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本研究通过SDS法从香蕉幼嫩叶片中提取基因组DNA,通过PCR的方法对ACS启动子2.5Kb的序列进行缺失改造,克隆到了长1185bp的ACS启动子片段,与GenBank报道序列相比较同源性为93.2%。经PlantCARE软件分析发现序列中含有多种调控元件,经预测ACS启动子可能被光、热、GA、乙烯或伤所诱导。为验证其果实特异表达活性,通过插入到pBI101.2中间表达载体上的GUS基因5’端前,构建了含GUS基因的瞬时表达载体pBACS。用基因枪法将pBACS转化到香蕉果实中,结果表明:用pBACS包被的金粉轰击的果实经GUS组织化学染色后,都出现兰色斑点,对照没有出现兰色斑点。因此认为GUS基因在香蕉果实成功实现瞬时表达。同时,通过改进的酚-氯仿提取法从乙肝病毒感染者的血清中提取总DNA,然后根据报道的序列设计特异性引物,同时在上游引物中引入了Kozak序列,经PCR克隆到HBsAg基因序列,长度为681bp。NCBIBLAST分析的结果表明,获得的HBsAg基因序列及推导的氨基酸序列与GenBank中所报道的序列的同源率分别为97.2%和97.4%。将经过改造的HBsAg基因替代pBACS中的GUS基因,成功构建了含有ACS启动子和HBsAg基因的香蕉树果实表达载体,为下一步转化香蕉,获得在果实中表达HBsAg蛋白的转基因植株奠定了基础。
In this study, genomic DNA was extracted from young leaves of banana by SDS method. The 2.5Kb sequence of ACS promoter was deleted by PCR. The ACS promoter fragment with a length of 1185 bp was cloned and compared with the published sequence of GenBank 93.2%. Analysis by PlantCARE software revealed multiple regulatory elements in the sequence, suggesting that the ACS promoter may be induced by light, heat, GA, ethylene or wounding. To verify the fruit-specific expression activity, the transient expression vector pBACS containing the GUS gene was constructed by inserting it into the 5 ’end of the GUS gene on the intermediate expression vector pBI101.2. The pBACS was transformed into banana fruit by particle bombardment method. The results showed that the blue speckles were observed in GUS histochemical staining of pBACS-coated gold powder, and no blue specks in the control. Therefore, GUS gene was successfully expressed in banana fruit. At the same time, the total DNA was extracted from the serum of hepatitis B virus infected patients by the improved phenol-chloroform extraction method, then the specific primers were designed according to the reported sequences. At the same time, Kozak sequence was introduced into the upstream primers and the HBsAg gene sequence was cloned by PCR. The length is 681bp. The results of NCBIBLAST analysis showed that the identities of the obtained HBsAg gene sequences and deduced amino acid sequences with those reported in GenBank were 97.2% and 97.4%, respectively. The modified HBsAg gene was substituted for the GUS gene in pBACS, and the banana tree fruit expression vector containing ACS promoter and HBsAg gene was successfully constructed, which laid the foundation for the next transformation of banana and obtaining the transgenic plants expressing HBsAg protein in the fruit.