Functional Analysis Of CiMYB4 Gene In Response To Cadmium Stress And Cloning,Expression Analyse Of Its Promoter In Chrysanthemum Indicum Var.Aromaticum

Chrysanthemum indicum is part of wild flower which is widely spread and has strong reproductive ability.It has been widely used in afforesting the barren hills as a dominant plant because of its rapid growth,strong stress resistance,resistance to extensive management.Therefore,the directional improvement of C.indicum by transgenic technology to improve its tolerance can make its application more meaningful and valuable in landscaping.Members of MYB transcription factor family can participate in the response to a variety of abiotic stresses,however,the studies on the response of MYB4 transcription factor to heavy metal cadmium(Cd)are few,so exploring its response mechanism to Cd can lay a theoretical foundation for revealing the action mechanism of CiMYB4 gene in the process of stress resistance,and provide more high-quality plant materials for afforesting the barren hills.Based on the CiMYB4transgenic tobacco and C.indicum obtained in the laboratory,they were treated with Cd treatment respectively,and the changes of physiological characteristics and the molecular response mechanism of Cd tolerance of different strains under Cd treatment were analyzed;Meanwhile,the promoter of CiMYB4 gene was cloned and analyzed.The main findings are as follows:1.After Cd treatment,CiMYB4 transgenic tobacco showed less damage,better growth status,and stronger cadmium enrichment and transport capacity than the wild type;The MDA content of CiMYB4 transgenic tobacco was lower than that of wild type,however,the activities of SOD,POD and CAT were significantly higher than that of wild type;The values of G_s、P_nand T_r of CiMYB4 transgenic tobacco were higher than those of wild type,and C_i was lower than that of wild type;The contents of chlorophyll a,chlorophyll b and total chlorophyll of CiMYB4 transgenic tobacco were higher than those of wild type.After Cd treatment,PCS1,GSH1 and HMA3 genes of CiMYB4 transgenic tobacco were up-regulated at a higher level comparing with wild type,while expression of ABCC1 did not find an obvious rule.The results showed that compared with wild type,CiMYB4 transgenic tobacco had stronger tolerance to Cd and stronger growth advantage.2.After Cd treatment,overexpression CiMYB4 C.indicum showed less damage,better growth status,and stronger cadmium enrichment and transport capacity than the wild type and RNAi strains;The MDA content of overexpression strains was lower than that of wild type,and the activities of SOD,POD and CAT were significantly higher than that of wild type.On the contrary,the MDA content of RNAi strains was higher than that of wild type,and the activities of SOD,POD and CAT were significantly lower than that of wild type;The values of G_s、P_n and T_r of the overexpression strains were higher than those of the wild type,and C_i was lower than that of the wild type.On the contrary,the values of G_s、P_n and T_r of the RNAi strains were lower than that of the wild type,and C_i was higher than that of the wild type;The contents of chlorophyll a,chlorophyll b and total chlorophyll of overexpression CiMYB4 C.indicum were higher than those of wild type and RNAi strains.After Cd treatment,PCS1,GSH1 and HMA3 genes of overexpression CiMYB4 C.indicum were up-regulated at a higher level comparing with wild type and RNAi strains,while expression of ABCC1 did not find an obvious rule.The results showed that compared with wild type and RNAi strains,transgenic CiMYB4 C.indicum had stronger tolerance to Cd and growth advantage.3.The promoter sequence 2001bp of CiMYB4 gene was cloned by chromosome stepping technique.Cis-acting element analysis showed that the promoter of CiMYB4 gene not only had the core elements necessary for the promoter,but also had many environmental signal factor response elements,such as light response,hormone response,stress response elements and MYC binding sites.According to the position of cis-acting element,the promoter 2001bp was divided into five 5’end deletion fragments,which were named P(2001bp),P1(1964 bp),P2(1299 bp),P3(954 bp)and P4(417 bp)respectively,and the Ca MV35s promoter was replaced.After transient transformation of Nicotiana benthamiana and GUS staining,it was found that all the five 5’end deletion fragments had certain activity.The deep staining of P and P3 showed that the 417 bp-954 bp region was the key region affecting the promoter activity.