| Soybean [Glycine max(L)Merr] as one of the important oil crops was applied in each fields of social life in China.However,the production of soybean was affected by underground insect,resulting in a large number yiled of soybean decreased,grub,one of the Coleoptera insects was harmed for soybean particularly.During soybean growth period,the roots were feeded by Coleoptera insect grub that effected soybean growth,resulting in yield of soybean decreased between 10.00% and 20.00% every year.In severe cases,it can reduced beyond 30.00%.Transformation of the insect resistant gene into soybean by using genetic engineering can improve the resistance ability of soybean against Coleoptera insect grub.In this reaserch,we obey the principle of Seamless cloning technology,using pCAMBIA3301 as basic plant expression vector,then insect resistant gene Cry8-like and soybean root-specific promoter fragment RSP were recombined to the basic vector,and Root-specific plant expression vector pCAMBIA3301-RSP-Cry8-like-nos with glufosinate resistant marker was constructed successfully.The root-specific plant expression vectors were transferred into two different soybean varieties ‘Ji Nong 28’ and ‘Ji Nong 42’ respectively by using agrobacterium-mediated method.After preliminary PCR detection of transformed soybean,then the seeds of positive plants were germed into soil in the artificial climate chamber.After that T2 transgenic plants were carried out Southern blotting detecting integration,qPCR detecting transcription level of them,and preliminary identification of insect resistance indoor.The main results were obtained as follows:1.By using pCAMBIA3301 as basic plant expression vector,insect resistant gene Cry8-like and soybean root-specific promoter fragment RSP were recombined to the basic vector,and Root-specific plant expression vector pCAMBIA3301-RSP-Cry8-like-nos with glufosinate resistant marker was constructed successfully by Seamless cloning technology.2.The root-specific plant expression vectors were transferred into two different soybean varieties ‘Ji Nong 28’ and ‘Ji Nong 42’ respectively by using agrobacterium-mediated method.After preliminary PCR detection of transformed soybean,detection of PCR showed that we obtained five transformed plants in T0 generation of ‘Ji Nong 28’ and two of ‘Ji Nong 42’,and transformed rates were 2.33% and 1.74%.3.The seeds of T0 generation positive plants were germed into soil in the artificial climate chamber,and detection of PCR showed that we obtained seventeen transformed plants in T1 generation of ‘Ji Nong 28’ and thirteen of ‘Ji Nong 42’,Part of T1 generation positive plants seed were germed into soil in the artificial climate chamber,detection of PCR showed that we obtained thirty-six transformed plants in T2 generation of ‘Ji Nong 28’ and twenty-five of ‘Ji Nong 42’.4.Southern blotting of T2 generation indicated that insect resistant gene Cry8-like was integrated into the genome of Soybean with single copy,while the intensity of hybridization signal is different.5.Fluorescence quantitative PCR was performed on transgenic plants with single copy hybridization signal in Southern hybridization,and the results showed that Cry8-like gene was expressed in roots of transgenic plants.Compared with non transformed plants,the relative expression of transgenic plants increased by 13.83 times,and the lowest increased by 1.33 times.6.The results of insect resistance to Holotrichia parallela larvae in laboratory showed that the ability of insect resistance was improved by transgenic plants.The highest insecticidal mortality reached 20%,and the highest inhibition rate reached 56.39%.The insect resistance of this experiment was related to the high level of expression of the target gene. |
