The Effect Of Pear Recombinant S-RNase Protein On Pollen Tube Growth And The Mining Of Pollen SFBBs Gene

Self-Incompatibility(SI)is a typical mechanism for preventing inbreeding and promoting genetic variation in flowering plants.Pyrus belongs to the apple subfamily of Rosaceae and is an important economic fruit tree in China.Its fruit setting is regulated by Gametophytic Self-Incompatibility(GSI).After self-pollination,affected by the style of S-RNase,the pollen tube can only grow to 1/3 of the style,and the insemination process cannot be completed,resulting in a low rate of results.GSI is controlled by the SI pistil determinant S-RNase.S-RNase has been shown to encode RNase,a protein with a similar function to cytotoxins,which specifically inhibits the growth of self-pollination tubes and prevents inbreeding.SFBBs are candidate SI pollen determinants,which can degrade non-self-S-RNase by forming SCF complexes and promote cross-pollination.The current research shows that the S site generally contains multiple pollen SFBBs genes,for example,6 pollen S F-box genes are isolated from the S4 locus,and 10 pollen SFBBs genes are isolated from the S2 locus.At present,only three and four pollen SFBBs genes have been reported in "Yali" S21 and S34,and many unknown pollen SFBBs genes are waiting to be discovered.Addionally,mechanism of action of S-RNase is still unclear.The mechanism of S-RNase expression in vitro can accelerate the related research progress.In this paper,two S-RNases from Yali were purified by in vitro expression technique,and the effects of these two recombinant S-RNases on pollen tube growth were analyzed.In addition,we constructed unknown SFBB gene at the S site of "Yali" pear by constructing a genomic library.The main results are as follows:1.It was confirmed that S-RNase was expressed in vitro to inhibit pollen tube growth.The prokaryotic expressed S-RNase protein has the same function as the style S-RNase protein and can inhibit pollen tube growth.After adding the recombinant protein for four hours,there was a significant difference from the control group.After one single recombinant protein was added,the pollen tube length was about half of the control,and the pollen tube length of the two recombinant proteins added was 1/3 of the control group.2.The genes regulated by recombinant S-RNases was screened.A total of 14 differential genes were found in the transcription data of recombinant protein-treated pollen.The functions of these genes mainly include carbohydrate transport and metabolism,post-translational modification.replication,recombination and repair,post-translational modification,protein turnover and chaperone proteins,inorganic ion transport and metabolism,intracellular transport secretion and vesicle transport.Three up-regulated genes(Pbr028631,Pbr031310,and Pbr034143)that may be involved in ROS production and a down-regulated gene(Pbr039542)that may be involved in the spread of S-RNase in vivo.It is speculated the protein encoded by the Pbr039542 gene may be involved in the vesicles and microtubule system,and that system will help diffuse the recombinant S-RNase.3.6 new SFBBs genes were found.Seven F-box genes with high expression in pollen were found from the genomic library.Sequence comparison and cluster analysis showed that 6 of the F-box genes might be SFBBs,while Pbr037090 was lower than each gene,and it contained a nucleotide sequence not found in other SFBBs,presumably it may not be SFBBs gene,possibly SLFL.