The Application Of Ckrc-like Mutants Screening System And Studies On Rw1(Y81)

It has been more than130years since auxin was discovered. With the efforts of scientists, a huge accomplishment in synthesis, signaling and polar transport of auxin has been achieved. However, our knowledge of auxin is still very poor. There exists a complicated network concerning the synthesis of auxin. As far as we know, the network is composed of several pathways that usually interact with each other. Unfortunately, many genes in the network are still needed to be discovered, and the regulations and interractions of the pathways are also unclear.As auxin participates in many important activities in plant growth and development and function redundancy of auxin biosynthesis and metabolism genes exists, it is hard to isolate mutants in which endogenous auxin declines.In present study, a screening system of auxin biosynthesis and metabolism was described, which was built in our laboratory based on the theory that cytokinin improves auxin synthesis while inhibits transport of auxin; the resulting uneven distribution of auxin in roots will in turn make mutants produce distinguishable phenotype with root curling in medium ith0.1μM tZ.Based on the screening system, we isolated17mutants from almost360,000seeds. Using biochemical complimentation, genetic allelic analysis, TAIL-PCR, Inverse-PCR, micoarray assay and genomic re-sequencing, seven of them were confirmed as auxl mutants, six ckrcl/taal mutants, one wei2mutant and one pin2mutant. The last mutant was unknown, which was then named as rwl (y81). The phenotype with root curling was found for rwl (y81) in0.1μM tZ, which, however, could be restored by IAA, NAA and2,4-D. When rwl(y81) was treated by auxin and cytokinin, the expression of primary responding genes of auxin and cytokinin (IAA1/IAA2and ARR5/ARR15, respectively) was found in the same level compared with the wild type, indicating normal signaling of auxin and cytokinin in rwl(y81). When cultivated in medium for seven days, rwl (y81) produced significantly shorter primary root and hypocotyl than wild type, the characteristic phenotype of low level of auxin, which was also proven by GC-MS analysis.Using genomic re-sequencing, four T-DNA insertions and almost15,000single nucleotide polymorphisms (including Col-2and Col-0) were observed. We are using co-segregating and map-based cloning to locate the mutated genes.It is known that the balance of IBA-IAA affects the equilibrium of IAA. As significant difference was found between rwl (y81) and the wild type in their responses to IBA, it can thus be concluded that rwl (y81) could likely take part in the IBA-IAA equilibrium process.Taken together, our results indicated that our screening system of auxin synthesis and transport was highly effective. The rwl (y81) mutant, which was selected from almost360,000seeds, should be an unrecognized mutant taking part in regulating auxin equilibrium.