Modification Of Plant Height Via RNAi Suppression Of OsGA20ox2 In Rice

In 1960s, a Green Revolution rose in the crop field. Dwarfism is caused by loss-of-function mutations in OsGA20ox2 gene controlling the biosynthesis pathway of the plant hormone gibberellin (GA). The semi-dwarf cultivars are resistant to lodging and increase grain yield. Up to today, these dwarf varieties have still been applied in agriculture.Rice is one of the most important crops. Some rice varieties such as QX1 are high in yield, potentially normal in flowering and reproduction, good quality with better agronomic characteristics. But their height are enough taller inclined to lodge due to rain or wind, which brings much difficulty in harvesting and grain quality. Therefore, it is very necessary to reduce their height without effect on other agronomic traits. Based on the better varieties, we can modify the specific trait of variety by biotechnological and molecular approach. In this paper, we used the RNAi approach to down-regulate the expression of the target gene OsGA20ox2 specifically. The main results were as follows:1. Based on the nucleotide sequence of OsGA20ox2 gene, three specific fragments were amplified by PCR as the template of genomic DNA of the rice Japonica QX1. Three different specific fragments of OsGA20ox2 gene as interfering regions were obtained and contained 597 bp, 531 bp, and 990 bp respectively. Each specific fragment was ligated into the intermediate vector pTCK303 in different configuration, including the constructs in sense orientation, in antisense orientation, and in hairpin structure. Thus nine vectors were constructed.2. Via Agrobacterium-mediated transformation of four rice lines including QX1, Zhongzuo8923, Zhongzuo0114, and Zhongzuo0201. 1215 transgenic lines were generated, among which included 465 plants of dwarf phenotype. The dwarf ratios of three hairpin-RNAi constructs pCH1CK (interfering fragment: 597 bp), pCH2CK (531 bp), and pCH12CK (990 bp) were 25%, 29%, and 36%, respectively. The genetic stability of three hairpin-RNAi constructs was good through successive two generations propagation.3. Dwarf lines from zhongzuo8923/ pCH2CK decreased to the 53%⁵8% of wild-type in height. Each internode of RNAi dwarf line was shorter than that of wild-type. Also, the dwarf plants were obtained from the hairpin-RNAi constructs pCH1CK and pCH12CK. The height of dwarf plants from pCH1CK was reduced to the 70%-84% of wild type plants, and the dwarf plants from pCH12CK was reduced to the 62%-70% of wild type plants. As a result, the interfering fragment was longer accompanied with the dwarf extent being stronger. Compared with the wild type, all the dwarf plants exhibited normal flowering and reproduction. As a result, the yield of RNAi dwarf lines was similar to that of wild type.4. The results of Southern blot analyses showed that transgenic dwarf plants had one or two or several copies. The analyses of RT-PCR and Northern-blot indicated that the expression of OsGA20ox2 gene was reduced in the RNAi dwarf lines compared with wild-type. The analysis of RT-PCR demonstrated that the expression of OsGA20ox1, OsGA20ox3 and OsGA20ox4 genes in the RNAi dwarf lines was similar to that in wild-type.5. When partial RNAi dwarf lines were treated with various concentrations of exogenous GA3, the elongation of the second sheath of dwarf lines was promoted. The seedlings were treated with 10μM GA₃ for five days, and the RNAi dwarf lines could restore up to the height of wild-type. Above of the results demonstrated that the RNAi dwarf lines responded to the exogenous GA₃.6. The analysis of endogenous GAs in seedlings of the RNAi dwarf lines by GC-MS system demonstrated that the level of the biologically active GA₁ in the RNAi dwarf lines was reduced to 32%-56% of that in the wild-type. This result provided the direct evidence that the dwarf phenotype of the RNAi dwarf lines was associated with the decreased contents of biologically active GA₁, which was caused by the inhibition of stepwise oxidation catalyzed by GA20ox2.7. Finally, the RNAi method may be useful for modulating plant stature in other crops without effect on other agronomic traits including grain yield, flowering, reproductivity.