Gene Cloning And Functional Analysis On A Rice Mutant CA648

The plant height and tiller number are two important valuable traits for their significant contribution to plant architecture. In recent years, many new findings resulted in molecular research related to rice plant height and tiller number have been obtained, while plant height and tiller number are manipulated by numerous genes or QTLs, it is far from declearing the mechanism underlying the development. In this desertation, a mutant from a rice activation tagging mutant collection (T-DNA inserted rice mutant) was identified and named as CA648, which displays a phenotype of dwarfism and multi-tillering. CA648 was employed to analyze its physiological, genetics and molecular characteristics. The aims are to clone the putative gene which causes the defects of plant archetecture in CA648, and further declare the gene’s function. The main results are as follows:1. Compared with wild-type ZH11, CA648 displayed multi-tillering and dwarfism phenotypes, plus other abnormal traits such as delayed flowering, norrower and shorter leaves, shorter panicle length. The plant height of CA648 is sensitive while the multi-tillering is resistant to the treatments of plant growth regulator as well as condition changes.2. The F1 plants by crossing CA648 with ZH11 displays moderate phenotypes in terms of the plant height, tiller number, length of main panicle, and the total and filled gain number. In the F2 population, all of those plants showing short stature and multi-tillering are resistant to PPT, and the segregation ratio is statistically 3:1, suggesting the defects in CA648 are co-segregated with T-DNA, possibly caused by a single mutated locus. Southern bolting further verified that CA648 conferring only one T-DNA inserted sequence.3. The flanking sequence analysis showed that the T-DNA insertion is localized in the middle of an unknown gene on the 8th chromosome, designed as OsDMT(Oryza sativa mutants with a characteristics of Dwarfism and-Multi-Tiller). Further analysis indicated that OsDMT gene has no intron and one OsmiR156 family member OsMIR156f gene is located in the downstream of it.4. Bioinformatics analysis indicated that OsDMT gene encodes a putative protein which containing only 66 amino acid residues and a conserved domain shared by the protease inhibitor I family (potato protease inhibitor type I). Constitutive overexpression of OsDMT gene in CA648 by CaMV35S promoter can rescue the mutant traits of CA648 to the architecture of wild-type. We also had carried out experiment of RNA/to knock down Os08g34258, and the obtained transgenic line showed similar traits of CA648.5. BLAST analysis showed that there is a tandem repeat sequence segment of 13003 bp including OsDMT gene in the japonica rice Nipponbare genomic DNA, while there is only one such a segment but no tandem repeat in indica rice 9311 genomic DNA. After further investigated whether a copy number variation (CNV) of this 13003 bp sequence segment including OsDMT gene exists in 154 diverse varieties, the result showed that all of 93 indica rice and most of (41) japonica rice varieties conferring only one copy of this sequence segmant, the other 13 japonica rice varieties (including ZH11) owning a tandem repeat sequence segment in their genomic DNA, therefore the 13003 bp sequence-segment including OsDMT gene can be regarded as a CNV in different rice varities.6. The transcripts of the mature OsmiR156 were higher in CA648 than ZH11 according to the result of stem RT-PCR analysis. Many transgenic lines from constitutive overexpression of OsMIR156f precursor in ZH11 by Ubiquitinl promoter display a dwarfism and multi-tillering trait similar to CA648. Expression pattern analysis showed that the OsMIR156f is only expressed in outgrowable buds but not in dormant buds. When OsMIR156f gene was constitutively overexpressed in ZH11 or Nipponbare by Ubiquitinl promoter, all transformants display an obvious increased tiller number and all axillary buds on elongated-node can outgrow and form panicles. While OsMIR156f gene was restrictively overexpressed in the stem by D18p promoter, the transgenic lines display a considerable increased tillers on the basal unelongated node but tiller buds on the elongated nodes remained in quiescent state. Eventually, transgenic plants and wild-type rice were sown in plots and were investigated into the yield related traits. Result showed that when OsMIR156f was restrictly expressed in stem by D18 promoter, the transgenic lines show moderately increased effective tillers without change in other traits. The above results provided theoretical basis for the improvement of plant architecture and yield of rice by means of OsMIR156f and OsDMT through the regulation of tillering.