Isolation And Functional Characterization Of MeCWINV1Promoter From Manihot Esculenta Crantz

Sucrose is the main form of the carbohydrates transportation and storage in higher plant, sucrose invertase plays an important role in sucrose transformation from "source" to "sink". Cell wall invertases is the key enzyme in sucrose metabolism in plant source and sink organs, which is mainly involved in sucrose unloading in phloem and controlling the composition of sugar in storage organs. Cassava is a main energy storage material of starch-rich roots, the amount of photosynthate produced by leaves transfers to the roots for starch accumulation is less than1/5, so it is significant for cassava starch content to improve the energy transferring rate from leaves to roots. In this study, we cloned the promoter of cassava cell wall acid invertase gene MeCWINV1, the core domain of this promoter was found by the means of bioinformation and5’-end deletion PCR. This study might be laid the basis for further understanding the regulation model and signal pathway of the cassava cell wall acid invertase genes and investigating their functions on sucrose unloading and regulating in starch accumulation, and provided the theory basis for cultivating high starch cassava varieties. The main results are as follows:We isolated an865bp promoter region upstream of the MeCWINVl gene through PCR methods from the cassava (Manihot esculenta Crantz) genome. Bioinformation analysis showed that it contained several CAAT boxes and TATA boxes, which are the typical sequence of eukaryotic promoter. The promoter sequence also had some cis-acting elements related stress such as light responsive module, drought inducible elements, heat stress responsive elements, GA3, SA, ABA-responsive elements.The expression level to different stress of MeCWINV1including five hormone treatments:50μM IAA,2mM SA,100μM GA3,100μM MeJA,100μM ABA and four abiotic stresses:high temperature stress at42℃, low temperature stress at4℃,20%PEG6000drought stress and10mM H2O2oxidative stress were analyzed in roots, stems and leaves of cassava plants by Real-time PCR. The results illustrated that the MeCWINV1gene had played a role in abiotic stress.In order to identify the core domain of the promoter, a series of5’-end deletion mutant promoters were obtained by PCR methods and ligated into pVKH plasmid. The core domain was confirmed primarily by transient expression in tobacco. Then the constructs were introduced into Arabidopsis mediated by Agrobacterium tumefaciens. GUS analysis showed that the GUS gene was mainly expressed in leaves and the CW1-4F (-476) mutant promoter contained the core domain of MeCWINV1promoter and several positive cis-elements, while the-749/-476domain might contain negative regulated elements and could reduce the activity of MeCWINVl promoter. GUS quantitative analysis showed that the CW1-4F (-476) mutant promoter had a higher activity and had some specificities in tissues and organs.