Functional Identification Of Zinc Transporter Gene AsZNT1 Upstream Regulatory Elements

With the development of various human activities, pollutants containing heavy metals migrate into the soil through different channels. Due to biodegradable and insoluble, heavy metals along the food chain ultimately do harm to the health of people. Currently heavy metal pollution has become one of the world’s problems to be solved. Whether it is from research field or a technical measure, bioremediation prospects are broad, Bioremediation includes many aspects. Applied the symbiosis of plants and microorganism to strengthen heavy metal tolerance and improve the efficiency of bioremediation of contaminated soil has become a new hotspot. Arbuscular mycorrhizal fungi are widely distributed in nature, with the majority of the higher plants form a symbiotic system, it not only can improve on a variety of plants to absorb nutrients, but also can enhance the host plant to abiotic stress resistance. And mycorrhizal fungi belongs to a class which is directly linked of soil and plant roots, therefore, research and utilization of mycorrhizal fungi restoration heavy metals contaminated soil has important guiding significance for agricultural development. Currently, the molecular mechanism regulation to explain the absorption and transportation of heavy metals is unclear. In this study, by analyzed and identified 5 ’upstream sequence of zinc transporter in Astragalus sinicus L. The main results are as follows:1. By inverse PCR primers were designed to amplify the promoter sequence upstream of 1.5kb.Through PLACE and plant CARE software predicted cis-elements and their locations, by sequence analysis discovered several important cis-elements present in As ZNT1 promoter, P1 BS related with phosphorus regulation, 34bp-motif involved in phosphorus and mycorrhizal fungi, MYCS-like, ZRE-like elements which may related with mycorrhizal and zinc.2. The constructing As ZNT1::GUS expression vectors, the promoter driving expression of full-length genes As ZNT1 mainly in the meristem region, growth areas, at the ripe young lateral roots organizations and vascular tissue of maturation zone, PAs ZNT1::GUS fusion protein is not found in the root epidermis and cortex. This may be suggest that As ZNT1 involved in transportation or absorption of zinc or other metals.3. Through a series of truncated experiments showed that the presence of a promoter in response to phosphorus starvation-related components P1 BS, and a new tandem repeats 34bp-motif involved in regulating the expression of genes As ZNT1.However, the MYCS-like、ZRE-like elements did not respond to gene regulation.4. By over-lap PCR specific deletion vector construct, we knockout specific components, further validation of known phosphate transporter factor cis-elements P1 BS and 34-bp motif of a newly discovered are the cis-elements can sense zinc concentration in the environment and mycorrhizal fungi infection, they involved in the regulate the As ZNT1 expression.