Construction Of RNAi, Overexpress PRGL Vectors And Transgene Research In Gerbera Hybrida

PRGL was isolated as a novel gene in flower development in Gerbera in our lab.It is presumed to be correlated with GA induced cell elongation and may be localizedin cell wall according to the analysis of its gene structure. Recently, techniques ofgene over-expression and knock-down in the research of gene function have becomeincreasingly important. By previous study in our lab, Zhang Miaobin established ashoot-regeneration system mediated by Agrobacterium tumefaciens. In the presentstudy, further experiments were conducted to optimize the transformation system inGerbera hybridy and PRGL over-expression and RNA interference vectors wereconstructed followed by transformation of RNAi vector in Gerbera. The main resultsare as follows:1. An optimized genetic transformation system mediated by Agrobacterium wasestablished. The data showed that hygromycin significantly suppressed the inductionof lateral bud and root under the selective concentrations. The optimal concentrationsfor root and bud growth were 8 mg/L and 10 mg/L, separately. Induction rate ofresistant buds varied between genotypes and T1 variety had a higher induction ratethan that of S5 variety. Delayed screening method was developed by selection ofhygromycin resistant shoots. It was found that 4 days was the preferable delaying timefor screening after 3 days co-culture with Agrobacterium. L-cysteine, which wasreported to promote Agrobacterium transformation, had less effect in our experiment.2. RNAi and over-expression PRGL plant expression vectors were constructedby RNAi universal vector pRNAi.0, and then introduced into AgrobacteriumLBA4404. RT-PCR was performed to amplify the targeting gene and the productswere ligated to pMD simple 18-T vector. Interference expression vector pRNAi.2 wasobtained later, embracing the site of MCS2 with antisense fragment and MCS1 withsense fragment. In addition, sense PRGL was inserted into MCS1 of pRNAi.0 to formthe over-expression vector pRNAi.3.3. Agrobacterium-mediated transformation of vector pRNAi.2 was carried outand 16 hygromycin resistant seedlings were obtained. Among them, 11 seedings were positive by PCR assay. It might be the result of PRGL integrating into theGerbera genome. Average transformation frequency reached 6.57% in ourexperiment. The phenotype analysis of transformants is still under performance.This research provided experimental foundation for further investigation ofPRGL function in Gerbera and the transformation system will be useful for geneticengineering in Gerbera.