Cloning And Construction Of Plant Expression Vector For KSTI3 Gene Of Soybean Kunitz-type Trypsin Inhibitor

Soybean protein is a kind of high-quality protein composed of balanced amino acids. It contains allkinds of amino acids, especially eight amino acids necessary for human, but also contains somecomponents that are not good for human and affect food quality, such as Trypsin inhibitor and Lectin.Nutritional value and processing quality of soybean can be improved by means of development of newvariety with null Trypsin inhibitor.In this paper, the full-length DNA fragment of soybean Kunitz-type trypsin inhibitor gene KSTI3 wasisolated by polymerase chain reaction (PCR), an antisense expression vector and RNAi expression systemwere constructed. Then one construction was introduced into soybean though an Agrobacteriumtumefaciens-mediated transformation and pollen tube pathway method in order to suppress the expressionof soybean trypsin inhibitor. The results of study were acquired as fellow:The genomic DNA were extracted from soybean young leaf by SDS method and CTAB method. Usingthe genomic DNA as template and 22bp long oligonucleotide with restriction site in 5'terminus as primerwhich based on a known sequence of soybean Kunitz-type trypsin inhibitor and ATG codon, the soybeantrypsin inhibitor gene was amplified by polymerase chain reaction (PCR).The PCR fragment was firstly cloned into pMD 18-T Vector and then transformed into E.colicompetent DH5αfor lac selection. The positive clones were confirmed by restriction enzyme digestion andsequencing. The sequence obtained was used for a Blast search to identify the homologous sequences fromthe GenBank database. Sequence alignment and analysis were performed using the DNAMAN software.The result indicated that the cloned fragment was consist of 670bp and shared 99.54ï¼…identity with theknown KSTI3 gene.The recombinant plasmid and plant expression vector pBI121 were digested by restriction enzymesBamH I and Xba I. The antisense expression vector pBIantiKSTI3 was constructed by T4 DNA ligase.After the identification of enzymes digestion, it had been proved that the sequence had been successfullyinserted into the vector.The fragment of anti-sense gene+plus-sense gene was cloned into the downstream of 35S promoterin the binary vector pBI121, which formed the recombinant plasmid pBIKSTI3. All constructions werefurther identified by restriction enzyme digestion. The plasmid pBIKSTI3 was mobilized intoAgrobacterium tumefaciens strain EHA105 and LBA4404 respectively by freeze-melting methods, and aplant expression vector was thereby obtained. The pBIKSTI3 vector was introduced into soybean though an Agrobacterium tumefaciens-mediatedtransformation. The better result for that was used the concentration value of OD₆₀₀: 0.5-0.6, the time ofincursion: 26 minutes. The transformed cotyledon nodes was co-cultured for 3 days, then delivered to theselection mediums containing Kan, Cef and Carb. By the means of PCR, the extrinsic segment be proved tobe conformed into the genome of soybean, and obtain two transformants.We also introduced it into soybean by pollen tube pathway method and obtained T₀ generationtransgenic seeds. They were detected by polymerase chain reaction. The result showed that threeKan-resistant plants had strong positive signals, and no signal was shown in wild type plants.