and histological analyses showed that osatrz2 leaves contained no chlorophyll but undeveloped plastids that were arrested at an early stage of transition from proplastids to chloroplasts, and the number and size of the plastids in callus cells was substantially decreased.9. RT-PCR and cosegregation test showed that the truncation of OsaTRZ2 transcript by T-DNA insertion into the seventh exon of the gene is the causal reason resulting in the albino phenotype. OsaTRZ2 was annotated as a tRNase Z with 365 amino acids in length.10. Evidences from genetic complementation and RNA interference analyses confirmed that disruption of OsaTRZ2 was responsible for the mutant phenotype.11. OsaTRZ2 is constitutively expressed in rice tissues, but is preferentially expressed in green tissues and meristem tissues, such as leaves, sheathes, and calli.12. OsaTRZ2 was subcellularly localized in chloroplasts.13. In vitro and in vivo assays demonstrated that OsaTRZ2 possess tRNA 3’-end processing activity.14. Inactivation of OsaTRZ2 resulted in severe suppression and moderate activation of transcription of plastid-encoded and nucleus-encoded RNApolymerases, respectively.