Cloning Of HBsAg Gene And Construction Of Banana Fruit -expression Vector

The United Nations Food and Agriculture Organization has ranked bananas as the world's fourth most important food crop. Bananas have four major advantages as an important food: the period of growth is shoit; they are grown in most of the tropical regions of the world; people like to eat them and greatly enjoy their taste; they are eaten uncooked. Now disease resistant bananas have been created, and the future of a stable crop can be ensured. This makes bananas an economically feasible choice for use as a vector for the production and delivery of oral vaccines. Plant science has joined with vaccine research to provide low cost protection against various diseases that plague the developing countries. To gain genetic transformation banana, First.genomic DNA was extracted from light foliage by SDS method.Zhen we clone ACC synthase gene .which can promote the expression of gene in the fruit of banana. 1200bp fragments of ACS promoter were amplified by PCR using specific primers, and then sequenced. Compared with the reported sequence in GenBank, similarity of DNA sequence was more than 93%, so right sequence were obtained and modified. And many important as-acting regulatory elements were found in the cloned sequence by PlantCARE software analysis. ACS promoter was predicted to be most probably i iduced by light, hot, gibberellin, ethenen or wound, to identify its fruit-expressior character, A transient- expression vecters (pBACS) were constructed. In the transient-expression vector, ACS promoter sequence is inserted in pBI101.2 pBACS were transformed into fruits of banana by bombardment. GUS histochemistry analysis indicated that GUS gene had been successfully transformed into the bombarded fruits. And the blue dots were found in the fruits. At the same time, no blue dots were found in the fruits bombarded by the microcarriers coated with ddH2O. This result illuminated that GUS was transiently expressed in the fruits, and they were of fruit-expression. The efficiency of transformation, caused by different bombardement distances (3.0cm, 6.0cm, 9.0cm), was also valued. Bombardment with 9.0cm is most efficient, 62.5% positive explants were obtained. Each sample was bombarded 2 times. When the distance increases from 3cm~9cm, the percent of positive explant also increase. Therefore, the distance of 9.0cm and bombardment are appropriate parameters to transform heterogeneous gene into foliage explant of banana.Over three hundred million people worldwide are infected with hepatitis B,which is caused hepatitis B virus(HBV),hepatitis B is a serious liver infection mat can result in jaundice, cirrhosis, and liver cancer. Up to now , there is no effective ways and means to cure the disease. Although an injectable vaccine now exists, its expense and requirement for refrigeration- makes it unavailable to more than one-third of the world's population, especially in poor countries where the vaccine is in urgent need. So plant-based vaccine is an attractive alternative. In this research, total DNA was extracted from the blood serum of people infected HBV by the modified single-step method of phenol-chloroform. Based on reported sequence of HBsAg, specific primers were designed to amplify its coding sequence by PCR. At the same time, Kozak sequence was added to the translation site, in order to strengthen initiation of translation. The fragment of cloned HBsAg is 681bp. NCBI BLAST analysis indicated that similarity of HBsAg sequence and the postulated ammonic acids sequence is 97 % and 97.4% respectively, compared with the reported sequence in GenBank. So the modified HBsAg gene with Kozak sequence was obtained.A perfect expression vector is essential for the heterogeneous gene to efficiently express in fruit of banana tree. GUS gene in pBACS was replaced by the modified HBsAg gene, so banana fruit -expression vector pBAH was successfully constructed. This research has establish the foudation for HBsAg-transgenic banana plants.