Construction And Transformation Of The IhpRNA Expression Vector Of Soybean Beta-conglycinin Beta-subunit Gene

Soybean mainly used to make soy, oil, vegetable protein and animal feed and so on is the main economic and food crops in our country.The total protein content accounting for40%of total quality in soybean, eight kinds of essential amino acids ofsoybean proteins in sufficient and complete components are high quality proteins. However, soybean protein is also a plant protein with strong antigen, of which soybean globulinof11S protein and beta-conglycinin of7S protein is allergy source of soybean.Although soybean globulin possesses smaller percentage in soybean total protein, it is one of main allergy original of soybean proteinwhich can causedigestive road allergy reactionof baby and the young animal. Beta-conglycinin is trimer compound composed of α, α’and β subunits in different permutations and combinations.And the molecular weight of β subunitwhich consists of416-amino acidis52kD. The research demonstrates thatβsubunit also has immunological activity and can act specific combination with specific antigen.Our research based on the core sequence of7S protein beta subunits gene (gene number:AB030840), respectively constructed security plant RNAi expression vectorwith filter mark of BADH and Bar gene through RT-PCR and genetic engineering technology, and then assistedAgrobacterium-mediatedmethod to transform soybean’Jinong28’ and ’Jinong27’. The transgenic plants were detected through molecular biology detections of PCR, and RT-PCR, and Southern-blot and so on and further to obtain positive plants. The vector was used to induce soybean β-subunit gene silencing, further to reduce the expression and content of β subunit. All of these provided foundations forenhancingthe quality of soybean protein, shortening the breeding time and looking for new methods of soybean breeding.Major findings as follows:(1) We obtained full fragment gene sequence and core gene sequence of beta-conglycinin of7S protein and constructed cloning vector of PMD18T-QUSN and PMD18T-HEXIN.(2) pCAMBIA-3301-7ap-NHZ-BADH and pCAMBIA-3301-7ap-NHZ-Bar vectors modified in Jilin Agricultural University Center for Biotechnologyas the basis, the antisense fragment of core sequence was inserted into promoter7ap and functional sequence of intervals NHZ, then the justice fragment of core sequence was inserted into functional sequence of intervals NHZ and NOS Terminator, respectively constructed two RNAi plant expression vectorwith salt resistance gene as a selection marker of BADH-7ap-β and herbicide resistance gene as a selection marker of Bar-7ap-β.(3) We obtained two safety Agrobacterium engineering bacteriaof RNAi plant expression vectorwith the BADH-7ap-(3and Bar-7ap-β.Through Agrobacterium tumefaciens into receptor soybean cultivar ’Jinong28’, the transgenic plants were detected through molecular biological detections of PCR, Southern, RT-PCR and so on. Finally we got4strainsof TO generation plants,5strains of Tl generation plantof BADH-7ap-βsafety RNAi plant expression vectors.