Construction Of Flower CDNA Library Of CMS Restorer Line R₉ In Sunflower And Characterization Of Restoration Gene Rf₁

Total RNA of young flower of sunflower restorer line R9 was isolated using guanidinium isothiocyanate and phenol梒hloroform. mRNA was prepared by PolyATract mRNA Isolation Systems(Promega).Full條ength cDNA was synthesized and then cloned into phage XExCe11 vector. After packaged in vitro, the capacity of the library was measured. The capacity of the library was 1. 6 X lO6pfu, and the recombination rate reached to 95. 2%.The molecular marker of nuclear restoration gene Rf1, random primer OPCO7, was found by JiJing et al. (1996). Based on the RAPD marker of gene Rf,, the specific polymorphic fragment (0.9kb) in the restorer line R9 was amplified. The amplified fragment was cut out from the gel under UV light, and then the 0. 9kb fragment was extracted with DNA extraction kit. After molecular weight detection, the 0. 9kb fragment was cloned into the vector pMDL8桾. Through white梑lue screening, white clones were picked out from LB/Amp/X梘al/IPTG plates. Through double digestion witb restriction endonuelease(BamHI~ Hindlll), the recombinant with the 0.9kb insert was found and named pRf~. Sequencing of the insert revealed that it is 860bp in length and maybe include apart of an ORE(open reading frame).After digestion ol pRf1 by restriction eridonuclcase( BamHI~ HindllI), the 860bp insert fragment was extracted from the gel and labeled with digoxingenin條l--dUTP(DIG).After the probe was identified by dot blot and southern blot hybridization, it was used to screen part of the cDNA library and one positive clone was obtained. Thin recombinant with the cDNA ~crt fragment was released frc the positive clone and was di 憐;tcJ with EcoRI; the eDNA fra, ent was 2kb in length. This uudyIIwill lay foundations for the detection of the Rf1 restoration gene and its expression study.