Apple ABCF Transport S-RNase Into Pollen Tube Effecting Self-incompatibility

Self-incompatibility is a widespread reproduction barrier in flowering plants, which can prevent inbreeding and promote outcrossing by rejecting self (genetically related) pollen while leaving non-self (genetically unrelated) pollen to fertilization。 The apple and other rosaceae fruit trees belong to the S’-RNase-based gametophytic self-incompatibility. As a secreted protein,S-RNase can resolve rRNA and tRNA and it is an important determinant in self-incompatibility. Here we got6factors that can interact with S-RNase by screening an apple pollen cDNA library and determined the ABC transport protein as our object eventually. The pollen tube is considered to uptake style material including S-RNase at the same time, but there has been no related experimental proof about how the S-RNase is transported. In this research, we used ’Rails Janet’as main material and did some research on how the MdABCF can function in transporting protein. Our major results are as follows:1. Using S1-RNase as a bait screening pollen cDNA library, we got6proteins that can interact with SI-mat. After cotransformed with S1-RNase and analyzed in the NCBI, we found one protein that has the conventional NBD domain and Tran domain of ABC transport protein family. The protein belongs to F type, so we named it MdABCF. Meanwhile, we found that MdABCF has many possible transmembrane domains.2. We transformed MdABCF-GFP and MdABCF-CFP into onion epidermal, maize protoplast and apple pollen tube, finding that MdABCF can express on the plasma membrane and be co-localized with the endoplasmic reticulum; S-RNase can interact with MdABCF on the cytomembrane by cotransform MdABCF-YFPn and S-RNase-YFPc vectors in agrobacterium into apple leaves; according the functional domain of MdABCF, we divided it into several fragments. We determined the Tran domain of MdABCF is the main fragment that can interact with S-RNase by yeast two-hybrid、pull-down and BiFC; In addition, we found that MdABCF has no S-specific in interacting with S-RNase by cotransformed with S1, S2, S5, S7,S9-RNase.3. We silented the apple pollen tube MdABCF expression by using an antisense oligo nucleotide and found that pollen could grow normally on the medium containing S-RNase. Used FITC labeled S-RNase treated pollen, found that the S-RNase fluoresce points will be blocked outside the pollen tube. Through the immunofluorescence analysis used S-RNase antibody, we found the S-RNase will gather outside the pollen tube membrance. The results indicated that MdABCF can regulate transportation of S-RNase into pollen tube. After treated with FITC-S-RNase,S-RNase could enter the control pollen tube but could not enter the pollen tube. This indicated that MdABCF really can regulate the transportation of S-RNase; what’s more, FITC-S1-RNase and RBITC-S2-RNase cultivated S2and S9homozygote respectively, and found that MdABCF can transport S-RNase into pollen tube non-selectively, and there was no competitive interaction between different S-RNase.4. Though the LatA, Oryzalin, Taxol treated pollen, pollen tube can growth even the S-RNase was added into the medium. Though FITC-labeled S-RNase treated apple pollen, we find out the most of S-RNase fluorescent points would be blocked outside the pollen tube membranes, only a little S-RNase could transport into pollen tube. The results indicated that the microfilament cytoskeleton will as an accessory factor attend into the S-RNase transport process; it can influence the transportion of S-RNase.5. Treated apple pollen used S-RNase can influence the pollen germination and pollen tube growths. Through the actin dye and anti-β-tublin to label the F-actin and microtubule of the S-RNase treated pollen tube, find out the microfilament cytoskeleton will be depolymerization into punctuate foci. Used Western blot to check the S-RNase accumulation in the pollen tube, and find out the S-RNase will be accumulated after treated. Pollen ion bombardment experiment analysis indicated that S-RNase uptaken will depend on the ER-vescile transport system. The results showed that S-RNase might influence the construction of microfilament cytoskeleton and eventually lead to self-incompatibility reaction.6. We silenced the homologous gene of MdABCF, PhABCF inpetunia. The self-incompatibility was broke in the transgenic plants, showing that MdABCF can hinder the transportion of S-RNase and then influence the self-compatibility.In conclusion, we got MdABCF transport protein by screening pollen factor that can interact with S-RNase. MdABCF can transport S-RNase across cell membranes and enter the vesicle of pollen tube with the help of microfilament, and then regulate the self-compatibility reaction.