CDNA Cloning Of A Zinc-Finger Gene And Its Genetic Transformation

Groundcover chrysanthemum(Chrysanthemum morifolium) plays a vital role in landscape greening for its various flowers,rich color and high ornamental value.In this study,a full-lenth cDNA clone of a zinc finger protein was obtained from groundcover chrysanthemum 'Sunlight', which had been screened for salt-tolerrance from a great number of chrysanthemum species or varieties.Moreover.CmSTZF gene was correlative with plant salinity-tolerance by its structure analysis and homology analysis.Moreover,CmSTZF transformant of tobacco exhibited highen resistance to salt-stress.Therefore screened groundcover chrysanthemum lines have the wide application prospect in the salinity area.The plant expression vectors of CmSTZF and OsISAP1 genes were constructed by Gateway clone technique.CmSTZF gene was transformed to chrysanthemums.The main results are shown as follows:1.A zinc finger protein full-lenth cDNA clone was obtained with RT-PCR from groundcover chrysanthemum 'Sunlight' treated under 150mM Na₂CO₃ stress.Its cDNA full-length was 794 bp and encodes deduced protein with 170 amino acid residues,which is about 18.1 KDa.Analysis of its amino acid sequence showed it has a typical C-X₂-C-X_(9-12)-C-X_(1-2)-C -X₄-C-X₂-H-X₅-H-X-C zinc finger structure which belong to AN1 type.It was named CmSTZF(Chrysanthemum morifolium stress-tolerance zinc finger)(GenBank accession no. DQ864730).It has high identity to stress related protein of many plants.Protein 3D structure simulation showed that the zinc finger domain is similar to that of rice.2.CmSTZF and OsISAP1 expression vector pH7WG2D-CmSTZF and pH7WG2D-OsISAP1 was constructed by Gateway clone technique.Transformation system of tobacco (Nicotianna tabacum) was established by A.tumefaciens LBA4404.Several positive transgenic plants,confirmed by PCR and PCR-southernwere obtained.Transgenic plants were more resistant than the control under Na₂CO₃ stress.Therefore,CmSTZF gene is closely related to plant resistance of salinity stress.3.Study of the efficient regeneration system.The regeneration frequency of 'Donglin 9' was 90%,in medium of MS+0.5 mg/L BA +1.5 mg/L NAA.The bud differentiation rate in upper young leaves of 'Donglin 9' was as high as 92%,with the differentiation rate being lower than 50%for leaves at middle and lower parts which near the roots.The obvious difference in differentiation rate of leaf explants was revealed among gene types of 'Donglin 9','Donglin11'. 'Donglin13','Sunlight','Pinkrose','Jinbuhuan','Donglinruixue','Fuli','Xuetao' and 'Qiuyan'.The highest differentiation rate 91.55%was obtained in leaf explants of 'Donglin 9', while the differentiation rate of 'Donglin13','Pinkrose','Jinbuhuan'and 'Donglinruixue' were less than 10%.After a 2-6d preculture in medium of MS+BA 0.5 mg/L+2,4-D 1.0 mg/L, the regeneration frequency of 'Donglin 9' was enhanced.4.A study of the efficient transformation system.Hygromycin reduced bud differentiation of leaf explants.'Donglin 9' lost ability to differentiation in the culture medium containing 10 mg/L hygromycin and once-differentiated buds died when soaked in solution at 20,25mg/L hygromycin.The efficient transformation system was as follows:the suitable concentration of Carb was 100 mg/L,the infection time was 30min,the co-culture time was 3d,the delay selection time was 15d,and the addition of Acetosyringone could improve the efficiency of transformation.5.Transformation system of CmSTZF in chrysanthemums for outdoor planting was established by A.tumefaciens LBA4404.Positive transgenic plants confirmed by PCR identification were obtained.