Zinc-Finger Gene Expression And Salinity-Tolerance Protein Identification In Groundcover Chrysanthemum

Groundcover chrysanthemum (Chrysanthemum morifolium) is one of the Perennial flowers which applied to landscape garden widely for its various flowers, rich color and high ornamental. Based on screened to various groundcover chrysanthemum under salinity tolerance, high resistance variety was further studied. In the study, we fractionated and analyzed different proteins relative salinity- tolerance and obtained a new zinc-finger protein gene (CmSTZF). Moreover, CmSTZF gene was correlative with plant salinity-tolerance by its structure analysis, homology analysis and gene transformation in tobacco. Therefore screened groundcover chrysanthemum lines have the wide application prospect in the salinity area. It is great significative which of CmSTZF gene and resistant resources of chrysanthemum in new varieties breeding.The result showed as follows:(1) For obtainning salinity-tolerance lines, 99 lines of groundcover chrysanthemum (Chrysanthemum morifolium) were screened using 25 mmol/L Na₂CO₃,NaHCO₃ and the both, complex salt, and different kinds of salinity tolerance lines were screened out. There were 12 lines tolerance to Na₂CO₃, 24 lines tolerance to NaHCO₃, and 15 lines tolerance to the complex salt. At last, using 50 mmol/L Na₂CO₃, 8 lines of the former screened groundcover chrysanthemum were fined out which were tolerant to Na₂CO₃. It contained 'Sunlight',Donglin 13,'Fuli','Xuetao','Jinbuhuan',D4^1-1,Donglin9 and D. pacificum.(2) By comparing the five lines of salinity-tolerance groundcover chrysanthemums and five lines of salinity-sensitive ones, the result showed that: (1) the content of proline,activity of SOD and POD in salinity-tolerance groundcover chrysanthemums were higher than that in salinity-sensitive ones, but the content of MDA was lower in salinity-tolerance ones. (2) To the salinity-tolerance groundcover chrysanthemum, the correlation physiological parameters to stress were different: the level of proline in 'Xuetao' increased quickly, while the level of SOD was lower; in Donglin 13, the condition was very different. The levels of proline and SOD were both significant higher than the control.(3) Zinc finger protein is an important transcriptional factor in drought and salinity tolerance of plants in recent years. Semi-quantitative RT-PCR applied to analyze the expression of zinc finger gene on salinity-tolerance and salinity-sensitive groundcover chrysanthemums. It indicated that the expression of zinc finger gene was higher in salinity-tolerance chrysanthemum 'Sunlight' than in salinity-sensitivity 'Verve'.(4) A new zinc finger gene CmSTZF (Chrysanthemum morifolium stress-tolerance zinc finger) (GenBank accession no. DQ864730) was cloned from cDNA of salinity-tolerance 'Sunlight' by RT-PCR. Its cDNA full-length is 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 has high identity to stress related protein of many plants. Protein 3D structure simulation showed that the zinc finger domain is similar with that of rice.(5) Recombinant expression vectors, pET14b/His-CmSTZF and pET14b/His-OSISAPl, were constructed and recombinant proteins were induced expression. The results indicated that the fusion CmSTZF protein of 23 KDa was similar with that of rice. It showed that CmSTZF gene could be expressed perfectly.(6) Two-dimensional liquid chromatography (2-D LC) applied to fractionate and analyzed total protein from 'Sunlight' leaves of the treatment under 150mmol/L Na₂CO₃ stress and the control, while Chromatography peaks of fraction were transformed gel graph by Proteo Vue software. 33 different protein points were found, and 11 of them are enhanced expressed (R/L>2). Then 6 remarkable different protein fractions analyzed and identified by mass spectrograph (MS). The result showed that related salinity-tolerance protein genes known were consisted in analyzed different protein: Serine/threonine kinase,Putative cytochrome P450,Putative calcium binding protein,Superoxide dimutase (Cu-Zn),Glutathione S-transferase,Putative Na⁺/H⁺ exchanger。(7) CmSTZF expression vector, pH7WG2D-CmSTZF was constructed by Gateway clone technique. Transformation system of tobacco (Nicotianna tabacum) was established. Moreover, transgenic tobacco plants had obtained, and two plants had identified by PCR and PCR-southern. Moreover, transgenic plants were more resistant than the control under Na₂CO₃ stress. Therefore, CmSTZF gene is close relative to plant resistance of salinity stress.