CAJ | 학술논문

在拟南芥中腺嘌呤磷酸核糖转移酶基因(APRT)突变导致植株雄性不育.本文首次报道从水稻(Oryza sativa subsp.indica)中克隆了基因APRT(GenBank登录号AY238894),并将其定位于水稻第4染色体的一个BAC克隆(AL606604)的58 000 bp至63 000 bp区域.该基因长4 220 bp(起始密码子至终止密码子),含7个外显子、6个内含子,编码的APRT蛋白长212个氨基酸残基,与其他物种来源的APRT序列存在很高的同源性.与大麦、小麦、拟南芥1型及其2型的该蛋白同源性分别为54.9%、54.9%、49.6%和59.5%.经保守结构域搜索发现该蛋白中存在APRT催化结构域.从DNA、mRNA两个水平分析了该基因与水稻温敏核雄性不育(TGMS)的关系,结果表明:受温度诱导,水稻"安农S-1"APRT基因的表达变化可能与温敏核雄性不育表现型具相关性.
재의남개중선표령린산핵당전이매기인(APRT)돌변도치식주웅성불육.본문수차보도종수도(Oryza sativa subsp.indica)중극륭료기인APRT(GenBank등록호AY238894),병장기정위우수도제4염색체적일개BAC극륭(AL606604)적58 000 bp지63 000 bp구역.해기인장4 220 bp(기시밀마자지종지밀마자),함7개외현자、6개내함자,편마적APRT단백장212개안기산잔기,여기타물충래원적APRT서렬존재흔고적동원성.여대맥、소맥、의남개1형급기2형적해단백동원성분별위54.9%、54.9%、49.6%화59.5%.경보수결구역수색발현해단백중존재APRT최화결구역.종DNA、mRNA량개수평분석료해기인여수도온민핵웅성불육(TGMS)적관계,결과표명:수온도유도,수도"안농S-1"APRT기인적표체변화가능여온민핵웅성불육표현형구상관성.
Adenine phosphoribosyltransferase (APRT) is the major enzyme that converts adenine into adenosine-3'-phosphate (AMP). APRT-deficient mutant caused by APRTgene mutation results in the male sterility in Arabidopsis thaliana L. In order to confirm the existence of rice APRTgene and to investigate its association with thermo-sensitive genic male sterile (TGMS) phenotype of rice, a APRTgene was identified from BLAST search of the rice genome database using APRTgene sequences from other plant species as probes. Further, the gene was cloned from rice and named APRT(GenBank accession number AY238894) using the combination of bioinformatic and experimental approaches. The rice APRT was located in the 56 000 bp to 63 000 bp region of a rice bacterial artificial chromosome (BAC) clone (AL606604) on chromosome 4 and was deduced by software from the positive DNA clone. Its cDNA was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) using primers designed according to the sequence of the putative gene. The full-length cDNA was obtained by rapid amplification of cDNA ends (RACE) procedure and was sequenced. Open reading frame (ORF) analysis indicated that the rice APRTgene encodes a peptide of 212 amino acid residues, including seven exons and six introns. Using reverse position specific BLAST (RPS-BLAST), the APRT domain was identified in the polypeptide. The homology comparison demonstrated that the polypeptide exhibits 54.9%, 54.9%, 49.6% and 59.5% identity with that from Hordeum vulgare, Ttriticum aestivum, and A. thaliana (APRT types 1 and 2), respectively.Comparing the sequence of APRTgene from TGMS mutant lines "Annong S-1" (Oryza sativa subsp. indica )with that from its corresponding wild type "Annong F" (Oryza sativa. subsp.indica ), we found that there are five single nucleotid polymorphism (SNP) sites in the gene of "Annong S-1", which locate mainly in the second intron. However, the result of cDNA sequencing showed that these SNP sites do not damage the successful splicing of intron 2. Qualitative RT-PCR and Northern blot indicated that the gene tran-scription in the "Annong S-1" young panicles that were verified to be the thermo-sensitive organ at the early stage of pollen fertility alternation is down-regulated by high temperature stress (28℃), which is the critical temperature causing "Annong S-1" fertility conversion. These results revealed that the change of expression pattern of APRTin young panicles of "Annong S-1" in high temperature conditions is perhaps related to the TGMS of "Annong S-1".