Cloning Of 4-coumarate: Coenzyme A Ligase (4CL) Gene And Expression Analysis In Galega Orientalis

Galega orientalis L. is a pasture species which is introduced to China at the end of the twentieth century. In this study, a 4CL-like gene was cloned from Galega orientalis. The gene expression pattern, and the response to abidic stress were studied. All of the research formed the basis for the implantation and application of Galega orientalis in China in future.The main research results achieved in this study are as following:1. Using RACE and RT-PCR methods, the full length cDNA, which encode 4-coumarate: coenzyme A ligase was cloned from Galega orientalis. The 1,916-bp full length Go4CL had a 1,653-bp ORF, which encodes 510 amino acids residues. It was indicated by the sequence analysis that the protein coded by Go4CL gene contains the AMP-binding conservative domain LPYSSGTTGLPK, a special conservative domains of the 4CL protein and the center for catalysis activation GEICIRG. Moreover, Go4CL protein has the high similarity with other homology 4CL protein.2. By the aid of Real-Time PCR, we detected that the expression of 4CL gene was the strongest in the root of Galega orientalis, and the least in leaves. Variety stress, such as PEG, ABA, MeJA and SA, all up-regulated the expression of Go4CL gene, which indicated that 4CL may play a role in the biotic and abiotic stress-resistance regulation pathway in plants.3. The sense and antisense expression vector of Go4CL was successfully constructed. These expression vectors were the foundation to regulate and control the expression quantity of Go4CL in the plant. These expression vectors also established the groundwork for breeding Galega orientalis with high resistance and with appropriate lignin content. We transformed the sense expression vector into tobacco with Agrobacterium tumfaciens LBA4404 containing an expression vector by Agrobacterium infestation. By PCR we detected the T0 generation transgenic plants and obtained the positive transgenetic lines successfully. In 17 positive seedlings of the T0 generation, there are 13 seedlings were positive in the PCR detection, the percent of successfully transgenic rate were approximately 77%.