马铃薯甲虫热激蛋白基因Ld-hsp70的克隆及温度胁迫下的表达特性

Cloning of Heat Shock Protein Gene Ld-hsp70 in Leptinotarsa decemlineata and Its Expression Characteristics under Temperature Stress

【目的】热激蛋白(heat shock protein,HSP)是一种在生物体内广泛存在且高度保守的蛋白质,在生物抗逆过程中发挥重要作用。当生物体遭受不利环境条件胁迫时,其迅速产生可保证生物体的正常生理活动。本研究以重大检疫害虫马铃薯甲虫(Leptinotarsa decemlineata)为研究对象,对其热激蛋白HSP70基因(Ld-hsp70)进行克隆,并对其在温度胁迫下的表达特性进行分析,明确HSP70在马铃薯甲虫温度胁迫中的作用。【方法】基于NCBI数据库检索筛选马铃薯甲虫HSP70的cDNA序列,利用RT-PCR和RACE技术对Ld-hsp70的全长cDNA进行克隆;利用DNAMAN软件对其全长序列进行拼接;采用生物信息学方法对Ld-hsp70及其编码氨基酸的序列特性进行分析;使用MEGAX软件的邻接法(neighbor-joining,NJ)构建马铃薯甲虫HSP70与其他昆虫HSP70的系统进化树;利用实时荧光定量PCR(qRT-PCR)技术对Ld-hsp70在不同温度胁迫条件下的表达模式进行分析;利用Primer 5.0软件设计试验所用的特异性引物。【结果】基于NCBI数据库获得3类马铃薯甲虫HSP70 cDNA序列,分别命名为HSP70a、HSP70b和HSP70c,经克隆、拼接获得其全长序列,分别命名为Ld-hsp70a、Ld-hsp70b和Ld-hsp70c,GenBank登录号分别为KC544268、KC544269和KC544270。序列分析表明,3个Ld-hsp70编码的氨基酸序列均包含了完整保守结构域和3段保守的HSP70家族签名序列,且在氨基酸C末端具有保守的亚细胞定位基序。Ld-hsp70a和Ld-hsp70c的氨基酸C末端具有保守基序EEVD,属于胞质型热激蛋白;Ld-hsp70b的氨基酸C末端具有保守基序KDEL,属于内质网型热激蛋白。系统进化树分析显示,Ld-hsp70a和Ld-hsp70b与其他物种的HSP70分别聚为一支,Ld-hsp70c与已报道的马铃薯甲虫HSP70聚为一支。其中,Ld-hsp70a与龟纹瓢虫(Propylaea japonica)的Pj-hsp70,Ld-hsp70b与稻水象甲(Lissorhoptrus oryzophilus)的Lo-hsp70,Ld-hsp70c与大猿叶甲(Colaphellus bowringi)的Cb-hsp70同源性最高。qRT-PCR结果表明,高、低温均能诱导马铃薯甲虫雌、雄成虫的3个Ld-hsp70的表达。不同温度胁迫处理1 h后,马铃薯甲虫雌、雄成虫体内3个Ld-hsp70的相对表达量未见显著变化;而4 h后,马铃薯甲虫雌、雄成虫Ld-hsp70a的相对表达量与对照相比分别在低温-10℃和高温44℃时显著上调至2.24和2.41倍,Ld-hsp70b和Ld-hsp70c相对表达量在胁迫4 h后与对照相比差异均不显著。另外,无论是雌虫还是雄虫,3个Ld-hsp70的相对表达量在同一温度不同的胁迫处理时间之间均无显著差异。【结论】Ld-hsp70a可以响应高、低温胁迫,可能在马铃薯甲虫抵御温度胁迫中发挥作用,而Ld-hsp70b和Ld-hsp70c对高、低温均不敏感,表明马铃薯甲虫3个Ld-hsp70在抗温度胁迫中发挥不同的作用。

【Objective】 Heat shock protein(HSP), a class of highly conserved proteins, is generally found in all the organisms, which plays an important role in response to stress resistance. In order to ensure the normal physiological activities of the organism, the expression of HSP can be generated rapidly under the adverse environmental conditions. The objective of this study is to clone heat shock protein 70(HSP70) genes of the important quarantine pest Leptinotarsa decemlineata(Ld-hsp70 s), analyze their expression characteristics under temperature stress, and to clarify the function of L. decemlineata HSP70 under temperature stress.【Method】The cDNA sequences of HSP70 genes of L. decemlineata were obtained based on the NCBI database. The full length cDNAs encoding HSP70 genes of L. decemlineata were cloned by RT-PCR and RACE technology. The full-length sequences were spliced by DNAMAN software. The sequence characteristics of Ld-hsp70 s were analyzed by bioinformatic methods. The phylogenetic tree with the homologous sequences of HSP70 from L. decemlineata and other insects was constructed using the neighbor-joining(NJ) method with MEGAX software. The expression profiles of Ld-hsp70 s were analyzed by qRT-PCR. The specific primers were designed using Primer 5.0 software.【Result】The cDNA sequences of three HSP70 genes of L. decemlineata were obtained from the NCBI database, and were named as HSP70 a, HSP70 b and HSP70 c, respectively. Three full-length sequences were obtained by cloning and splicing, and were named as Ld-hsp70 a, Ld-hsp70 b and Ld-hsp70 c, respectively. The GenBank accession numbers were KC544268, KC544269 and KC544270, respectively. Sequence analysis showed that complete conserved domain, three signature sequences of HSP70 family, signal sequences and motifs of subcellular location at the carboxyl(C)-terminal were found in amino acid sequences of the three Ld-hsp70 s. The conserved EEVD motif in the C-terminal of Ld-hsp70 a and Ld-hsp70 c indicated that they were cytosolic HSP70. The conserved KDEL motif in the C-terminal of Ld-hsp70 b indicated that it was endoplasmic reticulum HSP70. Phylogenetic tree analysis showed Ld-hsp70 a and Ld-hsp70 b were clustered together with HSP70 from other insect species, while Ld-hsp70 c was clustered together with the reported HSP70 from L. decemlineata. Ld-hsp70 a, Ld-hsp70 b and Ld-hsp70 c had highest homology with Propylaea japonica Pj-hsp70, Lissorhoptrus oryzophilus Lo-hsp70, and Colaphellus bowringi Cb-hsp70, respectively. qRT-PCR results showed that the expression of all three Ld-hsp70 s could be induced by high and low temperatures. No significant difference was observed in the relative expression of the three Ld-hsp70 s after exposure to different temperatures for 1 h. The relative expression level of Ld-hsp70 a was significantly upregulated by 2.24 and 2.41 folds after exposure to-10℃ and 44℃ f or 4 h in female and male of L. decemlineata, respectively. However, no significant difference was observed in the relative expression of Ld-hsp70 b and Ld-hsp70 c under temperature treatments for 4 h. Whether Ld-hsp70 s in female or male, no significant difference was observed of different treatment times at the same temperature.【Conclusion】Ld-hsp70 a in L. decemlineata can respond to temperature stress and may play an important role in the adaptation to adverse temperatures. Ld-hsp70 b and Ld-hsp70 c are not very sensitive to temperature stress, suggesting functional differentiation of the three Ld-hsp70 s in response to abiotic stress.