Isolation And Analysis Of Flanking Sequences From A Rice T-DNA Insertional Mutant Library And Functional Study Of OsRNase Z Gene Which Controls Leaf Color In Rice

Mutants play an important role in rice functional genomic research.Generating loss-of-function mutant is one of the most straightforward and efficient way to study gene function.The isolation of the flanking sequence tags(FSTs) is crucial in the application of rice T-DNA insertional mutant library.First,based on the vast FST data,we can have a deep insight into the T-DNA distribution pattern in rice genome and then elucidate the Agrobacterium-mediated T-DNA transformation mechanism;on the other hand,we can carry out reverse genetics studies for the corresponding genes according to the tagged gene information.In this study,we examined the transformation positive ratio of our insertional population by checking the T-DNA insertional positivity.TAIL-PCR method was employed to isolate T-DNA FSTs.144,427 T-DNA FSTs and 22,432 Tosl7 FSTs were collected from worldwide databases for analyzing distribution pattern in the rice chromosomes in this study.We revealed the DNA physical property and base composition features of the insertion sites through analyzing T-DNA insertion site nearby sequences. We screened 1,994 T₁-generation T-DNA insertion lines and obtained some leaf color mutations in seeding stage.We obtained two albinos and study OsRNase Z gene function that interrupted by T-DNA.The detail results were summarized as follows:1.The transformation positive percentage of 15,535 transformants was checked by using GAL4/VP16 fragment in T-DNA as a PCR marker,13,080(88.9%) out of them showed to be PCR positive.2.Employing the TAIL-PCR method,I independently isolated total 7,333 FSTs, including 3,343 T-DNA or vector bone sequences,5,110 rice genome sequences. 2361 of 3215 FSTs can be well mapped into the rice genome(E-value＜10^-5).A total of 144,427 T-DNA FSTs and 22,432 Tosl7 FSTs in this study were collected from several major institutes in the world.3.T-DNA and Tosl7 insertions were biased towards large chromosomes,not only in the absolute number of insertions but also in the relative density,and the insertion density is highly correlated with the chromosome size.Within chromosomes,T-DNA insertions occurred more densely in the distal ends,and less densely in the centromeric regions and the insertion number is significantly correlated with the distances to centromere;Tosl7 insertions mainly distribute in the distal ends of chromosome,and a few distribute in centromeric region and the middle part of chromosome.The number of T-DNA insertions in the chromosome is significantly correlated with the cDNA number in corresponding region,which did not observed in Tosl7 insertions.4.The distribution of T-DNA and Tosl7 insertions in different regions of genome show that T-DNA and Tosl7 insertions strongly disfavored transposable element (TE)-related sequences and favored genic sequences;Tosl7 insertions strongly disfavored intergenic but T-DNA showed a random distribution in this region.In the genic region,T-DNA and Tosl7 prefer to insert in the coding sequences,but not the regulating sequences;particularly,T-DNA and Tosl 7 prefer exons,rather than introns, in the coding sequence.Compared with the intergenic region,T-DNA and Tosl7 insert favorably in the genic region.5.We also analyzed the function genes which inserted by T-DNA and Tosl7.In TE-related genes,the distributions of Tosl7 insertions were relatively concentrated, insert hot spot may be there,but T-DNA insertions were relatively uniform and no insert hot spot were observed.Tosl7 insertions prefer retrotransposon genes than transposon genes,T-DNA have no insertional bias between them.T-DNA and Tosl7 insertions bias among the various classes of functional genes were observed: preferentially occurred in "Motor","Signal transducer","Transporter" and "Transcription regulator",but preferentially not occurred in "Binding" and "Nutrient reservoir".In addition,T-DNA also biased "Structural molecule" and "Translation regulator",not biased insert "Catalytic",but Tosl7 showed a random distribution pattern in the 3 gene classes.6.In ISNS bendability analyze result,the random CK showed an irregular curve.But in our ISNS,elevated bendability was observed at positions from -200 to 200 bp and the bendability peak is symmetric in this region,with the highest points at both -10 and 10 bp from the insertion sites.There was an abrupt drop in the bendability value at the insertion sites.A similar result was also observed in ISNS from other research groups.7.The GC skew and TA skew calculation exhibited several features in both ISNS from our lab and other groups:The GC and TA skews appeared to be inversely correlated; The two curves crossed each other at the insertion sites(point 0) at which both GC and TA skews were equal to 0,indicating complete symmetry at this point;From the insertion site to -800 bp upstream,the GC skew was positive and reached a plateau in approximately the region from -300 to -100 bp,indicating a extremely uneven distribution of G and C in the strand analyzed,whereas the TA skew was negative and formed a valley in a similar position in approximately the region from -300 to -100 bp.The reverse was the case from the insertion sites to 800 bp downstream, whereas no such features were observed in the 2000 random sequences.8.We screened 1,994 T₁-generation T-DNA insertion lines,observed some color mutations in seeding stage.We obtained two albino lines 03Z11EV57 and 03Z11DH29 which co-segregation with T-DNA insertion.9.The albino line 03Z11EV57 which harbored a T-DNA insertion in the first intron of a gene in chromosome 5.The gene codes a protein kinase domail cotaining protein, which ID is LOC_Os05g47770 in TIGR,length 6,339 bp and CDS 2,853 bp contain 6 exons and 5 introns,encoding a 951 AA protein,but no supported by FLcDNA. 10.Line 03Z11DH29 was selected for further detailed study,which harbored a T-DNA insertion in the seventh exon of OsRNase Z in chromosome 9.The ID of OsRNase Z in TIGR is LOC_Os09g30466 with a length of 3066bp.KOME provided the full-length cDNA information(AK070747):1,352bp in length,containes 8 exons and 7 introns,encoding a 365AA protein,which has 97%similarity with RNase Z of Arabidopsis,so we named OsRNase Z.11.We investigated the albino mutant and normal green seedlings by transmission electron microscope,which showed that the chloroplast of albino mutants had changed remarkably compared with normal green seedlings:the number of chloroplast decreased,the shape distorted,the thylakoid membrane disappeared and prolamellar body formed instead of thylakoid.At the same time,we measured the chlorophyll contents,the result suggested that the pathway of chlorophyll biosynthesis have been blocked in albino.12.Microarray based expression profile and RT-PCR data showed that OsRNase Z has a close relationship with the synthesis of chlorophyll.The expression level of OsRNase Z was positively correlated with the chlorophyll content,In the tissues with high content chlorophyll,the expression of OsRNase Z was high;correspondingly,in the tissues with low content chlorophyll,the expression of OsRNase Z was low or otherwise couldn't be detected.13.RNA interference(RNAi) technique was used to suppress OsRNase Z expression in the rice variety Zhonghua 11.About 30 percent of transgenic seedlings appear albino phenotype.A part of these mutant plants were used to analyze the expression of OsRNase Z gene.RT-PCR result revealed that the expression of OsRNase Z was suppressed significantly in the chlorophyll mutant plants.14.The construction of P_Ubi:â–³OsRNase Z-GFP was transformed into Arabidopsis protoplast by PEG-mediated transformation method.Observation of protoplasts under a confocal microscope showed that OsRNase Z protein is exclusively localized in chloroplast.