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...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 V), which is the critical temperature causing 'Annong S-1' fertility conversion. These results revealed that the change of expression pattern of APRT in young particles of 'Annong S-1' in high temperature conditions is perhaps related to the TGMS of 'Annong S-1'.展开更多
文摘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 V), which is the critical temperature causing 'Annong S-1' fertility conversion. These results revealed that the change of expression pattern of APRT in young particles of 'Annong S-1' in high temperature conditions is perhaps related to the TGMS of 'Annong S-1'.
