A Protein Complex Of The Sc Locus Controls Indica-Japonica Hybrid Male Sterility In Rrice (oryza Sativa L.)

Hybrids between indica and japonica rice?Oryza sativa L.?show a significant heterosis.However,hybrid sterility is the main barrier for the utilization of inter-subspecies heterosis.Therefore,cloning of the genes for hybrid sterility and elucidation of the molecular mechanism is very important for basic research and crop breeding.Dozens of loci controlling hybrid sterility have been found in rice,and Sc is one of them.This laboratory has cloned a candidate gene of the Sc locus using positional cloning strategy,and the results showed that the Sc-i locus consists of four tandem repeats,each containing Sc-i gene copy named as Sc-is,Sc-ia,Sc-ib1,and Sc-ib2,of which Sc-is is a pseudogene.Previous analysis at the indica Sc-i allele identified 3 more putative genes in each copy of the upstream region of Sc-ia,Sc-ib1,and Sc-ib2,which are named Sc-iP1,Sc-iP2 and Sc-iP3.In this study,gene knock out and gene silencing by CRISPR/Cas9 and RNAi,and transgenic over-expression,are used to analyze the functions of these genes.In addition,Yeast two-hybrid system and bimolecular fluorescence complementation?BiFC?were used to confirm the interaction between the proteins of these four genes.The results are as follows:1.RACE analysis was used to determine the coding sequence of Sc-iP1,Sc-i P2 and Sc-iP3.2.Knock out and RNAi-silencing of any one of the genes Sc-iP1,Sc-iP2 and Sc-iP3 in the hybrid F₁ rescued fertility of pollen grains carrying Sc-j.These results suggest that Sc-iP1,Sc-iP2 and Sc-iP3 also are necessary for the Sc-mediated hybrid male sterility.3.Over-expression constructs of Sc-i P1-OX,Sc-iP2-OX,Sc-iP3-OX,and Sc-iP1/P3/P2/Sc-i-OX were introduced into T65.These transgenic plants are valuable for study of gene function on hybrid male sterility.4.Sub-cellular localization tests indicated that Sc-iP1?Sc-i P2?Sc-iP3and Sc-i were localized in the nucleus.5.Yeast two-hybrid and BiFC analyses consistently showed that interactions occurred between Sc-iP1and Sc-iP2,Sc-iP2 and Sc-iP3,Sc-iP3 and Sc-i,and Sc-i and Sc-iP1,but did not occurred between Sc-iP1and Sc-iP3,or Sc-iP2 and Sc-i.These results demonstrate that a complex of the four proteins functions on the Sc-mediated hybrid male sterility.