Establishment Of Reporter Gene Expression Pattern Lines From An Enhancer Trap Mutant Library For Using Cis-element In Rice And Functional Study Of RID1 Gene Which Controls Flowing Time In Rice

The changing of gene expression pattern is a powerful tool for studying of gene function. This strategy can help researchers to know the temporal and spatial effects of the given genes, and discover their unknown functions. The GAL4/UAS two component ectopic expression system in this research is a recognized plan to chang the expression pattern of target genes, the tissue and time specific expression of the target gene can be put into practice by the genetic cross of pattern lines and target lines.The GAL4/UAS Enhancer Trap lines of our lab can be used as an original source of the pattern lines. A reporter gene expression profiling can be established by observing of the reporter gene expression. In the other hand, the traping lines can provide us a mutant library for studying of gene function. All of the the pattern lines and the rid1 mutant in this study come from the Enhancer Trap library. The main results are as follows:1. The introducing of pEGFP vector (modified by Dr. Wu Changyin) into the rice Enhancer Trap mutant library brings the differentiation rate to 57.35%. A positive detection of 4,416 lines shows the positive rate of those lines achieve to 91.21%. The pEGFP vector is fit for construction of mutant library in Zhonghua 11.2. A collection of 4,416 independent T0 plants from the pEGFP lines have been dissected for positive test and reporter gene expression pattern observation in different stages and tissues(without roots). The reporter gene expression ratio is about 60% in T0 plants.3. One hundred and forty specific expression T1 lines from T0 expression pattern library have been collected and grow in water culture pond for repoeter gene expression observation(within roots). The lost of the reporter gene expression in T1 generation compare to the T0 generation have been observed, and the rate of the lost is different around tissues.4. More selections have been done on T0 and T1 specific expression lines, and a number of undetermined pattern lines have been chosen.45 final pattern lines have been gathered from the undetermined lines, their expression patterns cover the main tissues of rice plants.5. Souther blot has been done in 143 strains from the 45 pattern lines for detecting the T-DNA copy number. The result shows that the copies of T-DNA have no distinctly segregation though the reporter gene expression patterns have stabilized by several generation's selection. Multiple copy lines still remain and few single copy strains arised.6. Several pattern lines from our screening have been used to cross with the target line (pGOC17 vector containing GUS reporter gene and miR156 gene controlled by UAS transformed into Zhonghua 11). Ectopic expression effect has been observed in the offspring of this target line crossed with our pattern lines. Our pattern lines can be well used in the GAL4/UAS ectopic expression system.7. Six lines with flower specific reporter gene expression pattern have been collected and the reporter gene expression of those lines in panicle development has been observed. The difference of the reporter gene expression pattern in time and location shows the differentiation of targeted genes.8. One hundred and six flanking sequences have been isolated and analyzed from 68 Enhancer trap lines.13 of those sequences show the same direction of reporter gene and targeted gene expression.9. Five hundred and seventy eight enhancer trap lines have been observed in normal cultivation conditions, and 177 mutant lines have been collected. The relativity of the mutation and reporter gene expression pattern has been discussed and 31 relativ lines have been found.10. The phenotype of a "never-flowering" mutant rid1 has been described detailedly. rid1 mutant plants stayed in vegetative growth stage long after the harvest of wild type plant, even 600 days after sowing. The statistics of leaf numbers shows that the mutant plant produces one leaf within every 10 days after the heading of wild type plant. Elongation of the internode and more nodes can be obersived in rid1 plants. SEM analysis indicated that the SAM of ridl can not chang from vegetative growth to reproductive growth state, and the "never-flowering" phenotype of rid1 have a direct correlation with this reason.11. More than 65% of the positive complementary plants can recover the "never-flowering" phenotype of rid1 mutant with a widely distributed heading date. The difference between plants even reaches to 111 days. The possible reason for this condition is the differential expression of RID1 gene led by position effect. 12. GFP observation of RID1::RID1:GFP plants shows the presence of RID1:GFP protein in young leaf and SAM, but our pre-data shows that the RID] mRNA only appears in the young leaf. The possible reason is the low expression level of RID1 gene and different detection methods, but we need more evidence.13. The "never-flowering" phenotype could be led into other rice varieties by crossing with rid] heterozygous plants since the rid1 mutant plants can not flower. The phenotype separation can be seen in F2 plant, indicated that the RID1 gene play an important role in other rice varieties.14. Over-expression of Hd3a and RFT1(FTL3) can recover the mutant phenotype of rid1 mutant separately and bring a very early flowering phenotype to it. These phenomenons indicated that the mutation of RID] gene inhibit the expression and accumulation of Hd3a and RFT1.