水稻Ds插入双分蘖突变体形成机理的分析

Analysis of the Generation Mechanism of a Ds-inserted Rice Mutant with Double Tillers at One Tillering Node

在水稻Ds插入突变株筛选过程中,发现1个在同一分蘖节形成大、小2个分蘖的双分蘖突变体dt1(double tillersmutant),两个分蘖均可正常抽穗形成有效分蘖。采用TAIL-PCR技术,从该突变体中克隆了Ds插入的侧翼序列,将该序列作为查询序列进行核苷酸序列数据库(NCBI-BLAST)在线比对分析,发现所克隆的Ds侧翼序列与3号染色体的克隆OJ1345H02(gi|21281466)序列同一性达100%。以FGENESH和GeneMark.hmm两种软件分别对Ds插入基因的结构进行分析,在外显子的数目、大小、位置等方面均得出高度一致的结果。同时,用NCBI Entrez server和Pfam等软件对该基因进行功能预测,推测的基因编码产物含保守的FH2结构域,为水稻类形成素蛋白。突变体后代Ds插入基因型分析显示,其后代出现分离,表明该突变体为Ds插入杂合体。

Insertion mutagenesis with T-DNA or transposable elements is currently one of the most powerful tools for screening various types of mutations in higher plants, and for analyzing functional information of genes related to those mutations. By using the Ac/Ds transposable elements system in rice, a great deal of Ds-inserted mutants may be efficiently generated, and used for functional identification of genes that influence important agronomic traits, by which the researches in rice functional genomics have been greatly promoted. As a key agronomic trait related to grain production in rice and other grass species and an important property parallel to branching in dicotyledonous plants, tillering in rice has attracted more research interests. In the present study, a rice tillering mutant was isolated from a collection of Ds-inserted mutant lines, and was designated dtl （double tillers mutant） according to its tillering phenotype of a major tiller and a minor tiller developed at the same filleting node. Both tillers were found to be productive with the ears normally emerged from each of them. 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, the results indicated that the Ds-flanking sequence showed 100% identity at the nucleotide level to the sequence of clone OJ1345H02 （gil21281466） of rice chromosome 3. The structure of the Ds-inserted gene was predicted by using software of FGENESH and GeneMark, respectively, and gotten highly similar in the number, size and location of exons within the gene. The results also showed that the Ds element was inserted into the region between transcription start site and the first exon, at 173 bp upstream of the first exon. Moreover, the product encoded by the gene was predicted by NCBI Entrez server and Pfam. It was revealed that the deduced product was a rice formin-like protein containing a highly conserved FH2-domain. Given the fact that the Ds element as big as 5.9 kb in size was inserted immediately into upstream of the first exon, it is suggested that Ds insertion probably make the gene loss its function, and thereby generate the rice mutant dtl. In addition, the genotyping for Ds insertion in the progeny plants of dtl mutant indicated that these progeny plants segregated into homozygeus and heterozygous types for Ds insertion and Ds free plants,which suggested that the dtl mutant was heterozygous for Ds insertion.