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Analysis Of Phenotype And Gene Identification Of The Arabidopsis Late-flowering Mutant Laf1

Posted on:2015-09-28Degree:MasterType:Thesis
Country:ChinaCandidate:J LiuFull Text:PDF
GTID:2180330431498877Subject:Cell biology
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Flowering is an important transition point from vegetative growth to reproductive growth in higherplant. Flowering of plants are subject to precise and intricate network regulation, and regulated by a varietyof endogenous and exogenous signals. The present study showed flowering time is regulated by factors thatinclude vernalization, photoperiod, autonomous pathway, GA and sugars.We have found a mutant laf1in the background of C24. This mutant showed delayed flowering timeand increased rosette leaves. Through statistical studies, we found that laf1flowered14days later than WT,and produced around20more rosette leaves than WT. In order to determine which flowering regulatingpathway the mutant gene was involved in, we treated the seeds at4℃for3days,4weeks and8weeksrespectively to simulate the vernalization conditions.With the increasing of vernalization time, the numberof rosette leaves and flowering time of laf1gradually reduced, but the vernalization treatment did notrecover the late flower phenotype, so laf1gene was sensitive to vernalization, but vernalization couldn’trestore the mutant’s phenotype, indicating that mutant gene does not function inflowering time throughvernalization pathway. To determine whether the mutant gene regulating the flower time through thephotoperiod pathway, the wild-type and mutant plants were kept under short-day (8h light/16h dark) orlong-day (16h light/8h dark) conditions respectively. With the extension of the light time length, the gapof the number of rosette leaves between wild type and laf1is gradually reduced. But laf1still shows lateflowering phenotype in both LD/SD conditions, which means that the mutated gene does not play a role inthe photoperiod pathway. Exogenous GA3was also applied to wild-type and the mutant, the flowering timeof wild-type and mutant were both earlier slightly after processing by GA3, but the treatment is still notrestored the mutant phenotype. Therefore, the mutate gene is out of the GA pathway. In conclusion, wespeculate that the mutated gene is involved in the autonomous pathway.Results of backcrossing experiments showed that laf1was a monogenic dominant gene. Because ofthe flowering time of F2plants were divergent when crossing C24with Ler, map-based cloning methodswere not applicable to determine the mutation site of laf1. Based on the above we decided to adoptbulk segregant analysis (BSA) combined with high-throughput sequencing means of mutants genome resequencing method to obtain mutation site. We have presumed the mutation site oflaf1was identified as the site of the nonsense mutation AT1G34095.Together with the late flowering phenotype of laf1, proteomics-related inevitable changes will alsooccur, so we used two-dimensional electrophoresis methods to analyze the proteins at the growth phase of47days (in this period C24is about to start flowering, but laf1still not enter the initial stage of flowering).We have got16up-regulated and down-regulated protein spots, and further assayed by mass spectrometry.The results showed that the expression of FLK gene which involved in autonomous pathway was increased,while the FRI was contrary. By RT-PCR for the expression of several autonomous pathway genes and FLCwere detected, only found that in the laf1FCA was a significant reduction when laf1about to blossom, theothers did not change significantly.
Keywords/Search Tags:Flowering time, Arabidopsis thaliana, high-throughput sequencing, two-dimensionalelectrophoresis
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