Isolation And Analysis Of Flanking Sequences Of Rice T-DNA Insertional Mutant And Characterization Of Osfbox Gene Which Controls The Semi-sterility Of Hemizygous Lines

Generating loss-of-function mutant is one of the most strait-forward and efficient way to study gene's bio-function.Agro-bacterium induced T-DNA integration not only disrupt the gene function of the insertion site,but also facilitate the gene cloning for the highly developed methods in isolating the sequences flanking the T-DNA. With a relatively random distribution pattern in rice genome,highly efficient transformation system,stable inheritance in progeny and simple genetic background, Agro-bacterium induced T-DNA transformation has been widely used in rice insertional mutant library establishment.The isolation of the flanking sequence tags(FST) is of great importance in the application of rice T-DNA insertional mutant library.On one hand,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 mechanism of Agro-bacterium induced T-DNA transformation;on the other hand,we can carry out reverse genetics study of the corresponding genes according to,the tagged gene information.In this study,I accounted the transformation positive ratio of our insertional population by PCR marker in T-DNA.By employing the TAIL-PCR method,30577 T-DNA FSTs were successfully isolated;T-DNA distribution pattern was analyzed, and several DNA physical property and base composition features of the insertion sites were revealed.With the tagged gene information,reverse genetics study was carried out here and a mutant which showed a semi-sterile phenotype in hemizygous lines was obtained.Cytological analysis of the mutant and functional analysis of the candidate gene Osfbox have been conducted.The main results are described as follows:1.The transformation positive ratio of 9024 transformants was checked by using GAL4/VP16 fragment in T-DNA as a PCR marker,7862(87.1%) showed to be PCR positive.2.Employing the TAIL-PCR method,I independently isolated 5100 FSTs,2609 (51.2%) out of which can be well mapped onto the dee genome.Combined with the FSTs isolated by colleagues,there are totally 30577 FSTs in our group, including 51.5%can be mapped.3.T-DNA 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(r= 0.74,P＜0.01).Within chromosomes,the insertions occurred more densely in the distal ends,and less densely in the centromeric regions and the insertion number is significantly correlated with the cDNA number in corresponding region(r=0.70,P＜0.01); T-DNA insertions strongly disfavored transposable element(TE)-related sequences(SR=—34.0) and favored genic sequences(SR=15.55).In rice genes, strong bias toward the 5' upstream(SR=12.8)and Y downstream regions(SR=10.3) of the genes were detected respectively,but the coding sequence region is observed to be disfavored(SR=—13.2).In the coding region,no insertional bias between intron and exon region was observed(χ~2 = 2.81,P = 0.09).4.According to the "molecular functions" of genes,T-DNA insertions bias among the various classes of functional genes were observed(χ~2=93.5,P＜0.000001): preferentially occurred in "Antioxidant"(SR =6.07)and "Catalytic"(SR=3.79), but preferentially not occurred in "Nutrient reservoir","Enzyme regulator", "Transcription regulator" and "Ligand binding and cartier"(SR between -2.1 to -3.8).In the other 5 gene classes,T-DNA showed a random distribution pattern.5.In ISNS bendability analysis 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 3 research groups.6.The analysis of ISNS GC content indicated that GC content is not a necessary factor for T-DNA insertion site.But The GC skew and TA skew calculation exhibited several features in both ISNS from our lab and 3 other groups:The GC and TA skews appeared to be inversely correlated with r＞-0.92;The two curves crossed each other at the insertion sites(point 0) at which both GC and TA skews were equal to 0;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, 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.7.In our T-DNA insertional population,T-DNA inserted in one loci as a "tandem repeats" was widely detected.The ratio of "direct repeat","5' end junction inverse repeat" and "3' end junction inverse repeat" can be up to 19.9%,10.5% and 25.4%respectively.40.9%are detected to have at least one tandem repeat. The introduction of vector backbone sequence into the rice genome are prominent as well,up to 49.6%and 29.7%of the transformants habor a backbone sequence in left and right border respectively.8.Reverse genetics study was carried out in a mutant which harboring a T-DNA insertion in the 3'UTR of an f-box gene in chromosome 6.The T-DNA hemizygous lines are semi-sterile,but both the T-DNA negative and homozygous lines are full fertile.This is similar to the semi-sterility of indica-japonica subspecies hybrids.The mutant phenotype is checked to be co-segregated with the T-DNA insertion.The candidate gene is named as Osfoox.9.The ID of Osfoox in TIGR is LOC_Os06g06050 with a length of 4441bp.The full-length eDNA information in KOME(AK065478) showed that:3723bp in length,containes 4 exons and 3 introns,encoding a 720AA protein.A potential F-box domain in 15-63 AA and Cysteine-eontaining LRR profile domain in 390-469AA were detected by Plantsp.10.Cytological analysis showed that tapetum degeneration retardation happened in tetrad stage,and the semi-sterility of T-DNA hemizygous lines are a consequence of both the undeveloped microspore and megaspore,which can pass the meiosis stage but are blocked in the following mitosis stage.11.By comparing the Osfbox gene sequence of 10 typical rice indica or japonica varieties,we found 18 base variations between the 2 subspecies,including 12 occurred in the 648bp upstream region from the transcription start site.In gene coding region,only 2 deletions were detected,resulting in a 3 tandem Glu and 1 Gly deletion respectively.12.Microarray based expression profile and RT-PCR data showed that Osfbox is a constitutively expressed gene.A higher expression level was detected in anther and pistil.In young panicle development stage 3-8,Osfbox expression level reaches the peak in stage 4 and then decreased.In situ hybridization result also showed that Osfbox is strongly expressed until tetrad formatted,but after the vacuolated pollen stage,no expression was detected in pollens.13.The Osfbox protein is specifically localized in cell nuclear.