Molecular Identification Of Ds Insertion Sites And Ds-tagged Genes In Rice Dwarf Mutant

Mutants are important germplasm resources for crop genetic analysis and functional genomics research.Insertional mutations based on transposable elements are an important pathway for high throughput mutants.Ac/Ds transposable elements are the most widely used genetic elements for rice mutant libraries.Plant height is an important agronomic trait that directly affect the high yield and quality of crops in rice and other gramineous crops.In previous studies,we screened a dwarf mutant with Ds inserted from the Ac/Ds insertion mutant strain of japonica rice cultivar Dongjin(Oryza sativa L.var.Japonica cv.Dongjin).Using the mutant,this study was used to characterize the Ds insertion site and Ds marker gene in the genome.The aim of this study was to provide reference for the study of high quality and high yield rice breeding.The results of this study mainly include the following aspects:1.Preliminary Morphological and Physiological Identification of Dwarf Mutant.Compared with wide-type rice,the dwarf mutant showed dwarf height and large flag leaf angle.The data of the plant height,flag leaf and spike length.The results showed that the plant height of the mutant line(average plant height 44.6 cm)was significantly lower than that in wild-type rice(average plant height 88.0 cm).The flag leaf angle of the mutant line(74.3°)was significantly larger than that of wild-type rice(20.9°).2.Based on TAIL-PCR technique,the flanking sequences of the Ds insertion site of the dwarf mutant genome were amplified and separated by the combination of different degenerate primers and specific primers.Through NCBI-BLAST analysis,we found that the Ds element inserted in a gene which coding divalent ion transporter protein on chromosome 3(gene= "Os03g0147400",protein_id= "BAS82290.1").3.The Ds-tagged gene contains three exons and two introns,with Ds is inserted in the third exon.This gene encoded a 469 amino acid with divalent ion transporter protein.The dwarf phenotype is derived from the insertion of the Ds element on the divalent iontransporter protein gene,The study illustrated that the gene was related to the divalent ions necessary for the normal growth and development of rice,and the normal plant height of rice was maintained by ion absorption,transport and distribution.4.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,the structure of the divalent ion transporter protein encoded by the dwarf mutant rice was changed by a mechanism called exon shuffling.The length of the second exon of the gene which encode divalent ion transporter protein had shortened from 1214 bp to 590 bp.The shorten exon contained parts of Ds sequences.5.Real-time quantitative PCR was used to detect the expression of Ds-tagged genes in mutants in different organs.The results of SPSS software showed that the expression of Ds-tagged gene was the highest in stem,followed by leaf,root and stamens.As a whole,we preliminary forecasted that the expression of Ds was the highest in the stem,and the Ds insertion induced the exonation of the related gene.The structure of the divalent ion transporter was changed and involved in the life activities of the rice of the necessary ions,functional molecules to absorb and transport obstacles,leading to rice plant phenotypic dwarf.