Perfection Of T-DNA Mutant Library And Functional Research Of Flowering Time Geness OsELF3 And CDF1

Rice is an important staple crop and also the model plant of monocotyledons and cereal crop for functional genomics research. Mutant is one of the most strait-forward and efficient way to study gene function. And then the large mutant library is a very important platform for large-scale, comprehensive and systematic gene research on rice whole genome. Our lab have established large rice T-DNA insertional mutant library transformed by Agrobacterium. Because Tos17 is active under tissue culture conditions, additional Tos17 insertions are translocated in the rice T-DNA insertional population. My study work is to enrich data platform of our T-DNA mutant library:we isolated the flanking sequences of insertional tags, T-DNA and Tos17, and built the flanking sequences database as the reverse genetic research platform of rice functional genome; we screened the T1 mutant lines under normal growth conditions and mainly collected hundreds of mutant lines related to panicle development. These rice mutant lines fitting to Mendelian separation ratio were selected for rice functional genomics research. Then we examined whether the mutant phenotype is co-segregation with the T-DNA or Tos17 homozygous insertion or not. We collected 9 co-segregation lines, providing research materials for the corresponding gene cloning and functional analysis. According to the late flowering mutant lines, we cloned corresponding genes and researched the function and mechanism of the genes in flowering time regulation network. The main results were described as follows:1. I have isolated 5,806 flanking sequences positioned on rice genome (E<10-5),2,372 flanked T-DNA insertion tags,3,434 flanked Tosl7 insertion tags, which enciched the mutant flanking sequene tags data and will benefit the reverse genetics strategy in rice gene cloning.2. Screening 11,418 mutant lines with ZH11 background, I observed 196 lines showed abnormal phenotype about panicle development. Through the further screening of two generations of 354 lines with sterility or abnormal panicle, identified homozygous type and heterozygous type. Positive screening 1600 lines with Nipponbare background,64 mutant lines were obtained. Reverse screening 377 lines, obtained 136 lines with abnormal phenotype. The hundreds of mutants provide materials of forward genetics for rice functional genome research.3. Through the screening of mutant lines, I obtained 9 co-segregated lines such as 07YY150, 07YY111,07YY026,07YY108,07YY040,08RS075,08RS142,08RS196,08RS218 with the mutant phenotype as lax panicle, sterility and reduced branches and spikelets, semi-sterility and abnormal top spikelets, stick shaped panicles, semi-dwarf and abnormal top spikelets, delayed heading-date, delayed heading-date, less tillering, dwarf and tiny seed. These lines of co-segregation will be used to corresponding gene cloning and functional studies. More detail analysis was done with the mutant line 08RS075 and 08RS142 to isolate and characte the corresponsive genes, analyze the function of the genes.4. Successful complementary test proved that the delayed-heading date of 08RS142 is due to the loss function of OsELF3. The mutant was designed as oself3. The mutant oself3 was about two weeks later than wild type in summer of Wuhan under natural long day growth conditions, but no obvious variation was observed under short day conditions. It proves that OsELF3 is the gene that controls flowering time of rice mainly under long day conditions.5. Successful complementary test proved that the delayed-heading date of 08RS075 is due to the loss function of CDF1. The mutant was designed as cdfl. The mutant cdfl was about two weeks later than wild type under long day conditions and short day conditions. It proves that CDF1 is the gene that controls flowering time of rice under short day and long day conditions.6. Statistical analysis about leaf emergence rate of wild type and mutants respectively showed that compared with the wild-type, oself3 and cdfl have no variation. Statistical analysis about agronomic traits of wild type and mutants respectively showed that compared with the wild-type, oself3 and cdfl have no variation. Under various light conditions, statistical analysis about the lengths of coleoptiles between wild type and mutants respectively showed that compared with the wild-type, oself3 and cdf1 have no variation. These proved that mutations of OsELF3 and CDF1, only affecting rice heading stage, do not affect the other traits, such as:rice growth rate, agronomic traits, photomorphogenesis and skotomorphogenesis.7. Semi-quantitative RT-PCR analysis showed that compared with the wild-type, the mRNAs of OsELF3 were reduced in oself3, the mRNAs of CDF1 were reduced in cdf1. Loss function of OsELF3 and CDF1 both influenced the expression of Ehdl, Hdl and Hd3a.8. Real-time quantitative RT (qRT)-PCR analysis of flowering-time related genes showed that both OsELF3 and CDF1 showed diurnal expression patterns with a peak at night and a trough at dusk under LDs and SDs. Compared with the wild-type, the rhythmic expression of Hdl, Ehd1, Hd3a were changed obviously in oself3 and cdfl.9. Real-time quantitative RT (qRT)-PCR analysis showed that compared with the wild-type, the rhythmic expression of circadian-clock related genes were changed obviously in oself3, but not in cdf1. These showed that OsELF3 and CDF1 maily affect the expression of main flowering time regulators:Hd1, Ehd1 and Hd3a. OsELF3 also affected the expression of circadian clock genes, but CDF1 didn’t affect the circadian clock genes.