Structural and functional characterization of a novel gene, Hc-daf-22, from the strongylid nematode Haemonchus contortus.
Structural and functional characterization of a novel gene, Hc-daf-22, from the strongylid nematode Haemonchus contortus.
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圆线虫新基因 Hc-daf-22 的结构和功能特征
DOI：
10.1186/s13071-016-1704-1
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发表时间：
2016-07-29
影响因子：
3.2
通讯作者：
Du A
中科院分区：
文献类型：
--
作者：
Guo X;Zhang H;Zheng X;Zhou Q;Yang Y;Chen X;Du A
背景：缠绕血吸虫是现代畜牧业的一种重要寄生虫，因为恶劣的环境条件可能会导致第四期早期幼虫滞育。方法：鉴定了一个新基因HC-daf-22，它是ce-daf-22和人类SCPx的同源物。通过基因组步行和RACE获得该基因的全长c DNA和基因组序列。以各发育阶段的qRT-PCR为模板检测转录水平，显微注射法检测5‘-侧翼区的启动子活性。结果：线虫HC-daf-22基因全长为6,939个核苷酸，由15个内含子组成，编码1,602个核苷酸(533个氨基酸，约59.3 kDa)，在转录水平上位于L3和L4处。HC-DAF-22蛋白由3-氧酰辅酶A硫解酶结构域和SCP2结构域组成，在进化上是保守的。与Ce-DAF-22的5‘-侧翼区相比，5’-侧翼区上游的1,548个核苷酸片段具有启动子活性。微量注射daf-22(Ok693)突变株的拯救实验显示，经油红O染色证实，被拯救的虫体大小和孵化仔数显著增加，脂肪颗粒显著减少或完全消失，表明HC-daf-22可以部分挽救ce-daf-22的功能。此外，含有HC-daf-22的RNAi可以部分沉默N2虫体内内源性的ce-daf-22，并模拟daf-22(Ok693)突变株的表型。结论：利用线虫作为模式生物分离了HC-daf-22基因，并对其功能进行了鉴定。我们的结果表明，HC-DAF-22与Ce-DAF-22具有相似的特性和功能，可能在螺旋藻的过氧体β氧化和发育中发挥重要作用。
Background:The strongylid nematode Haemonchus contortus is a parasite of major concern for modern livestock husbandry because hostile environmental conditions may induce diapause in the early fourth-stage larvae.Methods:A new gene Hc-daf-22 was identified which is the homologue of Ce-daf-22 and human SCPx. Genome walking and RACE were performed to obtain the whole cDNA and genomic sequence of this gene. Using qRT-PCR with all developmental stages as templates to explore the transcription level and micro-injection was applied to confirm the promoter activity of the 5'-flanking region. Overexpression, rescue and RNA interference experiments were performed in N2, daf-22 mutant (ok 693) strains of C. elegans to study the gene function of Hc-daf-22.Results:The full length gene of Hc-daf-22 (6,939 bp) contained 16 exons separated by 15 introns, and encoded a cDNA of 1,602 bp (533 amino acids, estimated at about 59.3 kDa) with a peak in L3 and L4 in transcriptional level. The Hc-DAF-22 protein was consisted of a 3-oxoacyl-CoA thiolase domain and a SCP2 domain and evolutionarily conserved. The 1,548 bp fragment upstream of the 5'-flanking region was confirmed to have promoter activity compared with 5'-flanking region of Ce-daf-22. The rescue experiment by micro-injection of daf-22 (ok693) mutant strain showed significant increase in body size and brood size in the rescued worms with significantly reduced or completely absent fat granules confirmed by Oil red O staining, indicating that Hc-daf-22 could partially rescue the function of Ce-daf-22. Furthermore, RNAi with Hc-daf-22 could partially silence the endogenous Ce-daf-22 in N2 worms and mimic the phenotype of daf-22 (ok693) mutants.Conclusion:The gene Hc-daf-22 was isolated and its function identified using C. elegans as a model organism. Our results indicate that Hc-daf-22 shared similar characteristics and function with Ce-daf-22 and may play an important role in peroxisomal β-oxidation and the development in H. contortus.