收稿日期: 2021-11-19
修回日期: 2022-03-12
网络出版日期: 2022-09-07
基金资助
内蒙古自治区农牧业科学院青年创新基金(2021QNJJM06);国家自然科学基金(31660710)
Polymorphism analysis of candidate genes for ivermectin resistance in Haemonchus contortus
Received date: 2021-11-19
Revised date: 2022-03-12
Online published: 2022-09-07
Supported by
Young Scientist Foundation of Inner Mongolia Academy of Agricultural & Animal Husbandry Sciences(2021QNJJM06);National Natural Science Foundation of China(31660710)
提取对伊维菌素敏感及耐药的捻转血矛线虫各50条单条虫体全基因组DNA,PCR扩增耐伊维菌素候选基因HCOI00378500、HCOI00506600和HCOI01200700的目的片段后测序,序列使用 Lasergene 7.0 软件中的SeqMan程序进行人工校正,采用EditSeq程序截取含有特定单核苷酸多态性（SNP）位点所在编码序列（CDS）区的DNA片段,采用MegAlign程序进行序列比对及单核苷酸和氨基酸多态性分析。结果显示,25个测序成功的敏感虫株中,HCOI00378500基因所有核苷酸位点无差异;35个测序成功的耐药虫株中,有8个虫株出现了共32个SNP位点,并且导致17个氨基酸突变,其中突变率最高的SNP占8.6%（3/35）。测序成功的40个耐药虫株中,HCOI00506600基因CDS区序列第75位碱基均发生突变（T > C）;测序成功的21个敏感虫株中,有7个虫株发生突变（T > C）;氨基酸由异亮氨酸（Ile, I）变为苏氨酸（Thr, T）。测序成功的38个敏感虫株中,HCOI01200700基因的38、101、125和134位点共有4个SNP;测序成功的13个耐药虫株中,32、48、71、86、101和131等5个位点共有6个SNP。HCOI01200700基因第38位呈G/A多态性（52.6%,20/38）,第125位呈T/C多态性（57.9%,22/38）,第134位呈C/A多态性（57.9%,22/38）,敏感虫株基因中至少有1个38G、125T或134C,耐药虫株基因中均为38A、125C或134A。提示HCOI00506600基因可作为为捻转血矛线虫耐伊维菌素相关候选功能基因,HCOI01200700基因可作为伊维菌素耐药性诊断的候选标记分子。
罗晓平 , 李军燕 , 高娃 , 耿万恒 , 王瑞 , 胡薇 , 乔蕾 , 邵国玉 , 杨晓野 . 捻转血矛线虫耐伊维菌素候选基因的多态性分析[J]. 中国寄生虫学与寄生虫病杂志, 2022 , 40(4) : 536 -539 . DOI: 10.12140/j.issn.1000-7423.2022.04.018
The whole genomic DNA of susceptible strains (Hc-S) and resistance strains (Hc-R) of Haemonchus contortus was extracted and the target fragments of candidate genes were amplified by synthesised primers and sequenced directly by PCR. Then DNA sequencing results were analysed using Lasergene7.0 software. EditSeq program was used to intercept DNA fragments containing the coding sequence (CDS) region where specific single nucleotide polymorphism (SNP) sites were located, and MegAlign program was used for sequence alignment and single nucleotide and amino acid polymorphism analysis. The results showed no difference in all nucleotide sites of the HCOI00378500 gene in 25 Hc-S samples, while among 35 Hc-R samples, 8 samples showed a total of 32 SNP sites with 17 amino acid mutations, of which SNPs with the highest mutation rate accounted for 8.6%. The CDS region of the HCOI00506600 gene sequence was mutated at the 75th base of all samples in 40 Hc-R (T > C), and 7 samples of the 21 Hc-S had mutations (T > C), in which the amino acid was changed from isoleucine (Ile, I) to threonine (Thr, T). Among the 38 susceptible strains successfully sequenced, there are 4 SNPs at sites 38, 101, 125 and 134 of the HCOI01200700 gene; among the 13 successfully sequenced resistant strains, there are 6 SNPs at sites 32, 48, 71, 86, 101 and 131. The 38th, 125th and 134th positions of the HCOI01200700 gene showed G/A polymorphism (52.6%, 20/38), T/C polymorphism (57.9%, 22/38) and C/A polymorphism (57.9%, 22/38), respectively. There was at least one 38G, 125T or 134C in the Hc-S, while 38A, 125C or 134A in the Hc-R. Two candidate positive molecules were analysed and identified, including a candidate functional gene HCOI00506600 and a candidate marker HCOI01200700 for the diagnosis of IVM resistance.
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