柞蚕NPV核多角体蛋白基因核苷酸序列分析及其mRNA转录起始点的确定

NUCLEOTIDE SEQUENCING AND TRANSCRIPTIONAL START SITE DETERMINATION OF THE POLYHEDRIN GENE OF ANTHERAEA PERNYI NUCLEAR POLYHEDROSIS VIRUS

采用DNA双脱氧法,对所克隆的载有ApNPV核多角体蛋白基因片段进行了核苷酸序列分析,并与AcNPV和BmNPV核多角体蛋白基因序列进行了比较。结果表明,ApNPV核多角体蛋白结构基因由735个核苷酸编码序列(编码245个氨基酸)组成,其序列与AcNPV和BmNPV的核多角体蛋白基因编码序列相比同源性较高,分别为79.6％和81.6％;但其5′端和3′端两侧翼序列与AcNPV和BmNPV相比差异显著,特别是控制该基因表达的5′端启动子部分调控序列(nt—2～—61):AcNPV与BmNPV完全相同,而ApNPV在此区域却有20个核苷酸序列发生变异,并且在对该基因表达起决定性作用的8个高度保守核苷酸序列(nt—44～—51),有两处发生自然突变。经核苷酸序列推测出的ApNPV核多角体蛋白氨基酸序列与AcNPV、BmNPV核多角体蛋白氨基酸序列的差异,同其三者之间的核苷酸序列的差异的比率降低10％。采用引物延伸法,对ApNPV核多角体蛋白mRNA转录起始点进行了测定,确定其位于该基因调控序列12个核苷酸高保守区的nt—50位点与AcNPV相似。

A 4. 0 kb DNA fragment of the genome of nuclear polyhedrosis virus of Antheraea pernyi contains the entire coding sequence of polyhedrin gene together with some of it's flanking 5'and 3'noncoding region has been cloned and sequenced by chain-temination method using both single-stranded and double stranded DNA as the templets. The ApNPV polyhedrin gene coding region consists of 735 bp nucleotides with 245 amino acids including the translation initiation codons. Comparisons of the coding sequence of ApNPV with AcNPV and BmNPV, a high homology , 79. 6%and 81. 6% was observed respectively. In contrast, however, the nucleotide sequences of ApNPV,BmNPV and AcNPV in the region of 5' (nt-1 to -198)and 3' (nt 739 to 879) .the homology are comparatively low ,especialy in the promoter region(nt-2 to -61 ) ,The sequences of AcNPV and BmNPV is exactly same,whereas ApNPV has 20 nucleotide sbstutions in this region and 2 nucleotides mutated in the high conserved 8 bp domain(nt-44 to-51 )which are the determinant for this gene expression. The predicted amino acid sequences of polyhedrin of ApNPV, AcNPV and BmNPV are more similar than that of their nucleotide sequences due to the nucleofide sequence silent mutations. The mRNA transcriptional start site was determined by primer extension, which located at nt -50 in the high conserved 12-mer nucleotide domain,similar as AcNPV.