Screening And Analysis Of Flowering Mutants In Rice

Heading date is an important character which decides the rice variety area and theseason compation. Heading date heredity has closely association to instructing breedingpractice, the improvement of varieties and the variety promotion. Making the heading dateearly is especially significant to enhance production and improve quality of rice. Themutant is good material to conduct the gene function research. In recent years, we haveobtained many mutants, including early heading mutants and serotinous mutants. It is veryfeasible to clone genes which control flowering time. Rice, the short date pattern plant,plays an important role on exploring flowering mechanism.In this research, early heading mutants are screened from T-DNA insertion labelmutants (including activation tagging and enhancer trapping lines), moreover, flankingsequences are separated and analysised. We have made the method of cloning gene forbetter. Experimental results are as follow:1. We investigate and screen to obtain about 350 flowering mutants from 50 000independent label mutants in the field. Early heading mutants are double to serotinousmutants. The efficiency of enhancer trapping is 0.1％higher than activation tagging. Mostof early heading mutants are at least a month earlier than CK, a few of them are twomonths earlier. Four serotinous mutants are a month later than CK.2. We use PCR-Walking to separate flanking sequences and improve this system. It issuggested that DraI is better than SspⅠ. In this research two enzymes DraⅠ和SspⅠare usedto get more flanking sequences. The volume of the second PCR is changed from 20μL to40μL does not reduce the final efficiency. We already obtain approximately 277 flankingsequences of T-DNA and 289 flanking sequences. Amplifying efficiency of flankingsequences is about 80％. We obtain 499 successful flanking sequences including 244T-DNA and 255 Tos17. Efficiency of flanking sequences is about 88％.3. Making preliminary analysis to the insertion site, we can known that Tos17 inclineto insert gene region and T-DNA does not have clear trend between gene region and spaceof genes. The constrast between the enhancer trapping and activation tagging libraryshowed the amplification efficiency of the enhancer trapping is better than activationtagging,but worse in acquiring successful sequences. 4. We explain that different results according to different insertion sites. T-DNAinsertion leads that gene function loses and expression of gene will be up or down. It mayactivate genes, whether 35S of activation tagging insert gene upstream or downstream.Particularize that different sites insert the same gene and different insertion sites ofmutants,etc.5. Analysis of gene and protein, such as kinase protein and Hd1. About 60 genes havebeen obtained after studying the gene function. 2 candidate genes of them already areconfirmed through separating experiment.Takeing advangtage of label mutants to clonegene has a long way to go and we think deeply.