Identification Of A Ds-tagged Lodging-susceptible Rice Mutant

Lodging is a major problem in rice production. It causes yield loss and reduces grain quality. So it is extremely significant to find out the factors that cause rice lodging and to clone genes that are involved in traits relevant to lodging. In this study, we take the Ds-tagged rice mutants as materials and do morphological, physiological and molecular comparisons between mutants and wild-types with an objective to identify possible causes for stem lodging, and provide references for further functional analysis of genes that are associated with stem lodging. In this study, two main aspects are contained:1. Morphological and physiological characterization of the lodging-susceptible rice mutant. By using the 100 plants randomly chosen from each population of mutants and wild-types, some morphological and physiological comparisons of the Ds-tagged mutant with wild-type rice plants were made based on measurements of plant height, and the internodal length, culm diameter, culm wall thickness and leaf sheath thickness of each internode. Moreover, the cellulose content and silicon content in roots, stems, leaf blades, leaf sheaths, panicles, and hulls were also determined, respectively. The anatomical differences in the number, shape, size of vascular bundles between the wild-type and the mutant plants were examined by paraffin section method.The results showed that there is no difference between the mutant and the wild-types in plant height and the internodal length. There are no prominent difference of first internodes under the panicles, clum wall thickness, and the R value between the mutant and the wild-type, while culm diameter of the wild-type is 1.24 times of the mutant; the 2nd, 3rd internodes have no difference in each examined parameters; while the culm diameter and the culm wall thickness of 4th internode of the mutant is much smaller than the wild-type, the R value has no difference. The wild-type and the mutant plants didn't exibit any difference in the number, shape, size, etc of vascular bundles through the gross anatomy with paraffin section method. The results showed that a significant reduction in cellulose content was observed in each organ of the lodging- susceptible mutant, and the silicon deposition and distribution in various organs of the mutant were found to be different from that of the wild-type. It is suggested that the reduction in cellulose content in each organ probably results in the insufficient mechanical strength in the Ds-tagged mutant plants, and thereby make them susceptible to lodging.2. Amplification of Ds-flanking sequence and structural and functional prediction of the Ds-tagged gene. The Ds-flanking sequence was cloned from this mutant by using TAIL-PCR amplification technique. The sequenced Ds-flanking sequence was used as a query to perform an online search for homologous sequence against nucleotide sequence database by NCBI-BLAST, and the Ds insertion site, and the chromosomal localization of Ds-tagged gene were analyzed, the structure and putative function of Ds-tagged gene were predicted by bioinformatics methods.The results indicated that the Ds-flanking sequence showed 100% identity at the nucleotide level to the sequence of clone Os07g0566200 of rice chromosome 7. The structure of the Ds-tagged gene was predicted by using software of FGENESH and GeneMark.hmm, respectively, both of which gave high similarities in the number, size and location of exons within the gene. The results showed that the Ds element was inserted into the region of the fifth exon of the gene. Moreover, the product encoded by the gene was predicted by NCBI Entrez server and Pfam. It was revealed that the deduced product was a member of rice PP2C protein family, which will affect the phosphatase of the serine/threonine. Given the fact that the Ds element as big as 5.9 kb in size was inserted immediately into the fifth exon, it is suggested that Ds insertion probably make the gene loss it's function, and thereby generate the rice lodging mutant.Take the morphological, physiological and molecular comparisons as a whole, we can draw a conclusion that the Ds insertion affects the phosphorylation of rice serine/ threonine protein, which probably involved in cellulose metabolism, and gives rise to the significant reduction in cellulose content of various rice organs, and eventually cause rice stem lodging.