Functional Analysis and the Application of the B class MADS box genes in Oncidium Gower Ramsey

• Main Authors: Chih-Liang Chien, 簡誌良
• Other Authors: 楊長賢
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/71101425183512917212

碩士 === 國立中興大學 === 生物科技學研究所 === 99 === To investigate floral formation in Oncidium ‘Gower Ramsey’, two paleoAPETALA3 (paleoAP3) genes, O. Grower Ramsey MADS box gene5 (OMADS5) and OMADS9 were characterized in our laboratory. The OMADS5 mRNA was absent in lips and was significantly down-regulated in lip-like organs in the peloric mutant flowers. In addition, the mRNA for OMADS9 was expressed in lip and petals and a high amount of OMADS9 expression was also observed in lip-like organs of peloric mutant flowers. This suggested a possible negative role for OMADS5 in regulating lip formation. To explore the function of OMADS5 in lip formation, we first cloned OMADS5 promoter with 2.3 kb by genomic walking. In addition, the vector pEpyon-32H containing OMADS5 was transformed into 45-day-old Oncidium protocorm-like body (PLB) via Agrobacterium tumefaciens. The transformants were selected by 8 μg/ml hygromycin G10 medium. To confirm that these plants were transformants, total DNA was isolated and analyzed by PCR from PLBs of four transgenic plants to find the appearance of the antibiotic resistant gene (hptII) and OMADS5 gene. We further used new PLB with high efficiency of transformation to perform the 35S::OMADS5 and 35S::OMADS9 transformation. Furthermore, to modify the floral shape of Oncidium, OMADS5 RNAi and OMADS9 RNAi as well as OMADS5/OMADS9 fused with either SRDX or VP16 sequence were constructed and transformed into Oncidium. In order to obtain a useful RNAi transferred construct in Oncidium, an α-tubulin intron from Oncidium genomic DNA was cloned. New Oncidium varieties are difficult to generate; hence molecular breeding is an attractive approach to create a new flower shape. It is expected that the manipulation of B function MADS box genes in this study will potentially convert perianths into differential shapes.