Cloning And Identification Of The Root Specific Promoter From Zea Mays

CaMV35S promoter is widely used in plant genetic engineering. Due to its constitutive expression pattern, foreign gene driven by CaMV35S promoter can express throughout all tissues of plant at every developmental stage, at a high metabolic expense to host plant. Too many resources have been distributed for foreign products expression in untargeted tissues, which result in great waste of both materials and energy. It is can also led to changes plant shape and influence plant growth. Therefore,researches on specific promoters, ideal alternatives to constitutive promoters, have been great important contents in plant genetic engineering. Driven by specific promoters, foreign genes are directed to express at special times, in given places of plant or even in desired quantities is regarded as an important way, and the first choice of high efficient and safety transgenic crops cultivation. Plant growth and development by gene regulation, the root specific promoter regulation of gene expression is one of the key components.Phytic acid, an anti-nutritional factors, while in plants it is the main form of phosphorus, the vast majority of animal digestion and absorption of the stomach can not sheets, but with the fecal excretion of phosphorus pollution caused by the environment. Through the role of phytase, phytate can be hydrolyzed to inorganic phosphate and inositol hydrolysis to release inositol and some trace elements, which can improve plant nutritional value of food and feed. So it has a special role in decomposition of phytate. Decomposition of cereal phytase present in the main storage material in the phosphate acid, phosphoric acid free to come out. Therefore difficult to absorb phytic acid for monogastric animals such as chickens and pigs absorption of phosphate..Using the root specific promoter to increase expression level of phytase in crops. Creation of transgenic crops with low phytic acid for improving the nutritional value of food, for human health is of great practical significance.In this study, the root specific promoter gene ZmGLU1P is cloned from Zea mays, and then identify the function of this promoter. On that basis we construct the expression vector of root specific promoter initiate PhyA gene seed coat specific expressed in maize. The expression vectors have been introduced into maize via pollen tube method and Agrobacteriumtum efaciens-mediated method. Expecting to provide an useful regulatory element for high level expression of PhyA genes which specifically expressed in the root. According to the promoter ofβ-glucosidase gene(GenBankDQ333310) sequence in maize, a pair of primers was designed. The promoter of root specific gene ZmGLU1P was isolated from the genomic DNA of maize P138 by PCR method. Then cloned into PMD18-T Vector. The result indicates the length of the clone sequence is 1846bp which shares a similarity of 99% with the published root specific promoter gene of Zea mays, the cloned gene contained typical TATA-box, CAAT-box and necessary regulatory motifs of root specific expression.Replace the plant expression vector PBI121 the CaMV35S promoter with ZmGLU1P, Connection with the GUS gene, to obtain recombinant expression vector ZmGLU1P-GUS plants.The plasmid ZmGLU1P-GUS was transformed into Agrobacterium tumefaciens EHA 105 competence cell by LN freeze thawing method. Then transferred to tobacco variety NC89 by Agrobacterium-mediated transformation. After cultivated in the selection mediums containing Kan, transformed tobaccos with ZmGLU1P-GUS were got. The transformed tobaccos were detected by PCR and southern blotting, The results showed that the transgenic plants were obtained successfully.In order to validate whether that cloned promoter has the function of specificity expression in the root, we analysed different tissues (including root, stem, and leaf) of transgenic tobacco plants by Histochemical staining of GUS activity. The result showed:there were non-coloration in stem and leaf of transgenic tobacco plants carrying the ZmGLU1P-GUS construct, but have the number of blue spot in the root, manifest the root specificity, markedly. So we confirmed that GUS gene controlled by this promoter gave specific-expression in the root.In order to exogenous gene having high performance to express in the control of the root-soecial promoter, and then carring out improvement to the maize quality, construction of plant expression vector PCAMBIA3301-ZmGLU1P-PhyA-NOS. Putting Nos fragment, ZmGLU1P fragment and PhyA gene sequence inserted into the plant expression vector PCAMBIA3301, so that the ZmGLU1P start downstream PhyA gene. The plant expression vectors have been introduced into maize via pollen tube method. Then, we obtained the transformed plants of To generation by pollen tube method and Agrobacteriumtum efaciens-mediated method and we used PCR and southern blotting in the research. The results showed that two transgenic plants were obtained.