棉铃虫核多角体病毒p10基因的序列及转录分析

Sequence and Transcriptional Analysis of the p10 Gene of Helicoverpa armigera Single Nucleocapsid Nucleopolyhedrovirus

为了利用棉铃虫核多角体病毒 (HaSNPV) p10基因的启动子构建重组病毒杀虫剂及表达系统 ,应用末端测序法和引物步移法测定了 p10基因的序列 ,并应用Northern杂交和引物延伸实验对该基因进行了深入的转录特性分析。HaSNPV p10基因被定位于基因组DNA的 115 4kb～ 115 6kb处 ,转录方向与多角体基因相反 ,在其上下游分别发现了 p2 6和 p74基因。转录分析结果确定了 p10基因是个极晚期基因 ,其转录从杆状病毒晚期转录保守序列GTAAG的第二个A开始 ,转录产物大小约为 430nt。HaSNPVp10基因最早可检测到的转录是在病毒感染后的 2 0h ,随后其转录产物量迅速放大 ,到感染后 72h达到非常高的水平。转录时相分析同时还检测到一个大小为130 0nt的产物 ,推测该产物为 p2 6和 p10通读的转录产物。对HaSNPVP10蛋白序列的分析表明 ,该蛋白第 6～44位及第 5 1～ 6 5位氨基酸序列处含多个典型的卷曲螺旋七聚体重复结构 ,两片段间相隔 6个氨基酸残基 ,在该蛋白序列的第 2 0～ 34位与第 5 1～ 6 5位存在L -X(2 ) -L -X(10 ) -L的亮氨酸重复。Helicalnet分析表明 ,HaSNPVP10的疏水氨式酸分布为两个集簇区 ,两者互为 180度转角。

As a prelude of the further use of the p10 gene promoter to construct genetic engineered viral insecticides and foreign gene exprssion systems, the HaSNPV p10 gene was identified and sequenced from both strands by using terminal sequencing and primer walking sequencing methods. The HaSNPV p10 gene was located at 1154-1156 kb on the genome in a reverse orientation to the polyhedrin gene, and was found flanked by the p26 gene and the p74 gene. The transcriptional characteristics of the p10 gene were analyzed by time course analysis and primer extension assay. The transcriptional analysis indicated that the HaSNPV p10 was a very late gene and its transcription was initiated from the second A of the late consensus transcriptional motif GTAAG. The transcripts of this gene had a size of 430 nt and were firstly detected at 20 h p.i. in infected Hz2e5 cells. The transcripts accumulated quickly and reached a peak at 72 h p.i. Another transcript, which had a size of about 1 300 nt, was also identified by using a p10-specific probe. It is possible that this product was a read-throught transcript of p26 and p10. The amino acid sequence of the P10 protein indicated that the N terminus of this protein contained two typical coiled-coil heptad repeat regions. A gap of six amino acid residues was found between these two regions. Furthermore, a L-X(2)-L-X(10)-L leucine repeat was found between the residues 20-34 and 51-65. Helical net model of HaSNPV P10 indicated that the distribution of the hydrophobic amino acids formed two separated clusters.