基于唾液蛋白组和唾液腺转录组信息克隆的HSP70基因及其表达分析.DOCVIP

基于唾液蛋白组和唾液腺转录组信息克隆的 HSP70基因及其表达分析基金项目：国家自然科学基金 基金项目：国家自然科学基金作者简介：杨 亚(1990-)，女，湖南娄底人，硕士研究生，主要从事兽医寄生虫研究.通信作者： E-mail： hn5368@163.com 杨亚1、徐兴莉1,2、刘进辉、程天印1* （1. 湖南农业大学 动物医学院 湖南长沙 410128；2.宜春学院，生命科学与资源环境学院，江西宜春，336000） 摘要 [目的]全长褐黄血蜱唾液中的HSP70蛋白的编码基因，测定HSP70基因在褐黄血蜱不同阶段、不同器官的表达水平。[方法]基于褐黄血蜱唾液腺转录组文库中的Contig2727设计引物，RACE法克隆3’端、5’端。RT-PCR法测定HSP70在褐黄血蜱不同饱血状态、不同组织的表达水平。[结果]褐黄血蜱HSP70全长分别为2363bp，ORF大小1965bp，编码蛋白655aa。RT-PCR分析显示，在全蜱，HSP70表达量是雌幼蜱饱血雌若蜱雄成蜱半饱血雌成蜱饱血雌成蜱；在中肠、唾液腺，半饱血雌成蜱唾液腺半饱血雌成蜱中肠饱血雌成蜱唾液腺饱血雌成蜱中肠。[结论] HSP70的表达量与发育程度呈正相关；唾液腺表达水平高于中肠。 关键词：蜱，Hsp70，RACE，RT-PCR Cloning and expression analysis of HSP70 gene isolated from saliva proteome and salivary gland transcriptome of Haemaphysalis flava Ya Yang1, Xing-Li Xu1,2,Liu Jin-hui1,Cheng Tian-Yin 1,* 1. College of Veterinary Medicine, Hunan Agricultural University, Changsha 410128, Hunan Province, PR China. 2. College of Life Sciences and Resource Environment, Yichun University, Yichun 336000, Jiangxi Province, PR China. Abstract: In this?study, we mainly focus on cloning the full-length sequence of gene encoding HSP70 protein in Haemaphysalis flava saliva, and analyzing the mRNA expression level of HSP70 gene at different stages and in different tissues of H. flava. Gene-specific sequencing primers were designed based on the Contig2727 from the salivary gland transcriptome dataset of H. flava. Rapid amplification of cDNA ends (RACE) was performed to amplify the 5’ and 3’ ends of the HSP70 gene. The expression levels of HSP70 in different tissues and at different developmental stages of H. flava were analyzed by real-time quantitative PCR (qRT-PCR). The full-length cDNA of HSP70 was 2363 bp and contained an ORF of 1965 bp encoding a protein of 655 amino acids. The expression levels of HSP70 at different developmental stages showed that the order from low to high was the larva female ticks, fully engorged nymph female ticks, adult male ticks, partially engorged adult female ticks and fully engorged adult female ticks. HS