Dissertation > Agricultural Sciences > Agriculture ( agronomy ) > Crop Genetics and Breeding and breeding > Biotechnology breeding methods

Cloning, Expression and Functional Analysis of STOP1 Transcriptional Factor

Author LiLi
Tutor LiKunZhi
School Kunming University of Science and Technology
Course Biochemistry and Molecular Biology
Keywords Acid soil A1 toxicity STOP1 AtMGT1 Malate
CLC S336
Type Master's thesis
Year 2011
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About 40%of world’s arable land in subtropical and tropical regions on the earth in developing countries is acidic soil. The growth of crops is limited under acid soil because of poor root development by rhizotoxicity caused by ions such as aluminum (Al3+), and a deficiency of essential nutrients including phosphorus (P), calcium (Ca), and magnesium(Mg). The most important limiting factor is Al3+. The acid soil is often improved with lime in agricultural practices, but the cost is high and subsoil cannot be improved. Therefore, Al tolerance in the acid soil would be solved by genetic engineering, which is effective way for solving the acid soil problem. The transcriptional factor STOP1 is involved in the signal transduction pathway for Al3+ responses as a critical transcriptional regulator. The AtMGTl, a member of the Arabidopsis magnesium transport family, involves in Mg2+transport and play important role in Al tolerance in higher plants. This study used the model plant Arabidopsis thaliana as the material to clone Al-resistant gene STOP1 and AtMGT1, and constructed the plant expression vector of STOP1 and AtMGTl by the gateway technology. Then, transformed these genes to the tobacco for verifying the functional characteristics. At the same time, separated the aluminum tolerance gene STOP1 from Tamba black soybeans, Large soybeans, Small soybeans and Small black soybeans and compared consistency, expression in plant tissue and apical malate secretion among them to explore the molecular mechanisms of plant tolerance to aluminum. The main results were obtained as following:(1) Separated the aluminium tolerance gene AtMGTl and STOP 1 from Arabidopsis thaliana by PCR and constructed plant expression vector of single gene PK2GW7.0-STOP1 and PK2GW7.0-AtMGTl and double gene GW3-STOP1-AtMGTl through the Gateway technology. At the same time, obtained single STOP1 and AtMGTl transgenic tobacco lines and double AtMGTl and STOP1 transgenic tobacco lines by Agrogacterium-mediated transformation.(2) The amino acid sequences of STOP1 among Glycine max cultivars, i.e., Tamba black soybean, Small black soybean, Large soybean and Small soybean, were highly homologous. The identity of the amino acids between Tamba black soybean and Small black soybean, Large soybean and Small soybean was 95%,97%and 95%, respectively. This indicates that there is the diversity of STOP1 in soybean.(3) Under different times gradient treated with 50μm Al, STOP1 expression in Tamba black soybean and Large soybean was up-regulated at first and then down-regulated with the time. There was the maximum expression at 0.5 hours for Tamba black soybean and at 2 hours for Large soybean. The expression of STOP1 in both Tamba black soybean and Large soybean was decreased with the increase of Al concentration, but Tamba black soybean decreased sharply compared with Large soybean. The expression of STOP1 in bothTamba black soybean and Large soybean was insensitive to La, Cu, Cr and Pb metals.(4) Under the different time treatment with 50μM Al, the amount of malate secretion in both Tamba black soybean and Large soybean root tip reached the maximum at 1,0.5 hour, respectively, then declined gradually. Both of them were insensitive to other heavy metals, but were consistent with its expression.The study suggested that STOP1 of soybean with different aluminum tolerance had more than 95%homology. STOPl was sensitive to Aluminium, but insensitive to the heavy metal. Soybean was promoted the expression of STOPl and the malate secretion under Al stress. At the same time, we separated two single genes STOP1 and AtMGTl from Arabidopsis and obtained transformed single STOP1 and AtMGTl as well as double STOP1 and AtMGTl tobacco lines. These transformed tobacco lines established the foundation for further studing STOP1 and AtMGTl functions and molecular mechanism of aluminium resistance.

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