Screening, Cloning, Sequencing And Specific Analysis Of Sterility Gene In Rice Mitochondrial DNA Of Nucleo-Cytoplasmic Interaction By RAPD

Plant cytoplasmic male sterility is one of hotspots in plant genetics. Moreover, rice cytoplasmic male sterility is the basis of breeding filial generation in rice. Since Sears set forth the theory of three types in 1945,exploring the genes of rice male sterility has become the current focus in plant genetics. Rice cytoplasmic male sterility is female parent genetic phenomenon, which is the main means to utlize filial superiority. Many results of research have showed that rice cytoplasmic male sterility was greatly concerned with rice mitochondrial gene group. Therefore, it is very significent to analyse the structure of rice mitochondrial gene group and disclose the molecular mechanism of rice cytoplasmic male sterility. 11-32 male sterile line, maintainer line and filial generation are used as samples in this experiment. We have screened their differential fragments and demonstrated the relationships with male sterility. The results show as follows:1. Optimize the reaction conditions of RAPDWe perform a series of experiments by changing the concentrations of Mg2+, dNTPs, Taq enzyme, sample DNA and primers.The results show the optimum conditions in 25 P 1 volume : Mg2+concentration 2.0mmol/l ; dNTPs concentration 200 u mol/1; Taq enzyme concentration 1.5U ; sample DNA concentration 0.5ng/ u 1 ; primer concentration 0.2 u mol/1.2. Screen rice mitochondrial DNA with 260 RAPD primersUnder the optimum conditions, 11-32 male sterility line, maintainer line and Fl hybrid mitochondria! DNA are screened by 260 RAPD primers from OPA to OPM. As a result, we find 22 differential fragments. Among them, OPH19-1800,OPJ09-400, OPJ18-1400,OPJ18-1000 and OPJ20-300 may have relations to rice male sterility.3.Reclaim and clone the differential fragments :OPJ18-1000 and OPJ18-1400Two specific fragments OPJ18-1000 and OPJ18-1400 are obtained from rice mitochondrial DNA of 11-32 and II-you 949 by RAPD under the optimum conditions in ultraviolet light. Then, they are recombinated into pGEM-T easy vector.4.DNA sequencing and specific analysis of tissueTwo specific fragments OPJ18-1000 and OPJ18-1400 are sequenced by ABI377 sequencer . The sequenced lengths of two differential fragments are 1068 bp and 1481 bp, respectively. The result of Southern hybridization demonstrates that the differential fragments are true. When hybridizing with II-32A, II-32B and II you 949 by OPJ18-1000 probe, we find OP J18-1000 has not the same effects on II-32B which has effects on rice II-32A and II you 949 cytoplasmic male sterility in transcription. Though RNA dot hybridization is negative, OP J18-1400 has a conservative domain of seven base pairs DNA sequence: 5-TTCCCTC-3, which is a homologous recombination hotspot domain in atp6 gene. It induces the formation of chimeric gene in mitochondrial DNA and causes rice male sterility further.