Isolation and characterization of an aluminum tolerance gene in Rice

Abstract

Aluminum (Al) toxicity is the major limiting factor of crop production in acid soil. Aluminum tolerance in rice is considered as a quantitative trait controlled by many genes. The objectives of this research were to isolate, clone, characterize the aluminum tolerance gene, and to develope codominant marker for rice B11 gene. Plant materials used in this research were four rice genotypes namely Grogol, Hawara Bunar, IR64, and Krowal, and the F2 segregating rice population derived from a cross between Al-sensitive rice genotype IR64 and Altolerant rice genotype Hawara Bunar. A rice Al tolerance gene was isolated based on a combination between rye-rice syntenic relationship and RT-PCR (Reverse Transcription-Polymerase Chain Reaction) approaches. The cDNA of rice Al tolerance gene was synthesized from mRNA isolated from Hawara Bunar after being stressed under 15 ppm Al at pH 4.0 for 24 hours. The Al tolerance gene was characterized by sequencing, bioinformatically analyzing, and introducing it to tobacco plant. The inheritance pattern of rice Al tolerance gene was analyzed by developing the CAPS (Cleaved Amplified Polymorphism Sequence) codominant marker of rice Al tolerance gene through a series of PCR, sequencing, and restriction enzyme analysis of the PCR fragment from IR64 and Hawara Bunar, followed by testing the marker in F2 rice population. This research has succesfully cloned an Al tolerance gene from Indonesian local rice genotype Hawara Bunar, called B11 gene. The B11 gene expression was induced by Al with higher expression level in Hawara Bunar than that of IR64. Transgenic tobacco plants carrying the gene were more tolerant to Al stress than that of nontransgenic tobacco plants. The B11 protein was similar to bacterial ribosomal L32 proteins and was predicted as a transcription factor with bZIP domain and C2H2-zinc finger like motif. Comparison between the B11 gene sequences of the IR64 and Hawara Bunar showed 8 SNPs (Single Nucleotide Polymorphisms). One of the SNPs located at nucleotide 668 causing AluI restriction site based polymorphism between IR64 and Hawara Bunar, which was then used as a basis of B11-CAPS codominant marker development. The marker segregation followed a single gene inheritance pattern. The marker might be used for MAS (Masrker Assisted Selection) in rice breeding programs to obtain Al-tolerant lines.