斯氏按蚊PPO1基因克隆及其与约氏疟原虫卵囊黑化关系的研究

CLONING OF PRO-PHENOLOXIDASE GENE FROM ANOPHELES STEPHENSI AND RELATIONSHIP BETWEEN PPO1 AND PLASMODIUM YOELII OOCYSTS MELANIZATION

目的克隆和分析斯氏按蚊前酚氧化酶基因与约氏疟原虫卵囊黑化关系。方法根据其他昆虫前酚氧化酶基因的保守氨基酸序列设计简并引物,以斯氏按蚊全蚊RNA为模板,进行RT-PCR扩增,PCR产物纯化,克隆入pUCm-T载体,测定插入片段序列,对序列进行BLAST检索和鉴定。根据序列设计相应基因的特异引物,然后分别从血淋巴细胞或中肠扩增目的基因,并进行不同食源条件下前酚氧化酶基因的半定量、原位核酸分子杂交定位及与约氏疟原虫卵囊黑化关系的分析。结果获得了1种斯氏按蚊血淋巴前酚氧化酶基因cDNA部分序列,即AsPPO1(600bp),分别与冈比亚按蚊PPO1和大劣按蚊PPO2基因很相似,其编码的氨基酸序列均包含PO保守的铜结合位点CuA(HHWHWHLVYP)序列。在中肠和血淋巴内也获得相同的目的基因片段,半定量分析显示约氏疟原虫感染或诱导卵囊黑化呈现之前的表达明显增强,原位核酸分子杂交技术也进一步证实血淋巴有AsPPO1mRNA表达。结论As-PPO1很可能与疟原虫感染或约氏疟原虫卵囊黑化相关。

Objective To clone pro-phenoloxidase gene from Anopheles stephensi and to analyze the relationship between PPO1 gene and Plasmodium yoelii oocysts melanization. Methods Pro-phenoloxidase gene was amplified by RT-PCR with degenerated primers designed based on conserved amino acid sequence of insect PPO, then target PCR product was purified and cloned into pUCm-T vector, the inserted fragments contained in the positive recombinant plasmids verified by PCR and digestion with restriction enzyme were sequenced. After that, the obtained sequences were blasted with Genebank sequence database. Special PPO1 gene was amplified with specific primers from hemocytes or midgut, respectively. The differential expression of SP was analyzed with semi- quantitative RT-PCR under different feeding conditions. PPO1 message ribonucleic acids were identified with hybridization in situ. Results One cDNA segments of PPOI（600 bp） similar to An. gambiae PPO1 and An. dirus PPO2 was acquired from An. ste- phensi. AsPPO1 had the conserved amino acid residues of coppa-binding site A domain （HHWHWHLVYP）. Also PPO1 gene segments were amplified from the hemocytes and midguts. Marked up-regulation expression of PPO1 in An. stephensi were observed after feeding Plasmodial infectious blood, or before inducible oocysts melanization. PPO1 mRNAs expression was also confirmed in the hemocytes and midguts by ISH. Conclusion AsPPO1 may be involved in the Plasmodial infection or melanotic encapsulation of P. yoelii oocysts.