Molecular Identification Of Ds Insertion Sites And Ds-tagged Genes In A Rice Mutant With White Palea And Lemma

Rice functional genomics research is a hot topic in the current research of rice molecular biology. Ac/Ds transposable elements are widely used in the construction of mutant library for the survey of functional genomics. In this study, A Ds-tagged rice mutant with white palea and lemma was isolated from Japonica rice(Oryza sativa L. var. Japonica cv. Dongjin) via the Ac/Ds transposition system. The genomic DNA sequences flanking the Ds elements were isolated by TAIL-PCR. The Ds insertion sites on chromosomes were identified and the structures and functions of Ds-tagged genes were predicted by using bioinformatic tools. Then, we isolated c DNA fragments derived from the 3’end of Ds-tagged genes by using RACE technology, aiming to analysis the structures and functions of the Ds-tagged genes. The research efforts were focused on aspects as follows:1. The morphological and physiological phenotypes of the mutant line were preliminarily examined. The lemma and pelea and small stems of the rice mutant were white at the heading stage, while other parts of the organization remained green. The data of the plant height, the length of the first leave, the width of the first leave were collected and analyzed by using SPSS software. The results showed that the plant height of the mutant line was significantly lower than that in wild-type rice. The length of the first leave of the mutant line was significantly higher than that in wild-type rice.2. The Ds flanking sequences were isolated from the mutant by using TAIL-PCR technique. Bioinformatics preliminary analysis indicated that Ds inserted in the rice mutant on chromosome 1, chromosome 2 and chromosome 3.The Ds tagged gene on chromosome 1(gene: OSJNOa234E14.3, protein ID: BAD69439.1) has six exons and five introns, with a Ds insertion in sixth exon. The gene encodes 258 amino acids with a CBS domain protein. The protein has a conserved tandem repeat CBS domain.The Ds tagged gene on chromosome 2(gene: OJ1058_F07.11, protein ID: BAD19323.1) has two exons and one intron and has a Ds insertion in the non-coding region of 3’end. The gene encodes 237 amino acids while presumably encoding product is an unknown protein.The Ds tagged gene on chromosome 3(gene: OSJNBa0054H04.28,protein ID: AAR89005.1) has four exons and three introns and has a Ds insertion in the fourth exon. Ds excised from the original integration site and left 8 bp of "CATCATGA" footprint sequence. The gene encoding 549 amino acids with a presumably product of oxidoreductase contains a conserved DIOX_N domain and 2OG-Fe(II) oxidoreductase domain. A homology search by using SWISS-MODEL homology software revealed a Leucoanthocyanidin dioxygenase in maize.3. RACE technology was used to validate the impact of Ds insertion to the structures and functions of the Ds-tagged genes. Bioinformatics analysis of the results showed that, compared with the wild type rice, new transcription products had been generate in the gene which encodes a CBS containing protein and in the gene which encodes an oxidoreductase. Both of the genes’ structural were changed.The length of the third exon of the gene which encode a CBS domain protein had elongated from 81 bp to 108 bp. The last three exons disappeared. In addition, the original 27 bp third intron had exonization to elongate the original third exon. The original 117 bp third intron sequences next to the original 27 bp third intron sequences had turn into 3’-UTR sequences. A homology search by using SWISS-MODEL homology software revealed a purative siganing transduction protein.The sequences of the terminal exon of the oxidoreductase gene shortened from 570 bp to 312 bp. The shorten exon contained parts of Ds sequences. The 3’-UTR sequences of the changed gene were resulting from the Ds element. The newly formed protein had reduced an α-helix and three β-sheets and some small parts of the random coil structures and also elongated an existed α-helix.The study illustrated that the rice mutant with white palea and lemma is a multi-copies Ds insertion mutant. Differences of gene expression products in transcriptional level revealed the events of exon shuffling and exonization happened in the Ds-tagged genes. These phenomena are helpful to deepen understanding of Ds transposition mechanism and the functions of associated genes in the rice mutant with white palea and lemma.