Cloning Of Storage Protein Gene Gy5 In Soybean And Construction Of The Plant Expression Vector

With the improvement of living standard and the development of foreign trade, people are paying more attention to improving nutritional quality of crops. At the beginning of 1990s, genetic engineering was used in this field and has got many achievements in recent ten years. These studies mostly focus on improving the content and component of storage protein, starch and lipid of seed.Crop seeds are the main source of plant protein for mankind, so improving the seed protein is very important. Soybean seeds contain more protein than any other cultivated commercial crops. Their storage protein accounts for over 40% of seeds dry weight. Soybean storage protein contains 8 kinds of human essential amino acids . It is an excellent source of dietary protein for people and animals. So soybean storage proteins have high nutritional value and commercial value.Globulin is the predominant seed storage protein in soybean, which is mainly composed of glycinin(HS) and B-conglycinin(7S). The content of sulfur-amino acid in glycinin is much higher than B-conglycinin, the content ratio(HS/7S)directly affects the quality and value of soybean storage protein. According to the above arguments, using genetic engineering technology clone the good soybean storage protein gene-US glycinin gene, then transfer it into the same or different plants to improve the storage protein content and quality, which has a good application prospect in the improvement of crop quality, and lays out a way for the production of special proteins.In this experiment, primers were designed according to the Gy5 cDNA sequence registered in Genebank. The total RNA of immature soybean seeds was extracted with improved Guanidine Thiocyanate method. The target fragment was amplified with RT-PCR and was cut by EcoRI/PstI, and ligated with pUC19 vector. This reconstructed plasmid was conducted into E.coli.DH5 a . The gene was sequenced and the results showed that the sequence contained 1704 nucleotides and only six nucleotides different from the Gy5 gene. Ligate this fragment with pCAMBIA1301, which is an expression vector with CaMV35s promoter. The recombinant was mobilized into Agrobacterium tumefaciens LBA4404 by the freeze-thaw method. Transform Gy5 into tobacco leaves. The Gy5 was integrated into acceptor, which was confirmed by resistant selection and PCR identification.This work cloned a good seed storage protein gene. It preliminarily confirmed that the reconstructed plant expression vector could make Gy5 integrated into acceptor. It also has laid on a foundation for further research on transferring the cloned gene into other plants in order to improve the content and quality of their seed storage protein.On the other side, the research has improved the isolated method of RNA from the plants with multitude of proteins, carbohydrates, et al. The extracted RNA was analyzed by ultraviolet spectrophotometer, the result was as follows: OD26o/28o=1.9~2.0, OD260/230>2.0; the content was 0.05mg/g. In the Agarose gel, 28S and 18S rRNA was recognized clearly and even the former was wider than the later. So this research improved the efficiency and quality of RNA extraction, it developed an efficient method for RNA extraction.At last, it was excellent to optimize RT-PCR in RNA quantity, primers density and annealing template in this research. It came to a conclusion: the optimum quantity is 1.5ug in 20ul reaction system; the optimum primers density is 1pmol/ul; the optimum annealing temperature is 50C. When using the products of RT-PCR as template proceed another PCR, the annealing temperature need be increased to 54C. The difference was probably caused because of the difference of the components in the system. So the RT-PCR annealing temperature was about lower 4C than normal PCR.