The Applied Research Of AAP1-Bar Bivalent Genes Expression By Root-specific Promoters In Creation Of New Maize Material

High-protein maize, which is a special type corn about high content of lysine in proteincomponent, is known as high-lysine corn.It is not only can be used as high-quality livestockfeed, but also has a great advantage in food processing. Since the standards of living improved,people’s food consumption has also turned to nutritious and wholesome direction. The dataindicates that consumption of high-protein corn can prevent "niacin" effectively.The aminopermease1(AAP1) gene,which separated out from Arabidopsis, widespread in Arabidopsis root,lateral roots, root hairs and root epidermal cells. It can increase the content of amino acid too.This study aims to make AAP1gene expressed in roots specifically by using specificity of theroot tissue.Thus it could increase the amino acid content of corn on the "source" of the level. Asa screening gene, we use Bar to obtain new corn materials in high-protein、 herbicide-resistant.In this study, we cloned Arabidopsis AAP1genes and analyzed genes function preliminarilythrough the expression of prokaryotic and eukaryotic. The results as follows:1.We extracted Arabidopsis RNA and reverse transcribed it into cDNA successfully,gotArabidopsis AAP1genes by PCR,AtAAP1protein molecular had a weight about52kDthrough bioinformatics analysis. The protein sequence had no signal peptide ortransmembrane domain.2.Based on the pET-32a we constructed AAP1prokaryotic expression vector throughrecombinant DNA technology, named pET-32a-AAP1. SDS-PAGE electrophoresis analysis,the result consistented with the predicted molecular weight of the protein.3.We used recombinant DNA technology to connect the purpose of gene with gene-specific rootpromoter RolD and plant gene expression vector PTF101.1with Bar,namedPTF101.1-RolD-AAP1.4.We transferred plant expression vector PTF101.1-RolD-AAP1into Agrobacterium by usingthree prohybridization successfully and transferred it into Arabidopsis through tidbits topinfection.By basta resistance screening, we got T1-generation seeds successfully, measuringthe amino acid content of root, stem, leaf and compared with the ordinary to determine therole of AAP1.5.We used bud tip transformation and gene gun bombardment to transfer target gene which fromthree prohybrid into corn B73respectively. After resistance screening and PCR testing, bud tip transformation has taken to get two positive seedlings; bombardment obtained resistantembryos, and we will further resistance screening.