Construction And Transformation Of Rnai Plant Expression Vector Of The Lipoxygenase And Kunitz Tripsin Inhibitor Genes From Soybean

Soybean is the main source of plant protein,but there are some inhibitive factors of nutrition that. which including Kunitz trypsin inhibitors (KTi) and Lipoxygenase. Which Limiting the nutritional value of soy protein and its food flavor. During the manufacturing process, we must through the heating method to remove its activeness. But, heating will decrease the nutrional values of soybeans.Therefore, cultivate new varieties of soybean Which missing lox and kti, and can improve the quality of soybean effectively.We also make good use of the hybrid and backcross breeding method to improve KTti and Lipoxygenase. But, the method can cost a long time for breeding and the germplasm is limited, so they have not reach the purpose of improving the quality of soybean. RNA Interference technique(RNAi) is the main method of transgenic breeding method, Which geting the favour of people. The objective of this study was using RNAi technology to build the plant expression vector Which inhibiting the expression of trypsin inhibitor and lipoxygenase and containing the bar gene. We use Agrobacterium-mediated to import it into soybean varieties of Jinong28, and lowered trypsin inhibitor gene and lipoxygenase. We expect a higher nutritional value of soybean varieties. The main results are listed as following:(1) In this study,we use pCAMBIA1301p7aP-GUS-KTiS as a template used seed-specific promoter upstream primer and the nox terminator down stream primer to clone KTi to insert into pCAMBIA1301-LoxRi, to Construct RNAi Plant Expression Vector of the Lipoxygenase and Kunitz Tripsin Inhibitor which No antibiotic selection marker and named pCAMBIA1301-KtiRi-LoxRi. And Identification of PCR and restriction, and its sequencing.(2) We use pCAMBIA3301as a template to clone bar from it,which using CaMV35S promoter upstream primer and camv35s terminator downstream primer and into pCAMBIA1301-KtiRi-LoxRi and named pCAMBIA1301-KtiRi-LoxRi-bar.And Identification of PCR and restriction, and its sequencing.(3)The ihpRNAseed-specific express edvector pCAMBIA1301-KtiRi-LoxRi-bar were introduced into EHA101respectively. After identification by PCR, the transcojugant was subsequently used to transform soybean cotyledonary node. The transformed explants were screened from barstar.These PCR and Southern blot analysis results of transgenic plant confirmed that the T-DNA region of the ihpRNA seed-specific expressed vector presented in the transgenic plants and integrated into the genome of soybean. hat dsRNA-expressing constructs were inherited in a Mendelian fashion. RT-PCR analysis was performed to detect the levels of endogenous Kti and Lox gene transcripts both in the developing embryos and leaves from the transgenic plants and non-transgenic plant. The soybean tefS1(elongation factor EF-1a) housekeeping gene was used as the internal control; both developing embryos and leaves presented the same level of the tefS1gene. The non-transgenic plant showed Kti and Lox gene expression both in developing embryos, while for the transgenic lines, Kti and Lox gene transcripts were obviously suppressed in the developing embryos. These results of RT-PCR analyses revealed that the accumulation of Kti and Lox mRNA was significantly reduced in the transgenic soybean seeds, and Kti and Lox gene transcripts were obviously suppressed cooperatively in the seeds of transgenic plants with pCAMBIA1301-KtiRi-LoxRi-bar.(4) In this study, we obtained10positive transgenic plants of To,26positive transgenic plants of T1,54positive transgenic plants of T2.