Molecular And Genetic Analysis For Osphr2 Regulation On Pi-Transporter Ospt2 In Rice

It has been known that OsPT2 is a member of rice phosphate transporter gene family. At the present time, exact function of OsPT2 and relationship between OsPT2 and other regulated genes involved in phosphorus signal, however, is still largely unknown. In order to make further study of these, we get OsPT2 overexpression transgenic plants and ospt2 mutant. We have investigated the affection of OsPT2 on phosphorus metabolism in rice by physiology and molecular biology ways, and have clarified the regulatory relation of OsPT2 and OsPHR2,OsPH02. The results are summarized as follows:1. OsPT2 was a member of rice phosphate transporter family, and located on chromosome 3. OsPT2 included one exon without intron. There was a PHR1 combination location at 343bp-335bp upstream of ATG. Protein structure analysis indicated that OsPT2 had 12 trans-membrane domains which exist in every member of this phosphate transporter family.2. OsPT2, OsPTll, OsMPT overexpression transgenic plants were obtained. Genotype experiment filtered OsPT11, OsMPT overexpression transgenic plants which changed less on phenotype, while overexpressed OsPT2 made dry weight decrease obviously compared with wild type, which made OsPT2 become a research concentration.3. cosegrigation experiment on two OsPT2 overexpressed transgenic lines displayed significant reduction in plant growth rate, tiller number and showed chlorosis and necrosis at the edge of leaves. Total P concentration of positive lines were 3.5 times of negative lines on shoot and 2.5 times of negative lines on root. Total P concentration and phenotype were cosegregated with gene expression.4. Wide region of Pi gradient hydroponics indicated the direct relation between Pi level in nutrition solution and dry weight together with total P in plant:with the increase of Pi level in nutrition solution, Pi toxicity became more serious, and with the decrease of Pi level in nutrition solution, toxicity phenotypes could be rescued.5. Narrow region of Pi gradient hydroponics indicated that under low Pi level, even if OsPT2 overexpression transgenic plant could absorb more Pi in some degree, the growth rate could not recover and exceed wild type.6. Identification of ospt2 mutant indicated that T-DNA inserted at 569bp upstream of ATG of OsPT2 and the inserted T-DNA was single copy. RT-PCR displayed the insertion decreased OsPT2 expression remarkablely.7. Plant height, primary length, tiller number and dry weight of ospt2 mutant were suppressed in some degree compared to wild type, and the suppression to tiller number and dry weight was more obvious under lOppm Pi than 0.5ppm Pi, more obvious in shoot than in root.8. Available Pi and total P concentration in ospt2 mutant did not change obviously compared with wild type, while total P content decreased compared with wild type.9. Analysis of Affymetrix chip indicated overexpression of OsPHR2 and ospho2 mutant induced expression of OsPT2, OsPT8 and OsPT3.10. Real-time PCR showed OsPT2 was positived regulated by OsPHR2, and was negatived regulated by OsPHO2.11. In homozygous ospt2/OsPHR2(O) F3 lines, available Pi concentration of homozygous hybridization reduced 70% compare to over-expressed OsPHR2 plant, so OsPHR2-mediated OsPT2 overexpression might play an important role in Pi excessive accumulation.12. In homozygous ospt2/ospho2 F3 lines, available Pi concentration of homozygous hybridization reduced 30% compare to ospho2 plant, so OsPHO2-mediated OsPT2 overexpression might contribute a part to Pi excessive accumulation.13. Yeast one-hybrid experiment and gel shift analysis displayed that OsPHR2 protein interact with OsPT2 promoter directly14. OsPHR2 regulated OsPT2 by two pathway:OsPHR2 directly regulated OsPT2 or OsPHR2 regulated OsPT2 through OsPHO2 cis-element.