The Construction Of The Expression Vectors For Splicing Sweet Protein Gene And Transformation In Arabidopsis Thaliana

Sweet foodstuff makes up the major diet of people. Up to now, most of sweet tests come from carbohydrate, such as sucrose and some synthesized sweet tastes. However, it has been identified that more sucrose for people easily elicit diseases, such as obesity,diabetes,high blood pressure, and coronary heart disease. Meanwhile, the synthesized sweet tastes have been popular for a long time, but now they are now dropping out from market as their not good sweet taste and suspicion of eliciting cancer. So the searching for new sweet tastes has been becoming hotter. Sweet proteins have now become people's favor tests, as they are green foods, high sweetness and health.Currently, six sweet proteins were found in the world. One of species Capparis masaikal levl only grows in sub-tropic areas of china. The seeds contain sweet protein mabinlin. The sweetness is 400 folds of sucrose. Mabinlin has 4 homologues, which mabinlinⅡkeeps sweetness well in 80℃for 48 h, so it has attracted people. The cDNA of mabinlinⅡgene is 468bp. The mabinlinⅡpreprotein is 155aa. The mabinlinⅡmature protein is 105aa consisting by A strain (33aa) and B strain (72aa) through non-covalent bond. Thus mabinlinII mature protein processes from the preprotein after translation.The objectives of this research are the studies of the sweet expression of mabinlinII gene, and the identification of the effects of the joint peptide,signal pepdide,C-terminus peptide and N-terminus to the protein sweet. we obtained the results as the follow:1) The mabinlinⅡgene was cloned from DNA of Capparis masaikal levl by PCR. The gene sequence from the DNA has only 2 different bases with the gene sequence cloned from RNA, so it indicates that mabinlinⅡhas no introns .2) The mabinlinⅡDNA segment of joint peptide,signal pepdide,C-terminus peptide and N-terminus has been omitted by using gene splicing by over extension-gene (SOEing).3) Three constitutive expression vectors of pVKH-35S-MBL -pA,pVKH-35S-MBL'-pA,pVKH-35S-(S1-S2)-pA were constructed by combination of MBL gene or the recombinant gene segments with 35S promoter, and inserting into the vector pVKH respectively. Three seed specific expression vectors of pVKH-Pm-MBL -pA,pVKH- Pm-MBL'-pA,pVKH- Pm -(S1-S2)-pA were constructed by combination of MBL gene or the recombinant gene segments with 35S downstream of seeds special expression promoter Pm, which was cloned from up stream of mabinlin II gene, and inserting into the vector pVKH respectively.4) Construction of the bi-expression vector of pVKH-Pm-S1-LegA-S2-pA, in which Mabinlin II gene segment of A strain and B strain separately combined with seed specific promoter, then inserted into pVKH through vector PUC and PBS.5)The six recombinant expression vectors of pVKH-35S-MBL–pA, pVKH-35S-MBL'–pA, pVKH-35S-(S1-S2)-pA, and pVKH-Pm-MBL–pA, pVKH- Pm-MBL'–pA, pVKH- Pm -(S1-S2)-pA were introduced into Agrobacterium by using the electric transformation method.6)The six recombinant expression vectors of pVKH-35S-MBL–pA, pVKH-35S-MBL'–pA, pVKH-35S-(S1-S2)-pA, and pVKH-Pm-MBL–pA, pVKH- Pm-MBL'–pA, pVKH- Pm -(S1-S2)-pA were transformed to Arabidopsis thaliana by using vacuum infiltration method.7)Selecting the transformants of the six expression vectors in MS medium with Hygromycin, then transferring 10-20 independent lines to soil respectively and selected the T2 seeds.8)Identified the transformant of the six expression vectors by PCR. The selection of sign locus, homozygous transgenic lines; and the identification of the expression of the MBL gene or the recombinant gene segments is going to be done.