Construction Of RNAi Expressed Vector Of Kunitz Trypsin Inhibitor And Soybean Agglutinin Gene From Soybean And Its Transformation Of Soybean

Soybean has a high nutritional value because its contains rich proteins and fats. It is one of the main food crops, but also the main oil-bearing crops for human beings. Soybeans are also the main feeding raw materials in breeding, the reason is that the content of Essential amino acids of soybean protein is balanced and is good water-soluble,so these proteins are easy for animals to digestion and absorption. In industry, soybean protein can be used for fiber, adhesives etc. synthetic, so soybean becomes to the important industrial raw material. However, there are many nutritional inhibitory factors influence the quality of soybean. For example, in this study, The two nutritional inhibitory factors of soybean trypsin inhibitors and soybean agglutinin I concerned are the main nutrition inhibitory factors, because the content of them are too high in soybean proteins. At present, there are three main methods for improving the quality of soybean:①Physical and chemical methods②Traditional breeding method③Transgenic breeding method. The first method can destroy the nutrients of soybean, and the second method can cost a long time for breeding and the germplasm is limited, so the two method before have not reach the purpose of improving the quality of soybean. But the transgenic breeding method can overcome these disadvantages, and can improve the quality of soybean effectively. It becomes the focus of human beings' attention.RNA Interference technique(RNAi) is the main method of transgenic breeding method, it gets the favour of people. The principle of RNAi is that the homologous double-stranded RNAs(dsRNA) can be used to silence the expression of target genes in a variety of organisms and cell types. It has many advantages, such as efficiency,pleiotropi,specificity etc. Because of these advantages, it was used to improve the quality of many corps, for example, rice,wheat,soybean etc. So in this study, I used the RNAi technique to inhibit the two main nutrition inhibitory factors of soybean trypsin inhibitors and soybean agglutinin, expected to get the more nutritional soybean varieties. The main results are listed as following:(1) Extract the general RNA of 'Jinong 17 soybean', and reverse it to cDNA.And use the cDNA as the template, design two pairs of specificity primers, soybean trypsin inhibitors KTi gene and soybean agglutinin SBA gene were amplified by PCR.(2) Extract the general DNA of 'Jinong 17 soybean', and use the DNA as the template, design a pair of specificity primers, the seed specific promoter 7αp was amplified by PCR.(3) Extract the plasmid DNA of AHLG, and use the DNA as the template, design a pair of specificity primers, the green fluorescent protein GFP gene was amplified by PCR.(4) The four fragment above was cloned to pMD18-T Vector, and get four restructuring cloned vectors:pMD18-T-KTi,pMD18-T-SBA,PMD18-T-7αP (p-7αP)and pMD18-T-GFP (p-GFP)。(5) According to RNAi principle and based on the vector of pCAMBIA1301, the KTi gene seed specific promoter vector pl301-7αP-KTiZ-GFP-KTiF was constructed, design a pair of specificity primers, and the whole ihpRNA component of KTi gene was amplified by PCR. The whole fragment was cloned to pMD18-T Vector, and then get the restructuring cloned vector: pMD18-T-KTiRNAi.(6) According to RNAi principle and based on the vector of pCAMBIA1301, the SBA gene seed specific promoter vector p1301-7αP-SBAZ-GFP-SBAF was constructed, cut the p1301-7αP-SBAZ-GFP-SBAF and pMD18-T-KTiRNAi by one enzyme, the connection was restructuring cloned vector pKTi-SBA-RNAi with two small stems of RNAi.(7) First, make a connection between the KTi gene and SBA gene. And then the big fragment KTi-SBA was inserted into the both sides of GFP in forward and reverse direction, respectively. Finally, the restructuring cloned vector pCAMBIA3301-KTi-SBA with one big stem of RNAi was getted.(8) The plasmid DNA of pCAMBIA3301-KTi-SBA vector was introduced into soybean via the Agrobacterium. The DNA of regeneration plants were extracted for PCR analysis, the results of eight of the plants show positive. It can preliminaryly identify the RNAi construction vector of pCAMBIA3301-KTi-SBA has already transformed into soybean genome.(9) The tow plasmid DNA of pKTi-SBA-RNAi and pCAMBIA3301-KTi-SBA vectors were introduced into soybean via the pollen tube pathway.The seeds of T0 generation of soybean were getted for detection.