RPA文献解读.ppt

复制蛋白A（RPA1a）是水稻减数分裂和体 细胞DNA修复所必需的，但不是DNA复制和 同源重组所必需 华中农业大学作物遗传改良国家重点实验室和国家植物基 因研究中心（武汉）摘要Replication protein A (RPA), a highly conserved single-stranded DNA-binding protein in eukaryotes, is a stable complex comprising three subunits termed RPA1, RPA2, and RPA3. RPA is required for multiple processes in DNA metabolism such as replication, repair, and homologous recombination in yeast (Saccharomyces cerevisiae) and human. Most eukaryotic organisms, including fungi, insects, and vertebrates, have only a single RPA gene that encodes each RPA subunit. Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), however, possess multiple copies of an RPA gene. Rice has three paralogs each of RPA1 and RPA2, and one for RPA3. Previous studies have established their biochemical interactions in vitro and in vivo, but little is known about their exact function in rice. We examined the function of OsRPA1a in rice using a T-DNA insertional mutant. The osrpa1a mutants had a normal phenotype during vegetative growth but were sterile at the reproductive stage. Cytological examination confirmed that no embryo sac formed in female meiocytes and that abnormal chromosomal fragmentation occurred in male meiocytes after anaphase I. Compared with wild type, the osrpa1a mutant showed no visible defects in mitosis and chromosome pairing and synapsis during meiosis. In addition, the osrpa1a mutant was hypersensitive to ultraviolet-C irradiation and the DNA-damaging agents mitomycin C and methyl methanesulfonate. Thus, our data suggest that OsRPA1a plays an essential role in DNA repair but may not participate in, or at least is dispensable for, DNA replication and homologous recombination in rice.结果1、osrpa1a突变体的观察 2、OsRPA1a基因的分离和描述 3、遗传互补和通过RNA干扰检测OsRPA1a 4、 osrpa1a中雌配子体发育在大孢子母细胞减数分 裂时中断 5、 osrpa1a中花粉母细胞减数分裂受影响 6、 osrpa1a对DNA诱变剂高度敏感 7、 osrpa1a突变体中有丝分裂不受影响Figure 1. Phenotypic characterization of the osrpa1a mutant. A, A wild-type plant (left) and osrpa1a mutant (right) at maturity. B and C, Spikelets from wild-type (B) and osrpa1a mutant (C) plants are morphologically identical. D and E, Viability of mature pollen grains of wild type (D) and the osrpa1a mutant (E) as assessed by I2-KI staining. Bars = 100 mm. F and G, Structure of mature embryo sacs from wild type (F) and osrpa1a mutant (G). AN, Antipodals; PN, polar nucleus; SY, synergids.Figure 2. Analysis of T-DNA tagging of OsRPA1a and the expression of OsRPA1a. A, Structure of OsRPA1a and the position of the T-DNA insertion (inverted filled triangle). The intron (line), open reading frame (black box), and 5# and 3# untranslated regions (white boxes) are indicated. The positions of the primers (RTL and RTR used in B and C; O1, O2, and LBT3 used in D) are indicated by arrows. B, Spatial and temporal expression pattern of OsRPA1a in leaf (L), sheath (Sh), stem (S), root (R) from a booting plant and panicles at various developmental stages according to their length (P1–P9). P1: approximately 0.5 cm; P2: approximately 1.0 cm; P3: approximately 2.0 cm; P4: approximately 3.5 cm; P5: approximately 4.5 cm; P6: approximately 11 cm; P7: approximately 16.5 cm; P8: approximately 19 cm; P9: approximately 22 cm. C, RT-PCR analysis of OsRPA1a expression in panicles from wild type (WT) and osrpa1a mutant (M). Actin is used as the reference for the mRNA level in B and C. D, Genotyping of the OsRPA1a T-DNA-tagging progeny.Figure 3. Transgenic complementation of the osrpa1a mutants. A, Fertility of the 17 complemented osrpa1a lines of the T0 generation. B, Fertility of the progenies of a single-copy insertion complemented osrpa1a line of the T1 generation. A wild-type plant was used as control (CK). The average fertility (mean 6 SE) was based on three panicles selected randomly from each plant in A and B. C, Genotyping of the plants used to obtain the data shown in B.Figure 4. Formation and development of the embryo sac in wild-type (A–J) and osrpa1a mutants (K–O). A, Archesporial cell (arrowhead) formation stage. B, Megasporocyte (arrowhead) formation stage. C, Megasporocyte meiosis stage. The megasporocyte undergoes two meiotic nuclear divisions to form a linear tetrad of megaspores (arrowheads). D, Functional megaspore formation stage. Three megaspores nearest the micropyle (arrowheads) degenerate to preserve the chalazal megaspore as the functional megaspore. E, Mononucleate embryo sac formation stage. F to H, Embryo sac mitosis stage. The functional megaspore undergoes three mitotic divisions to yield a two-nucleate (arrowheads) embryo sac (F), four-nucleate (arrowheads) embryo sac (G), and eight-nucleate embryo sac (H), respectively. I, Eight-nucleate embryo sac development stage. J, Mature embryo sac stage. The polar nucleus (PN), synergids (SY), and antipodals (AN) are indicated by arrowheads.表皮下的珠心组织细胞→孢原细胞→大孢子母细胞→细胞减数分裂→4个大孢子→3个大孢子退化， 只有一个形成有功能的大孢子→3次有丝分裂→1个卵细胞、2个极核、2个助细胞、3个反足细胞（ 七胞八核）K, Megasporocyte formation stage of osrpa1a mutant. In th。