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J Integr Plant Biol, 2003, 45 (11): 1319-1328, Research Article
Cloning of the APRT Gene from Rice and Analysis of Its Association with TGMS
LI Jun*, LIANG Cun-Yang*, YANG Ji-Liang, XING Quan-Hua, YANG Dian-Er, DENG Qi-Yun, WENG Man-Li, WANG Bin**
doi:
Abstract

Adenine phosphoribosyltransferase (APRT) is the major enzyme that converts adenine into adenosine-3'-phosphate (AMP). APRT-deficient mutant caused by APRT gene mutation results in the male sterility in Arabidopsis thaliana L. In order to confirm the existence of rice APRT gene and to investigate its association with thermo-sensitive genic male sterile (TGMS) phenotype of rice, a APRT gene was identified from BLAST search of the rice genome database using APRT gene 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 APRT gene 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 APRT gene 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 APRT in young panicles of “Annong S-1” in high temperature conditions is perhaps related to the TGMS of “Annong S-1”.

水稻基因A P R T 的克隆及其与温敏核雄性不育的关系
李 军  梁春阳 杨继良  邢全华 杨典洱 邓启云 翁曼丽  王 斌

(1.中国科学院遗传与发育生物学研究所,北京 100101; 2.湖南杂交水稻中心,长沙 410125)

摘要: 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 sativasubsp.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。
关键词: APRT 基因;温敏核雄性不育(TGMS);水稻
通讯作者。Tel: 010-64889378; E-mail: <bwang@genetics.ac.cn>。

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