Functional Analysis Of Rice Phosphate Transporter Genes Through RNA Interference

The genome sequencing of rice(Oryza sativa),a model plant of monocotyledon,has been finished.Rice functional genomics has become one of the research focuses in plant biology.One of the key mechanisms of rice to adapt the phosphate deficiency is enhancing expression of some phosphate transporter genes.In the rice genome,a total of 13 sequences can be identified as belonging to phosphate transporter(Pht1) family.Understanding of the physiological functions of phosphate transporters is essential for dissecting the molecular mechanism of phosphate uptake,translocation and utilization by plants.In our previous experiments,we observed that two rice phosphate transporter genes in Pht1 family,OsPT2 and OsPT6,were expressed abundantly under P starvation condition.In the present study,one of the rice cultivars belong to the subspecies of Japonica for genome sequencing,Nipponbare,was chosen for knockdown of OsPT2 and OsPT6 through the technique of RNA interference(RNAi).The suppression of target genes,isotopic absorption,mRNA expression of most Pht1 family members,dry weight,total phosphate uptake,root configuration,seed germination ratio in the RNAi knockdown T1 generation were measured in comparison to the wild type to explore the functions of OsPT2 and OsPT6 gene on phosphate absorption and transportation.The main results obtained were as follows:The mRNA expression of OsPT2 and OsPT6 were effectively suppressed but not to a complete level by transformation of the targeted genes with RNAi vector construction basing on pTCK303.There were very large differences of the suppression of the targeted gene among different lines treated with low Pi concentration.The inhibition rate of OsPT2 mRNA expression in r2-1 and r2-2,the two OsPT2 RNAi transgenic lines were 78.2%and 25.7%in the roots,41.2%and 22.7%in the shoot,respectively.While the inhibition rate OSPT6 mRNA expression in r6-1,r6-2 and r6-3,the three OSPT6 RNAi transgenic lines were 85.3%,77.5%and 45.5%in the roots,73.9%,70.1%and 71.4%in the shoot, respectively. The knockdown of either OsPT2 or OsPT6 by the RNAi also affected the expression of other Pht1 family members in the rice treated with low P solution.In the r2-1 mutant,the expression of OsPT1,OsPT6,OsPT8,OsPT12 relative their expression in the wild type were down regulated,and OsPT3,OsPT4,OsPT5,OsPTT,OsPT9,OsPT10 were up regulated in the roots;meanwhile,OsPT1,OsPT5,OsPT6,OsPT7,OsPT9 were down regulated,and OsPT3,OsPT4,OsPTS,OsPT10,OsPT12 were up regulated in the shoot. In the r6-1 mutant,the expression of OsPT1,OsPT2,OsPT3,OsPT5,OsPT7,OsPT8,OsPT9,OsPT12 relative their expression in the wild type were down regulated,and OsPT4,OsPT10 were up regulated in the roots;meanwhile,OsPT1,OsPT2,OsPT3,OsPT4,OsPT5,OsPTT,OsPT8,OsPT9,OsPT12 were down regulated,and only OsPT10 were faintly up regulated in the shoot.Knockdown the expression of either OsPT2 or OsPT6 decreased the total phosphate absorption,dry weight,total root length,total root surface area,root volume,root numbers under low P supply condition.In addition,the germination ratio and speed of the r6-1 seeds were markedly lower than that of the wild type seeds.The results indicated that OsPT2 and OsPT6 played very important roles in the uptake and translocation of Pi,as well as in regulation of root growth in the rice.The regulated expression of many other Pi transporters by knockdown of OsPT2 and OsPT6 there was a possible functional interaction among OsPT2,OsPT6 and other members of Pht1 family.