Study On The Regulation Mechanism On RNAi Expression Of β-Conglycinin In Soybean

Soybean is widely utilized in the food and feed industries. However soybean contains many anti-nutritional factors such β-conglycinin that limited its extended application. β-conglycinin is the main allergen responsible for soybean allergy, and many results show that the antibody response generated against soy protein was mainly reactive towards the β-conglycinin. To eliminate antigens from proteins in soybean, current strategies include thermal processing, enzyme treatment, the use of chemical agents, and traditional breading techniques. But these means methods often have some limited effects. With the development of molecular biology and genetic engineering, RNA interference（RNAi） has become a widely used technology, and which provides a new way for improving soybean quality.β-conglycinin is a soybean seed storage protein that consist of three primary subunits: α′-subunit,α-subunit,β-subunit. Lowering content of β-conglycinin by inhibiting the expression of mRNA in each subunit is the base method to eliminate soybean allergy. Our research inhibits expression of α′-subunit,α-subunit,β-subunit, α′-subunit and β-subunit by RNA interference（RNAi） to analyze the effect of β-conglycinin knockdown in soybean by PCR, real-time quantitative PCR, ELISA and major agronomic trait, and the effect of RNAi technique on the expression of three major subunit genes of β-conglycinin and its effect on the improvement of soybean quality traits. The main results are as follows.1.Two functional fragments was clonned for the ihp-RNAi expression vector structure, and RNAi expression vector containing the β-conglycinin α-subunit gene was constructed successfully. Then it was transformated into soybean JN 27 via Agrobacterium tumefaciens-mediated method. The results of PCR showed that the vector can be expressed in soybean, 11 strains of PCR-positive plants in T0 generation, 4 strains of PCR-positive plants in T1 generation, 6 strains of PCR-positive plants in T₂ generation were obtained. The results of Southern blot showed that the expression vector of α-subunit gene RNAi was integrated into the soybean genome with 1-2 copies. The results of real-time quantitative PCR suggestted that the target transcripts were inhibited 82.3%, 85.1%,70.6% in the transgenic strains. Enzyme Linked immunosorbent（ELISA） was used to detect the content of β-conglycinin. The results showed that the β-conglycinin content was decreased between 46.42% 63.07%.2.The plant expression vector of β-subunit RNAi was transformed into JN27 Soybean cotyledon via Agrobacterium tumefaciens-mediated method. The results of PCR showed that the vector can be expressed in soybean, and 10 strains of PCR-positive plants in T0 generation, 13 strains of PCR-positive plants in T1 generation, 6 strains of PCR-positive plants in T₂ generation were obtained. The results of Southern blot suggested that 2 strains of PCR-positive soybean showed positive signals, suggesting that the expression vector of β-subunit gene RNAi vector was integrated into the soybean genome with one copy. The results of real-time quantitative PCR showed that the target transcripts were inhibited 77.5%,82.8% on two transgenic strain. Enzyme Linked immunosorbent（ELISA） was used to detect the content of β-conglycinin. The results showed that the β-conglycinin content was decreased between 48.11%⁵8.73%.3.α′-subunit transgenic lines were grown in the field and seed harvested from the lines were analyzed,Southern blotting analysis revealed that α′-subunit vector was integrated into the five T₄ soybean genome with 1-2 copies. The mRNA of α′-subunit was inhibited 43.56%⁸8.6% on the transgenic strains as determined by real-time quantitative PCR. Enzyme Linked immunosorbent（ELISA） was used to detect the content of β-conglycinin. The results showed that the β-conglycinin content was decreased between22.67% ⁶9.57%.4.A bivalent expression vector of the β-conglycinin α′-subunit gene and β-subunit gene was constructed and then transformed into JN28 Soybean cotyledon via Agrobacterium tumefaciens-mediated method. The results of PCR showed that the vector can be expressed in soybean, and 11 strains of PCR-positive plants in T0 generation, 11 strains of PCR-positive plants in T1 generation,4 strains of PCR-positive plants in T₂ generation were obtained. The results of Southern blot showed that 3 strains of PCR-positive soybean showed positive signals, revealing that the vector was integrated into the genome with 1-2 copies. The mRNA of β-subunit were inhibited 76.80%、86.1%、78.4% on the transgenic strains as determined by real-time quantitative PCR, and the α′-subunit were inhibited 63.2%, 62.19%, 74.6%. Enzyme Linked immunosorbent（ELISA） was used to detect the content of β-conglycinin. The results showed that the β-conglycinin content was decreased between 46.8%⁶6.09%.5.Determination of protein and fat content of T₄ five transgenic soybean plants by near infrared grain analyzer showed that protein was decreased between 0.35%¹.47%. However, Fat content was increased between 0.33%¹.52%. The main agronomic traits of the T₄ transgenic soybean plants were not changed. Investigation agronomic traits of the T₂ transgenic soybean plants which obtained from the previous study.It was showed that all of them were not changed.