Purple-fleshed Sweetpotato IbBADH Gene Clone And Function Analysis

Sweetpotato as a multi-nutritional food seem as an important crop plant for solving developing country food security. The purple-fleshed sweetpotato as a new cultivar is popular with its special color and health-promoting function in recent years. Though it is more economically valuable, the average yield is lower than the normal yellow or orange-fleshed ones. Thus using genetic modification to analyze and promote their abiotic stress response ability is an cost saving and environment-friendly way to enhance crop yield. Under the trend of global warming, The salinity soil and dramatic changing temperature are now causing reducing yield of crops and bring serious problems to agriculture and environment. Genetic engineering to modify the plant response to abiotic stress has already show its advantages in solving these problems. The ideas to achieve salinity tolerance using genetic engineering methods at present mainly include:achieving ions compartmentalization, accumulating compatible solutes and elevating salinity response gene expression level. The compatible solutes like glycine betaine are low toxicity chemicals when large accumulated in plant cells through genetic engineering. They protect the cell from abiotic stress like salinity, drought and extreme temperature. The simple modify target which induce multi tolerance to stresses made the genetic modify strategy practicable and applicable compare with other catabolism pathway modifications. The betaine aldehyde dehydrogenase(BADH) which catalyze betaine aldehyde to produce glycine betaine(GB)is the key enzyme in GB accumulating and has been widely used in stress tolerance in recent decades worldwide. Over-expression of BADH in plants can accumulate large amount of GB and achieve multi tolerance to stress like salinity and cold temperature, most of which have a better growth condition or yield than wild type controls under such abiotic stress.In this study, through purple-fleshed sweetpotato powdering and RNA extraction, the BADH full length coding sequence(CDS)was cloned and bioinformatic analysis was processed, the whole CDS constitute1505base-pair nucleotide and code a505amino acid protein. With web based prediction the protein has typical BADH family conserved domain and NAD binding site. The multi sequence alignment and neighbor-joining model phylogenetic tree shows that the protein has a very close phylogenetic relationship with BADH protein in Lycium barbarum. And through membrane protein prediction, the protein is not an membrane anchored protein. Then as construct the prokaryotic expression vector with His tag, large amount of protein synthesized in Rosetta cells and purified thorough affinity chromatography with Ni-column and enzyme activity was detect under340nm light wave length with ultraviolet spectrometry,the results show that the IbBADH enzyme has the ability to catalyze glycine betaine aldehyde to form glycine betaine. The supernatant of IPTG induced and ultrasonic processed bacterial which had resuspended can detect enzyme on SDS-PAGE electrophoresis proves the protein is solvable.With the CDS of IbBADH insert into p1300-GFP vector and transfer into Tobacco protoplast, the figure observed under Confocal laser scanning microscopy (CLSM) show that the protein is localized in chloroplast which is in accord with subcellular localization prediction and also reasonable as previous research proved the GB has the close relationship with light capture system II protein complex in chloroplast like a molecular chaperone. As the CMO gene(the gene code for the first step enzyme of glycine betaine catalyzation) was proved expression in chloroplast in plants which made the choline oxidation to GlyBet more accessible with regulating IbBADH gene expression in sweetpotato.(if the protein is in cytosol or other plastid,the betaine aldehyde need to transport through the chloroplast membrane to be oxidized) And at last the real-time qPCR experiment of field purple-fleshed sweetpotato and Hoagland cultured shoots analysis show that the IbBADH gene expression correlated with photosynthesis reaction, which had a significant difference after salt stress treated. In all, these studies show the IbBADH is a functional protein for GB accumulation and is important for understanding and metabolic engineering of GlyBet accumulation to achieve salinity and/or other abiotic stress tolerance in purple-fleshed sweet potato which is a potent strategy to enhance yields.