Cloning Aed Functional Analyses Of Tissue-specific Promoters AtML1,AtCER6 And MtML1

Drought restricts agricultural development in our country.Plant cuticle wax content increased to improve the drought resistance.Accumulation of cuticle wax content in the plant,Thereby improving pasture resistance in desert region,is one of the effective ways to solve the drought problem in agriculture.Epidermis-specific promoter is an important tissue-specific promoter.Epidermis-specific promoter can drive the waxy synthesis related genes specifically expressed in plant cuticle,thereby increasing the plant cuticle wax content.But the information regarding epidermis-specific promoter in relevant research is limited.In the present study,we clone a 5' sequence fragment of At L1 and ACER6 from Arabidopsis thalinaa DNA AND MtMLl from Medicago truncatula DNA.According to leaf transient expression of these three promoters in Nicotiana tabacum and transforming Arabidopsis thaliana of AtCER6 and MtML1 promoters by Agrobacterium-mediated floral dip method.The following conclusions were obtained.1.A 5' flanking sequence of AtML1,AtCER6 and MtML1,with a length of 2162 bp and 1209 bp from genomic DNA cloning of Arabidopsis thaliana and of 2150 bp from genomic DNA cloning of Medicago truncatula.By plantCARE online software analysis indicated that the 5' flanking sequence of AtML1,AtCER6 and MtMLl contain conserved element,and a variety of light response element.while a 5' flanking sequence of AtML1 contain epidermal-specific element.The three sequences were predicted as AtMLl,AtCER6 and MtMLl promoters.2.Using GUS as a reporter grne three expression vectors were constructed through gateway technology named as AtML1::GUS,AtCER6::GUS and MtML1::GUS..3.Using Nicotiana tabacum leaf as the recipient,transient expression research of three promoters was performed.GUS histochemical staining by Agrobacterium-mediated transformation indicated that the three promoters have GUS staining.4.Using AtCER6::GUS and MtML1::GUS expression vectors by Agrobacterium-mediated genetic transformation of floral dip in Arabidopsis,trough BASTA resistance,we got transgenic plants 4 lines from AtCER6 promoter,and 6 lines from MtMLl promoter.Results of transgenic Arabidopsis thaliana GUS staining indicated that AtCER6 and MtML1 promoters are epidermal-specific promoters.They can drive GUS gene expression in shoot epidermis.The study cloned AtMLl,AtCER6 gene promoter from Arabidopsis thaliana and MtML1 gene promoter from Medicago truncatula.We verify their functional feature in the tobacco leaf preliminary and further analyze their epidermis-specifity in the Arabidopsis thaliana.By regulating the wax deposition improved resistance of the pasture and crops could provide a solid foundation.