Construction Of RNAi Expression Vector Of α’-subunit Gene In β-conglycinin From Soybean And Expression

The soy protein is the major source of vegetable protein in human beings and animals, which is about40%in the soybean, and90%is seed storage protein.7s and11s protein are the main component of the soybean storage protein. Studies showed that regulating the ratio between the two could improve soybean protein processing adaptability, increased11s protein content and decreased7s protein content could improve the nutritional quality of soy protein. The main component of the7s protein is β-conglycinin, it is a soy antigen protein, which can cause digestive tract allergic reaction for infants and young animals,α’, a and β are three subunits of β-conglycinin, which are all the potential food allergens factors.β-conglycinin has good thermal stability, reduced the effect of antigen activity by conventional heat treatment is very limited, therefore, through breeding means to reduce β-conglycinin content is the main way to reduce their activity. So far, it has been produced β-conglycinin subunits missing material by natural variation or artificial mutation breeding way, and its molecular basis has been in-depth studied. However, by means of genetic engineering to reduce the β-conglycinin subunits content has been rarely reported.In this study, according to the RNA interference principle, cloning400bp fragments of the core conserved sequence of β-conglycinin a’-subunit gene. Two ihp-RNAi efficient expression vector p3301-PFNZ and p3301-PFNZ-BADH were constructed separately by genetic engineering, one selection marker is herbicide-resistant Bar gene and the other is resistancesalinization BADH gene, and a bivalent expression vector p3301-α’+β was contructed too, Bar gene is the selection marker. The two ihp-RNAi expression vector of a’-subunit gene was introduced into soybean by Agrobacterium-mediated and pollen tube pathway. After resistance screening, the transgenic plants were detected by PCR, Southern blot, RT-PCR and ELISA. Using RNA interference to study the expression and regulation of β-conglycinin a’-subunit gene and soybean quality improvement laid the foundation for the application of genetic engineering techniques.The main results are listed as follows:Four fragments were cloned for the ihp-RNAi expression vector structure:seed-specific promoter7αp, a’-subunit gene sense and antisense fragments and functional spacer sequences intron-s. On the base of the plant expression vector pCAMBIA3301, the seed-specific promoter7αp, antisense fragment of a’-subunit gene, functional spacer sequences intron-s and the a’-subunit gene were inserted into pCAMBIA3301one by one, construct the ihp-RNAi expression vector p3301-PFNZ with herbicide-resistant Bar gene as selection mark. The salinity gene BADH was cloned, and the Bar gene in the p3301-PFNZ was replaced by BADH gene after being single enzyme by Xhol I. The direction of BADH gene was detected by EcoR I. The security-type expression vector p3301-PFNZ-BADH was constructed successfully.The (3-subunit gene of β-conglycinin Has been cloned, and its sense fragment was inserted into the downstream of promoter7αp and its antisense fragment was inserted into upstream of terminator nos, and their inserted direction were detected by restriction endonuclease digestion. The bivalent ihp-RNAi expression vector p3301-α’+β of a’-subunit and β-subunit gene was constructed successfully.The two monovalent expression vectors were transformed into soybean via agrobacterium-mediated method.15To positive plants of the salinity of the generation of "Jinong27" and10To positive plants of anti-glufosinate of "Jinong28" were obtained through resistance screening and preliminary PCR detection, the transformation efficient was2.1%.The two monovalent expression vectors were transformed into soybean via pollen tube pathway.780seeds were obtained, including salinity seeds:125of "Jinong27",112of "Ji Xindou",143of "Tongnong13" anti-glufosinate seeds:123of "Jinong27",131of "Ji Xindou",146of "Tongnong13". Through resistance screening and PCR detection, the positive seedlings were obtained, including salinity positive plants "Jinong27":10,"Ji Xindou":7,"Tongnong13":5; anti-glufosinate positive plants:"Jinong27":8,"Ji Xindou":6,"Tongnong13":5; average transformation efficient was3.2%.Through PCR detection of transgenic offspring,45positive plants of the T1generation via agrobacterium-mediated method were obtained, including25positive salinity T1generation plants of "Jinong27" and20positive anti-glufosinate T1generation plants of "Jinong28". To further determine whether the T-DNA of expression vectors were inserted into the soybean genome, the T1positive plants were extracted from "Jinong27" and "Jinong28" for Southern blot analysis respectively. The results of positive plants have appeared hybridization signal, and the non-transgenic plants no hybridization signal, thus it proved that the two monovalent expression vector T-DNA region have been indeed integrated into the soybean genome. RT-PCR results showed that the transgenic plants, α’subunit gene mRNA level was significantly lower than non-transgenic plants, RNA interference mechanism played a role in the post-transcriptional level. The ELISA result showed that the7S protein content and immune activity in the transgenic soybeans decreased, the average decline rate is about10%, the RNA interference in protein expression level also played a role.