Construction Of RNAi Expression Vector Of The Soybean Agglutinin And Lipoxygenase Inhibitor Genes And Its Transformation In Soybean

Soybean(Glycine max), as one of the most important economic and oil crops inChina and even the world, is the major source of plant protein. It has a very highnutritional value with the balance of amino acids. However, there are also manyantinutritional factors in soybean, which seriously reducing the quality andnutritional value of soybean. Soybean lipoxidase and soybean agglutinin is mainlytwo kinds of antinutritional factors. Soybean lipoxidase reduces the soybean oilcontent. It can catalyze the unsaturated fatty acids of catalytic oxidation to producesome small molecules of smelly, making the bean products with beany flavor. Thestructure of typical tetramer of Soybean agglutinin can agglutinate animal red bloodcells and other cells, damage the normal structure of small intestine, have a badinfluence on the digestion and absorption, immune system, internal system of peopleor animals, which seriously influencing the use efficiency of soybean protein. So ithas become a top priority on how to remove or reduce the content of soybeanlipoxidase and soybean agglutinin in today’s breeding. It has a series of problems toimprove the quality of soybean by conventional breeding methods currently, such aslong breeding period, high cost of seed breeding, limited resources, narrow geneticbasis resources and so on. Molecular breeding is able to take genetic operations atmultiple levels, such as from phenotype to molecular level thereby improve genesefficiently and directionally, as well as to improve the efficiency of breeding and tosave the cost of breeding.In this study, the RNAi expression vector of the Soybean agglutinin andLipoxygenase inhibitor genes, which was promoted by P7P and using herbicide asselection, was constructed by digestion and cloning. Then the obtained expressionvector was introduced into to soybean cultivars Jinong28viaAgrobacterium-mediated transformation and the pollen tube path way transformation,and detected by PCR, Southern blot, and Real-time quantitative PCR.Major findingswere as follows:(1)Through a series of digestion and connection, the RNAi expression vector of the Soybean agglutinin and Lipoxygenase inhibitor genes was successfully obtained usingthe basic expression vector pC1301-LoxRi and pCAMBIA3301-KTi-SBA providedby our laboratory.(2)Soybean strains were transformed by Agrobacterium-mediated method, got6strains of T0transformed，got23strains of T1transformed，got73grains of soybeanseeds. By genetic transformation system mediated with pollen tube pathway, got532grains of soybean seeds of jinong28, got14strains of T1transformed, got27strainsof T2transformed.(3)PCR and Southern blot results showed that the transgenic plants were obtainedsuccessfully. The results of Real-time quantitative PCR demonstrated that theexpression of Soybean agglutinin and Lipoxygenase in transgenic soybeans wassignificantly inhibited in seed. The expression quantity of Soybean agglutinindecreased35.9%-47.2%by agrobacterium-mediated method and24.0%-47.5%bypollen tube path way. At the same time, the expression quantity of Lipoxygenasedecreased32.8%-56.1%and51.7%-59.1%.