Research On The Regulation Of Small G Protein In Potato Defense Reacting System

Small GTPases, are monomeric guanine nucleotide binding proteins related to the subunit of heterotrimeric G proteins. In plants, a unique class of Rho-like proteins, were cloned and termed"Rop"(Rho-related GTPase from plant). Till now, 28 Rop genes have cloned from Arabidopsis, maize, rice and alfalfa. The functions of ROPs have been studied and it was found to be invoved in a diverse array of cellular responses such as cell polarity development, endocytosis and H2O2 production.Potato late blight caused by Phytophthora infestans and very famous for the Irish Famine. Now, for the lacking of resistance cultivars and the variation of the race of Phytophthora infestans, molecular potao breeding has become a major way for controlling the disease. In this study, unravel the mechanism of ROP-GTPase in the establishment of their defense system to potato late blight is our main task. Arabidopsis gene AtRop1 funciton in Shepody will be tested its function on the resistance to potao late blight.Used Zihuabai and Shepody, we compared the activity variation of potato defense enzymes such as CAT, POD and SOD under vivo and vitro conditions. Our results suggested that the tendency of CAT, POD and SOD activity was the same under vitro and vivo condition. Therefore, the detached potato leaves could be used for further resistace analysis.The regeneration system of six potato varities was tested in advance. The results showed that the callus in different potato varieties may need different medium, the callus induction is Atlantic 100%, Favorite 95.83%, Sharpdi 96.96%, Zihuabai 100%, Desiree 100% and Hutou 83.87% respectively in the appropriate medium. Through different concentrations of 6-BA,NAA,ZT and GA3 to induce adventitious buds, The results indicate that the best adventitious buds medium is different in variety potatoes, the highest adventitious buds induction in Atlantic, Favorite, Sharpdi, Zihuabai, Desiree and Hutou are 18.18%,13.04%,17.86%,15.00%,44.44% and 47.83% respectively。Arabidopsis gene AtRop1 was fused with GFP tag and driven by CaMV 35S promoter. Via enzyme digestion and sequencing, we confirmed that GFP AtRop1 has been inserted into the expression bionary vector. Through freeze-thaw method, we transformed the expression vector into Agrobacterium LBA4404 for further experiments.Using Agrobacterium-mediated transient expression system, we primitively test the function of AtRop1 in potato leaves. After inoculating with the spore of Phytophthora infestans (106 piece per millilitre), we observed that the disease lesion at DN-AtROP1 the expression site is much smaller than control; DAB staining results revealed that there has more H2O2 accumulation in DN-AtROP1 infiltration sites, around the DN-AtROP1 infiltration sites, the mycelium growth was also inhibited. These results suggested that AtROP1 do play roles to the resistance to Phytophthora infestans infection and it achieved this goal via accumulating H2O2.Using Agrobacterium containing p1300-GDR(P1300GFP-DNAtRop1) transform potato leave disc and selected with hygromycin. Through PCR-Southern, RT-PCR and western dot blot, we confirmed the positive transgenic potato lines.After inoculating with Phytophthora infestans in the positive transgenic DN-Rop1 potato lines, we found that there do have much more H2O2. Accumulation in transgenic potato leaves. And the mycelium growth was also inhibited at the surface of transgenic potato leaves, suggesting that AtRop1 was involved in the establishment of potato defence system via accumulating H2O2, and its regulation way is negative.Via RT-PCR with degerate primers, we cloned potato small G protein called StRac1. Its cDNA is 633bp.Its function domain is rather conserved compared with the other small G proteins. The evolutionary tree analysis put StRAC1 into Arabiodopsis small G protein class IV, has high similarity with AtROP10 and AtROP11. Comparing the amino acid sequence with the other animals and plants small G proteins, the results showed that StRAC1 has the highest homology with the tobacco NtRAC4 (99%). Using RT-PCR, we found that StRAC1 highly express stalk, next to it is young leaves, tubers and old leaves. Fusing StRac1 with GFP, we construct the expression bionary vector for future transformation, so as to get transfenic potato lines for resistance analysis.