Cloning Of Genes Related To The Hydrolysis And Synthesis Of (1,3-1,4)-β-glucan And Agrobacterium Tumefaciens-mediated Transformation Of Barley (Hordeum Vulgare L.)

Barley (Hordeum vulgare L.) ranks fourth among the cereal crops, and is grown and used widely around the world. In recent years, the research of barley transformation has made great archievement. The research progress and application of barley transformation technology and the investigation advancement of the physiological and biochemical function, synthesis and hydrolysis mechanism of barley (1,3-1,4)-β-glucan were introduced in this paper. To establish an efficient Agrobacterium tumefaciens mediated barley transformation system and explore the reverse-genetics approach to regulate the (1,3-1,4-β)-glucan content of barley, barley (1,3-1,4)-β-glucanase (EC3.2.1.73) isoenzyme EⅡI and barley cellulose synthase-like gene HvCslF4 were cloned and transfered to barley (cv. Golden promise) by Agrobacterium tumefaciens mediated infection with Immature embryos(IE) based on the development descripted above. Main results achieved were shown as follows:1. Barley (1,3-1,4)-β-glucanase(EC3.2.1.73) isoenzyme EⅠand EⅡfull-length CDS sequences(1005bp) were obtained through reverse transcript-PCR. To generate 1301E over-expression vector, EⅡCDS was cloned into pCAMBIA1301 plasmid under the control of 35S promoter by enzymatic excision and conjunction; To generate 1301DE endosperm and root specific over-expression vector,434bp length barley Hor-D promoter was obtained from barley genome by PCR and cloned into 1301E vector by replacing the 35S promoter. The two vectors was used in Agrobacterium tumefaciens mediated barley transformation experiment as follows.2. Agrobacterium tumefaciens-mediated transformation method including a co-cultivation step for Agrobacterium tumefaciens and Immature embryos on solid medium was used to conduct barley transformation experiment. Six 1301DE transgenic regeneration plants were produced from approximate 200 IEs, denoting the transformation efficiency was 3%, only one feitile line(T1) was acquired at last, which has single transgene copy verified by Southern blot. The transgene separation ratio in T1 generation(3.5:1) convicted by genome PCR, which complies with the Mendelian rules, and there's detectable transgene mRNA expression in root of T1 plant proved by RT-PCR.3. Barley Cellulose Synthase-Like Gene HvCslF4 full-length CDS was obtained by Reverse Transcript-PCR and cloned into modified pH7WG2D plasmid which has no report gene under the control of 35S promoter using Gateway(?) System to generate pH7WG2D-35S:Hv4 vector. Another Agrobacterium tumefaciens mediated transformation method based on the infection of immature embryos(IE) include a co-cultivation step with Agrobacterium tumefaciens in liquid medium was conducted in this barley transformation essay, but no transgenic lines were gotten at last. However, the exploration of the operation and procedure of Agrobacterium tumefaciens mediate barley transformation assays which including diffirent co-cultivation methods provide reliable data and useful experience for the establishing of high performance Agrobacterium tumefaciens transformation system of barley, meanwhile, the acquisition of 1301DE transgeneic seedline offers potential study basis for down-regulating the content of (1,3-1,4)-p-glucan in barley endosperm via reverse-genetics approach. Further research for completing the Agrobacterium tumefaciens transformation system of barley and clarifying the impact manner and activity mechanism of (1,3-1,4)-β-glucan content of barley that exerted by the transgene Hor-D:EII will be carrying out subsequently to supply vigorous support for molecular breeding of barley and promot the development of reverse-genetics research of barley, and enhance the overall biotechnology level of our country ultimately.