| Utilization of male sterility in plants make the mass production of hybrid rice,sorghum,rape seeds become possible,and cytoplasmic male sterility is crop heterosis utilization,recurrent selection and population improvement tools,is an important material to study pollen development,cytoplasmic and nuclear cytoplasmic interaction..Although the corn mainly adopt the method of artificial emasculation for hybrid seed production,but with the increase of labor costs,many large seed companies started using sterile seed production.In the three types of sterile cytoplasm of maize,C type cytoplasm is a common type of infertility.In the previous study,the Rf4 of the C cytoplasmic male sterility restorer gene of maize was cloned,which included two functional domains,including SBP and ANK.To elucidate the biological function to restore gene Rf4,this study constructed a c DNA Library of maize tassel at different developmental stages,and by using the yeast two hybrid technology of Rf4 two functional domains of the interacting proteins were screened,the main results were as follows:1?By extracting maize tassel RNA,reverse transcription for c DNA,the c DNA Library of maize male spike was constructed by using SMART technique.Will c DNA library cloned into the p GADT-7 vector was constructed a full-length c DNA library of mixed samples of maize tassel in the different growth time for yeast two hybrid,the initial library titer was 1.08 x 1011 cfu/ ml,storage capacity for 1.08 x 107 cfu no-load rate is 0%,colony PCR insert and the average size of the fragment above 1.0 kb.The detection of the plating showed no contamination of c DNA library,which showed that the c DNA library was able to meet the needs of screening of the target protein and storage.2?RF4 gene was used as a template,PCR amplification,recycling,enzyme digestion,connection and other steps will be two functional domains SBP and ANK and yeast two hybrid bait vector p GBKT-7 connection,get the bait plasmid BK-SBP and ANK-BK.By sequencing,the bait vector and BK no-load were transferred to Y2 H yeast strains,and using SD seletive medium to validate the toxicity and self activation of validation.The results showed that in SD /-Trp culture medium,the size and number of the clone is no obvious difference between transferred to the no-load and transferred to SBP-BK bait vector,the bait protein had no toxicity.,transferred to the colon of transferred ANK-BK bait vector is significantly smaller than transferred no-load,the bait protein have toxicity and couldn't go on with the experiment.In SD/-Trp/-His/-Ade medium,no colony growth,indicating the vector no self activation.3?Mating Yeast method was used to screen the library of functional domain SBP,screening in SD selective medium,through the expression of AUR1-C,ADE2,HIS3,MEL1 four reporter genes,a total of 44 positive clones were obtained.Protein function prediction and analysis were carried out on NCBI,and 4 possible candidate proteins were identified according to the results of sequencing and bioinformatics analysis.They are mitochondrial membrane protein Tom40,ACP synthase,TCP-1 chaperone protein,and chaperone protein that promotes the activity of ATP... |
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