Generation Of T-DNA Insertional Mutants And Isolation And Characterization Of A Homologous Chromosome Pairing Gene PAIR3 In Rice

Rice is an important and model cereal crop for functional genomics research.Mutant is one of the most strait-forward and efficient way to study gene function and then generation mutant library is a very important technical platform for large-scale, comprehensive and systematic gene research on rice whole genome.Agro-bacterium induced T-DNA transformation has been widely used in rice insertional mutant library establishment for a relatively random distribution pattern,highly efficient transformation system,stable inheritance in progeny and simple genetic background.In the study,we established rice mutant library by T-DNA,then we screened the T₁ mutant lines under normal growth conditions and indentified whether the mutant phenotype is co-segregation with the T-DNA homozygous insertion or not.Aim at the co-segregation lines,we then isolated and indentified the corresponding genes.The main study results were described as follows:1.Generating independent T-DNA insertional mutants as many as 30,579 with workers.2.Screening 9,193 mutant lines,I observed 15.4%(1417/9193) lines showed abnormal phenotype and obtained 3 co-segregated lines such as 03Z11UG73,03Z11UL95 and 03Z11UF78 with the mutant phenotype as albino,semi-dwarf and complete sterility respectively.More detail analysis was done with the mutant line 03Z11UF78 and to isolate and character the corresponsive gene PAIR3.3.The mutant segregated from 03Z11UF78 was designed as pair3-1.Compared with wild type,pair3-1 showed no differences during vegetative and floral,flower development stages;however,in the heading stage the mutant failed to release the pollen out and resulted completely sterility at mature stage.The mutant pollen failed to stain with KI-I₂ indicated that the mutant was male sterility.Pollinating the flowers from pair3-1 with wild-type pollen did not produce seed indicated the mutant was also female sterility.4.Co-segregation analysis in T₁ and T₂ generation of 03Z11UF78 showed that the mutant phenotype was co-segregated with the T-DNA homologous insertion.5.Another allele mutant 05Z11HE71,designed as pair3-2,was searched out from our mutant library.The mutant phenotype was also complete sterility and co-segragated with the T-DNA homologous insertion in T₁ generation. 6.The transgenic plants with double-chains RNAi vector caused lower fertility and twenty-one plants among sixty primary transformants were complete sterility. Semi-quantitative RT-PCR analysis showed that the fertility of RNAi transgenic plants was consistent with the mRNA level of PAIR3.7.RT-PCR and RACE indentified the structure of PAIR3.PAIR3 was located at LOC_Os10g26560 at TIGR(The Institute for Genomic Research) and the whole gene was 6,930bp(from transcript start site to stop site).The FLcDNA(full length cDNA) was 2,963bp and consisted of eleven exons,interrupted by ten introns and coded a putative protein with 844 aa(amino acid).BLASTN analysis showed that PAIR3 was a novel gene with no homolog according to the current datebases.Online network protein sequence analysis predicted thatα-Helical coiled-coil motifs were located near the C terminus of the peptide of PAIR3.8.Plastic sections analysis with anthers development showed:Compared with the wild-type anther development,pair3 parietal layer cells developments were normal. The microspore mother cells and the young microspores were no obvious differences; however,all the mutant pollen was severely shrivelled and had no nutrients in the cytoplasm at the pollen mitosis stage and the mature pollen stage.9.Paraffin sections analysis with embryo sac development showed:Compared with the wild-type embryo sac development,pair3 showed the first defects at the functional megaspore formation stage.It was the megaspore nearest the chalazal did not enlarge to develop into functional megaspore as WT did but degenerated following the three non-functional megaspores near the micropyle.Finally in the mutant ovule,there was no embryo sac structure formation instead by some remnants of nucellus.10.Chromosome behavior in pollen meiosis was characterized through directly crushing and staining meiocytes.The results showed:There were 24 univalents at diakinesis in pair3 and the univalents were random segregated subsequently.We conclude that the PAIR3 gene affects homologous chromosome pairing and normal chromosome segregation in both male and female meiocytes,leading to complete male and female sterility in pair3.11.Semi-quantitative PCR analysis showed:The PAIR3 gene was expressed in panicles but not at all or very low level in vegetative organs such as stem,leaf and leaf sheath, in agreement with our finding that the mutant phenotype was observed in the reproductive organs with complete sterility but not in the vegetative organs.The PAIR3 transcripts were not detectable in the young panicles of pair3-1 and pair3-2 mutants,confirming their status as loss-of-function mutant.The PAIR3 gene expression was detected in all kinds of panicle developmental stages according to different panicle lengths with a pattem which was first increased from panicle initiates,reached a peak in panicles(4.5cm to 11 cm) in which some male meiocytes enter meiosis and then decreased until to a very low level in heading panicles.12.In situ analysis showed:A little PAIR3 signals were first detected in the sporogenous cells and the primordial of the parietal layers at the premeiosis stage.Subsequently, the signals were detected exclusively in the meiocytes,dyads,tetrads,and the early young microspores,but were undetectable in the parietal cells of the anther.The PAIR3 signal was absent from vacuolated pollen,mitotic pollen,and mature pollen, but a strong signal was detected in the degenerating tapetum.A little PAIR3 signals were first detected in the archesporial cells and the primordial of the ovule at the early stage.Subsequently,the signals were detected exclusively in the whole ovule tissues including the integument,nucellus,funiculus,embryo sac,and the inner layer cells of the carpel wall during embryo sac development.However the signal was most marked during or just after the megasporocyte meiosis stage.The PAIR3 gene was preferentially expressed in the meiocytes especially during the female and male meiosis stages indicated that the PAIR3 protein may work in the meiocytes.The expression pattern of PAIR3 corresponded well with the mutant phenotype.13.To date five meiotic gene(PAIR1,PAIR2,MEL1,OsDMC1,OsRAD21-4) had been reported in rice.All the five genes showed no different expression in WT and pair3-1 indicated PAIR3 may not work on regulating these genes expression.