Transformation And Functional Analysis Of Soybean SCTF-1 Gene Encoding A C₂H₂-typezinc Finger Protein

As a high oil crop,soybeans are grown widely around the world, they have high economic value and the large market demand. However, the natural conditions have some influence on yield of soybean, low temperature is one of the important factors affecting the growth and yield of soybeans. Therefore, How to use molecular biology techniques developed and improved soybean varieties will attract more and more attention.Zinc finger protein is a transcription factor that has an important relationship with the growth of plants and tolerance under abiotic stress, and the study of C₂H₂ zinc finger protein is one of the more extensive study. The researchers in domestic and foreign of cold-inducible zinc finger protein plants are mostly concentrated in the petunia, rice and tobacco,but not a lot for C₂H₂-type zinc finger protein from soybean.The analysis of SCOF-1 is clearer, SCOF-1 is induced by the ABA and hypothermia, its overexpression induced COR gene while enhancing without cold acclimation GM crops resistance to the cold. SCTF-1 gene used the sequence information of SCOF-1 protein as a probe for cloning. In this research, the expression vector p CPB-SCTF-1 was built,introduced into the recipient species "Ji Nong 28" soybeans via agrobacterium-mediated transformation and pollentube pathway transformation.Testing these plants by PCR,southern bloting,real-time PCR and cold tolerance analysis of transformed soybeans, providing a theoretical basis for using gene.The main results: 1. The recombinant expression vector p BI121-SCTF-1 as a template, primers with restriction sites amplified fragment, the fragment was purified and double digestion by 1%agarose gel electrophoresis, constructed expression vector p CPB-SCTF-1. 2.The overexpression vector p CPB-SCTF-1 was transformed into soybean "Ji Nong 28" via agrobacterium-mediated, which obtained 2 strains positive plants of T0 generation.After seeds growing, 4 T1 positive plants were obtained through PCR detection. The overexpression vector p CPB-SCTF-1 was transformed into soybean "Ji Nong 28" via pollen tube pathway, obtained T0 generation of transforming seeds 65 of "Jinong 28", got 6 strains of T1 transformed after generation-adding. 3.Southern blotting showed that SCTF-1 was integrated into the genome of recipient soybean as a single copy. The relative expression of leaves are the highest and stems are the lowest. 4.The quantitative real-time PCR showed that the expression of introduced SCTF-1 gene was increased obviously in root, stem and leaf compared to the non-transformed soybean. Compared with normal temperature,the expression of target gene under the low has increased. 5. Under the stress of low temperature, compared to non-transformed plants, the relative electrical conductivity of transformed plants were significantly lower from 21.10% to 23.09%; the content of malondialdehyde（MDA） of transformed plants was reduced from 10.56% to 11.74%, the peroxidase activity of transformed plants were increased from 25.02% to 30.38%. There is no atrophy, wilting and rolling in the leaves of transgenic plants. Therefore, the cold tolerance of transformed plants was significantly enhanced by overexpressing of SCTF-1 gene.