The Combinational Vectors Construction And Genetic Transformation Of PSD1 Candidate Gene, A Key Gene For Floral Development In Rice (Oryza Sativa L.)

Because of the advancement in molecular biology and genetics, developmental biology has become one of the most noticeable fields in the biology. It has made great progress on the flower developmental research of eudicot plant, while it still needs more works for that of monocot plant. The study of the key and related genes of rice foral development can make us further understanding about its molecular mechanism.Pistil-stamen degeneration 1(psd1) is a homeotic mutant in rice floral organ development, which was found in a double haploid popular from the cross between Taiwanjing and Gui630．Genetic analysis revealed that psd1 mutant was caused by a recessive mutation in a single nuclear gene. We gained a 50kb candidate region for this gene in long arm of 2^nd chromosome of rice by fine-mapping method. Based on above results, we went further as follows:(1) A MADS gene was found after all genes by function prediction in candidate region. This MADS gene has only one copy in the genome; the protein has 250 amino acids and belongs to MIKC type of II type MADS transcriptional factor family in eukaryote; further belongs to AGL6-like subtribe of SEP gene family and has two conservative fragments in C region with other AGL6-like protein: ECEPTLQIGY and ATSQGAAGGENNFMLGWV. Sequencing analysis found the MADS gene has a 2915bp deletion and a 852bp insertation in the promoter and 5' region of psd1 mutants by amplification of genomic sequence in wild type and mutants. Then this MADS gene was chose as candidate.(2) For understanding this MADS gene expression position, the combinational vector pCG1, which GUS gene transcription was controled by 3560bp upstream promoter came from of the MADS candidate gene, was constructed based on the binary vector pCAMBIA1391Z. Then it was introduced into the calli come from seed of PSD1 homozygote and wild type Nipponbare. The vector pCG1 has a high transformation ratio in Nipponbare, gained 43 resistant calli. But a low transformation ratio appeared in PSD1 homozygote, gained 7 resistant calli, because calli became so wet in screening culture.(3) For over-expression analysis, two combinational vector pCE1 and pGE1, which PSD1 ORF transcription was controled by 35S and Ubi promoter respectively, were constructed based on the binary vector modified pCAMBIA1301& pGA1611． Then two combinational vectors were introduced into the calli come from seed of PSD1 homozygote. Because the calli in screening culture also became so wet and growth was inhibited, gained 2 resistant calli for pCE1 while pGE1, 1 resistant calli lastly.(4) For functional identification, the combinational vector pCF1, which PSD1 ORF transcription were controled by 3560bp upstream promoter came from of MADS candidate gene, was constructed based on the binary vector pCAMBIA1300．Then the pCF1, pCE1 and pGE1 were introduced into the calli come from seed of PSD1 heterozygote selfing. We just gained 6 resistant calli for three vectors (the pCF1, 4 resistant calli; pCE1, 1 resistant callus; pGE1, 1 resistant callus).