Purification Of Rubisco, Cloning Fragments Of Large And Small Subunit And Research On Protein Turnover Of During Grain Filling

Wheat is the most important crop in the Northwest of China. However,with contradiction between the decreasing cultivated lands and the more and more population becoming sharp,how to increase the yield is the urgent problem that agriculturists should resolve. At present,most studies were focused on improveing,breeding wheat variety ,fertilizer application and watering to get high yield. But it is limited. The protein turnover during the states of grain-filling was studied under five cultivation modules, and one of the modules that was advantageous to area of middle of Shaanxi Province be found. RubisCO,a protein which content account for 50 percent beyond the soluble protein content in flag leaves,has a important role during protein turnover.Therefore, the multiclonal antibody was prepared and fragments of large and small subunit were cloned as well, which establishing basis for exploring its function during protein turnover.The following results were obtained: 1 ) The purified RubisCO was obtained through four steps from Tris-HCl buffer,ammonium sulfate precipitation,gel filtration and ion exchange chromotagraphy; there is a single band showed in Native-PAGE and two bands in SDS-PAGE ,which molecular is 55KD and 14.8KD respectively. The multiclonal antibody was producted for the following experiments。2)The fragments of large and small subunit of RubisCO in wheat was cloned by PCR,which length was 620bp and 313bp respectively. Both them are located in extron,can be used as a probe in Southern blotting and Northern blotting. also,they can be used widely because of the high homology wit other species. 3)Among five cultivation modules(No mulch,straw mulch, plastic film mulch ,furrow and irrigation), the module of 8kg/mu nitrogen fertilizer level under plastic film mulch is more suitable than others in middle of Shaanxi Province. In this cultivation module, the protease activities of flagleaves is higher, the contents of soluble protein and free amino acid decrease thoroughly. So improved the efficiency of protein turnover. 4) More nitrogen fertilizer and water in soil could accelerate the improvement of leaves area, chlorophyll and photosynthesis, supply abundant source for protein turnover. 5) Western blotting showed that RubisCO crude extraction have four degraded bands with the inhibition of EDTA or PMSF, while have only two degraded bands with Leupeptin. This result provided more evidence that RubisCO is degraded by thiol protease.