Cloning And Characteristic Of F3H Promoter And CRY1 Gene In Turnip (Brassica Rapa L. Ssp. Rapifera)

Biosynthesis of anthocyanidinmany induced by blue light is common in plant. Cryptochrome(CRY),a receptor of blue light,plays a key role in the biosynthesis.In this study, CRY1 gene and promoter sequence of flavanone 3-hydroxylase(F3H) gene in turnip 'Tsuda' (Brassica rapa L.ssp.rapifera) were cloned,and their function was studied.The works in this study were part of investigation on the mechanism that CRY regulate the biosynthesis of anthocyanidinmany.1 Cloning and transient expression of F3H promoter in turnip 'Tsuda'With primers designed according to the sequence of Brassica rapa subsp,pekinensis clone KBrS001M03 which including F3H gene,and total DNA of turnip 'Tsuda' as templat,the promoter of F3H was cloned by PCR.The results of blast and analysis of bioinformation showed the cloned sequence was upstream sequence of F3H and has characters of promoter. The statistical analysis of elements in the sequence was carried out with software Plantcare. The elements such as CAAT box and TATA box in eukaryote promoters were found.The cloned sequence was replaced into Gateway vector pKGWFS7 at the position upstream of Gus to induce the expression of Gus in excised hypocotyl and cotyledon of turnip 'Tsuda' seedling.The results showed that Gus was expressed both in hypocotyl and cotyledon. Moreover,either in light or in dark,the cloned sequence of F3H promoter can induce expression Gus stably.So,it is concluded that the cloned sequence had function of promoter.2Cloning of CRY1 gene and its function1)Sequence and function of CRY1CRY1 gene of turnip 'Tsuda' was cloned by RT-PCR.Homology analysis of cloned sequence suggested the sequence was homologous with CRY1 of Brassica napus and Arabidopsis thaliana.The expression of this gene in light with different wavelength was studied.The results suggested the expression was steady and not influenced by wavelength. The interaction of CRY1 and COP1 in turnip 'Tsuda' was confirmed by two hybrid system.2)Construction of vector CRY1-dsRNAi and its genetic transformationTo investigate the function of CRY1 by RNAi,the factors influence the regeneration and genetic transformation of turnip 'Tsuda' were studied.With cotyledonary petiole and hypocotyl explants,the regeneration frequencies of turnip 'Tsuda' cukivars were examined.To achieve a high-frequency regeneration system,the hormone combination of thidiazuron(TDZ) and naphthaleneacetic acid(NAA) was compared with combination of benzyladenine(BA) and NAA on shoot regeneration.The results show that cotyledonary petioles were the best explant and that Murashige and Skoog(MS) medium containing 7.0mg/L TDZ and 1.0 mg/L NAA was suitable recipe for getting high-frequency shoot regeneration.Based the recipe,the effects of AgNO₃ concentration,seedling age,pre-culture time of 2,4-Dichlorophenoxyacetic acid (2,4-D) were investigated to optimize the shoot regeneration system.The results suggseted that petiolate cotyledon with seedling age of 5 d cultured in MS medium containing TDZ 7.0 mg/L+NAA1.0mg/L+AgNO₃ 5.0 mg/L followed by pre-culture with 2,4-D 1.0 mg/L for 2 d can be induced with highest-frequency regeneration.The highest shoot formation rate was about 90%.The rooting percentage of shoots was 100%on MS supplemented with 0.1 mg/L indole-3-butyric acid(IBA).The 95%rooted shoots survived in a greenhouseThe pivotal factors,which influenced the transformation frequency,were compared,and the parameters of transformation system were optimized.It was showed that selective medium containing 300 mg/L carbenicillin,which inhibited growth of Agrobacterium,led to production of more shoots resistant to hygromycin;the time required for infecting and co-culture with Agrobacterium was 20min and three days respectively;and the efficiency of screening with 20 mg/L hygromycin was highest.Molecular analysis of seedlings resisting to hygromycin showed transformants were positive.With Gateway clonging system,expression vector,CRY1-dsRNAi was constructed with primers designed acording to the sequence of CRY1.The vector was transformed to turnip 'Tsuda' mediamed by agrobacterium successfully.