Primary Functional Analysis Of The Flowering Time Geng RID1 In Rice

We have identified a never-flowering rice mutant(rid1) caused by a T-DNA insertion at the RID1(Rice Indeterminate 1) locus in our previous study. In this study, we investigated the functions of RID1 by over-expressed, special tissue-expressed, in situ hybridization and in vitro expressed RID1 protein. Our results provide a basis for studying the flowering molecular mechanisms in rice. The detail results in this study were summarized as follows:1. The genomic sequence and full-length cDNA of RID1 gene have been cloned. Two over-expressed vectors named Ubq:RIDlg and Ubq.RID1c were constructed based on them. Then these two vectors were transformed into rid1 by Agrobacterium tumefaciens-mediated transformation.109 regenerated plants were generated through transformation with Ubq:RID1g vector.20 out of them are positive transgenic plants, and 9 among them are flowering naturally. For Ubq:RID1c vector,80 plants were regenerated and 7 are positive transgenic plants 6 among them are flowering naturally. This indicated that the over-expressed vectors are functional and the full-length cDNA of RID1 gene is also functional.2. The expression level of flowering gene RID1,Ehd1,Hd1,Hd3a,RFT1 has been detected by RT-PCR in the corresponding over-expressed transgenic plants. The results indicated that the RID1 gene was over expressed in which resumed flowering plants. And we found that the expression of RID1 gene couldn't be detected by RT-PCR in two negative-transgenic plants named #68 in Ubq:RID1g and #27 in Ubq:RID1c transformation accidentally, but both of them were flowering naturally. We also detected the expression of RID1 gene in the offsprings of #27 by RT-PCR, the results indicated that all of the generations are flowering without the expression of RID1 gene, meanwhile the expression of the key flowering gene Hd3a could be detected.3. By using the Hd3a promoter which is fuctional specally in mature leaf tissues, we constructed a vector named Hd3a::RID1c. The vector was transformed into rid1 by agrobacterium tumefaciens-mediated transformation.10 out of the 41 regenerated plants were positive-transgenic plants,2 among them were flowering naturally. We analyzed the expression pattern of RID1 gene in the positive-transgenic plant #28 by RT-PCR, and detected the expression of RID1 in mature leaf, leaf sheath, shoot and root tissues. The results indicated that RID1 gene haven't been expressed specially.4. We constructed a vector named Oshl::RIDlc by using Oshl promoter which is fuctional specially in meristem tissues, the vector was transformed into rid1 by agrobacterium tumefaciens-mediated transformation.8 regenerated plants have generated through transformation, and 7 are positive-transgenic plants, all of the positive-transgenic plants are flowering naturally. We analyzed the expression pattern of RID1 gene in the positive-transgenic plant#6 by RT-PCR, and found that the RID1 expessed in mature leaf, leaf sheath, shoot and meristem tissues. The results indicated the RID1 was not expressed specially.5. A rice phloem tissue specific promoter rpp16 was used to constructed a vector named Rpp16::RIDlc. The vector was transformed into rid1 by agrobacterium tumefaciens-mediated transformation.31 regenerated plants have been collected, and 19 among them are positive-transgenic plants with 9 out of them are flowering naturally. We analyzed the expression pattern of RID1 gene in the positive-transgenic plant#8 by RT-PCR, the results indicated that the RID1 expessed in leaf, leaf sheath, shoot tissues which are in rich of vascular bundle, and we almost can't detect the expression of RID1 gene in the meristem and root tissues which are lacked of vascular bundle. We also detected the expression of key flowering gene in positive transgenic plants by RT-PCR, the results indicated that the RID1 gene which expressed specially in rice phloem can promote rice flowering and regulate other flowering genes.6. Two expressed vectors named pGEX-RID1C60 and pGEX-RID1M80 have been constructed and expressed in E.coli BL21 (DE3) using GST expression system in order to preparate antibody. Only RID1C60 protein was expressed successfully.7. In order to detect the expression partten of RID1 accurately, in situ hybridization method has been employed in detecting the mRNA of RID1 gene in rice SAM and immature leaf tissue from the plant which is about 30 days after sowing. The results indicated that the mRNA of RID1 gene was under detected level in SAM and immature leaf tissue both in ZHl 1 wide type and ridl mutant.