棕尾别麻蝇贮存蛋白基因克隆及寄生对其表达的影响
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摘要
本文成功克隆了编码棕尾别麻蝇Boettcherisca peregrina芳基贮存蛋白及LSP-2贮存蛋白亚基的cDNA序列,对其表达模式做了分析,并研究了丽蝇蛹集金小蜂Nasonia vitripennis寄生对其组成和含量的影响,结果如下:
1棕尾别麻蝇贮存蛋白亚基基因的克隆与序列分析
序列分析结果表明,所克隆的编码棕尾别麻蝇Arylphorin蛋白亚基的cDNA全序列总长为2,534 bp,开放阅读框长2,346 bp。预测该蛋白质亚基分子量约为95.42 kDa,等电点约为5.01。SignalP在线分析结果表明,该蛋白质含有一段长15aa的信号肽。分析该蛋白质亚基的氨基酸残基组成,发现其芳香族氨基酸含量约占23.81%。所获得的编码LSP-2蛋白亚基的全长cDNA序列长度为2,235 bp,其开放阅读框长度为2,106 bp。预测该蛋白质亚基的分子量约为82.96 kDa,等电点约为5.94。该蛋白质亚基亦含有一段长度为19 aa的信号肽序列。分析该蛋白质亚基的氨基酸残基组成,发现其芳香族氨基酸含量约占11.13%(小于15%),不同于Arylphorin蛋白亚基。
ClustalW分析及系统进化树构建结果表明,棕尾别麻蝇的Arylphorin及LSP-2亚基分别与红头丽蝇Calliphoria vicina的两种贮存蛋白最为相似,并在系统进化树中与其聚为一类。在不同昆虫的贮存蛋白亚基之间存在较高的相似性,说明贮存蛋白具一定保守性,分类学亲缘关系越近的昆虫其贮存蛋白保守性越高。
2棕尾别麻蝇脂肪体贮存蛋白基因表达的日龄变化及寄生对其影响
通过预备实验,确定了用于棕尾别麻蝇脂肪体贮存蛋白基因表达半定量RT-PCR分析的反应体系及条件,并确定反应起始模板的用量。半定量RT-PCR结果表明,编码Arylphorin亚基和LSP-2亚基基因的转录表达水平与随日龄变化密切相关。其中,编码Arylphorin亚基基因的表达趋势为,于1龄幼虫开始转录表达,表达量逐渐上升,在3龄幼虫时达到最高值,尔后于预蛹期显著下调,初孵幼虫、蛹期及成虫期均无转录表达;编码LSP-2亚基基因的表达趋势基本与编码Arylphorin亚基基因的表达趋势相同,只是其在1龄幼虫期亦无转录表达,其转录表达滞后于Arylphorin亚基基因。另外,半定量结果还显示,非寄生蛹与寄生蛹中的脂肪体均不转录表达贮存蛋白基因,表明寄生对于寄主脂肪体转录表达贮存蛋白基因并无影响。
3丽蝇蛹集金小蜂寄生对寄主蛹可溶性蛋白与芳基贮存蛋白组成和含量的影响
本文研究了棕尾别麻蝇蛹被丽蝇蛹集金小蜂寄生后其脂肪体和血淋巴中可溶性蛋白及芳基贮存蛋白组成与含量的变化。寄生蛹脂肪体可溶性蛋白及芳基贮存蛋白的组成与未寄生蛹相比没有明显变化,但含量均低于未寄生蛹。寄生蛹血淋巴中可溶性蛋白的组成与未寄生蛹相比差异不大,其含量低于未寄生蛹。与脂肪体不同,寄生后12 h时寄生蛹血淋巴中芳基贮存蛋白亚基的免疫印迹结果出现2条免疫条带,这表明其可能先于未寄生蛹发生降解,而芳基贮存蛋白的含量与未寄生蛹相比差异多不显著。
Two cDNA sequences of hexameric storage protein,SpeAry and SpeLSP-2,were cloned from Boettcherisca peregrine and their gene expression patterns were revealed. Then the effects of parasitization by Nasonia vitripennis(Hymenoptera:Pteromalidae)on the expressional and transcriptional levels were investigated.The results are as follows:
1 Cloning the cDNAs of SPs subunit in B.peregrine and sequence analysis
By using the homology searching software of GenBank,full length of the cloned Speary cDNA was found to be 2,534 bp with 2,346 b.p of open reading frame.The molecular weight of the SpeAry deduced by amino acid sequence was 95.42 kDa.The calculated isoelectric point was 5.01.Utilizing online software,SignalP,the signal peptide was consisted of 15 amino acids at the 3'- end excluding the poly(A)-tail.The deduced primary structure indicated a high content of aromatic amino acids up to 23.8%.Full length of the cloned SpeLSP-2 cDNA was 2,235 bp with 2,106 bp of ORF.We also found a polyadenylation signal peptide with 19 amino acid at the poly-A initiation site.The putative protein encoded by the SpeLSP-2 gene was with a molecular weight of 82.96 kDa, an isoelectric point of 5.94.The primary structure with a content of about 11.1%aromatic amino acids which was less than 15%,showed a strong homology with other LSP-2 gene sequences.
Clustal W and phylogenetic tree analysis showed that S.peregrine arylphorin was most closely related with CviAry in amino acid sequence and should belong to the same cluster. Furthermore,SpeLSP-2 was most closely related to CviLSP-2.According to the taxonomy that if the evolutionary relationship was nearer and the conservative were higher.SPs of S.peregrine and other insect storage proteins had relatively high similarity,from which we can conclude that they were highly conservative.
2 The transcriptional dynamics of the two SPs from B.peregrine with instar and its changes after parasitization.
By pre-experiment,we determined the conditions for Semi-quantitative RT-PCR of the fat body genes of B.peregrine.The results of showed the transcriptional levels of SpeAry and SpeSP2 had strong correlation with instar.The transcription level increased gradually from the first to the third instar,then decreased significantly in prepupae,and disappeared in late pupae and adult.The transcription trend of SpeSP2 was similar with that of SpeAry,with exception that there was no transcription in the first instar.This revealed that transcription of SpeSP2 was later than that of SpeAry.In addition,there was no transcritption observed in the fat body of either parasitized or non-parasitized pupae, from which we can conclude that parasitization had no influence on the transcription.
3 Effects of parasitization by Nasonia vitripennis on the compositions and contents of soluble proteins and arylphorin in its host B.peregrin pupae
In this article,the changes in compositions and contents of soluble proteins and Arylphorin,in fat body and hemolymph or B.peregrine pupae parasitized by N.vitripennis are investigated.The composition of soluble proteins and Arylphorin in fat body and hemolymph of parasitized pupae are similar to those of non-parasitized pupae.In contrast, their contents in both fat body and hemolymph of parasitized pupae are significantly lower than those of non-parasitized ones.Similarly,the composition of soluble proteins in hemolymph of parasitized pupae is likely to that of non-parasitized ones,while their contents are lower.In contrast to fat body,12 h after parasitization the hemolymph of parasitized pupae possess two bands reacting with the polyclonal antiserum against arylphorin in the western-blotting membrane.It is implicated that the Arylphorin in hemolymph might be broken down,which is earlier in parasitized than in non-parasitized pupae.The difference in the content of arylphorin in hemolymph is not significant between parasitized and non-parasitized pupae,except 12h after parasitization.
1棕尾别麻蝇贮存蛋白亚基基因的克隆与序列分析
序列分析结果表明,所克隆的编码棕尾别麻蝇Arylphorin蛋白亚基的cDNA全序列总长为2,534 bp,开放阅读框长2,346 bp。预测该蛋白质亚基分子量约为95.42 kDa,等电点约为5.01。SignalP在线分析结果表明,该蛋白质含有一段长15aa的信号肽。分析该蛋白质亚基的氨基酸残基组成,发现其芳香族氨基酸含量约占23.81%。所获得的编码LSP-2蛋白亚基的全长cDNA序列长度为2,235 bp,其开放阅读框长度为2,106 bp。预测该蛋白质亚基的分子量约为82.96 kDa,等电点约为5.94。该蛋白质亚基亦含有一段长度为19 aa的信号肽序列。分析该蛋白质亚基的氨基酸残基组成,发现其芳香族氨基酸含量约占11.13%(小于15%),不同于Arylphorin蛋白亚基。
ClustalW分析及系统进化树构建结果表明,棕尾别麻蝇的Arylphorin及LSP-2亚基分别与红头丽蝇Calliphoria vicina的两种贮存蛋白最为相似,并在系统进化树中与其聚为一类。在不同昆虫的贮存蛋白亚基之间存在较高的相似性,说明贮存蛋白具一定保守性,分类学亲缘关系越近的昆虫其贮存蛋白保守性越高。
2棕尾别麻蝇脂肪体贮存蛋白基因表达的日龄变化及寄生对其影响
通过预备实验,确定了用于棕尾别麻蝇脂肪体贮存蛋白基因表达半定量RT-PCR分析的反应体系及条件,并确定反应起始模板的用量。半定量RT-PCR结果表明,编码Arylphorin亚基和LSP-2亚基基因的转录表达水平与随日龄变化密切相关。其中,编码Arylphorin亚基基因的表达趋势为,于1龄幼虫开始转录表达,表达量逐渐上升,在3龄幼虫时达到最高值,尔后于预蛹期显著下调,初孵幼虫、蛹期及成虫期均无转录表达;编码LSP-2亚基基因的表达趋势基本与编码Arylphorin亚基基因的表达趋势相同,只是其在1龄幼虫期亦无转录表达,其转录表达滞后于Arylphorin亚基基因。另外,半定量结果还显示,非寄生蛹与寄生蛹中的脂肪体均不转录表达贮存蛋白基因,表明寄生对于寄主脂肪体转录表达贮存蛋白基因并无影响。
3丽蝇蛹集金小蜂寄生对寄主蛹可溶性蛋白与芳基贮存蛋白组成和含量的影响
本文研究了棕尾别麻蝇蛹被丽蝇蛹集金小蜂寄生后其脂肪体和血淋巴中可溶性蛋白及芳基贮存蛋白组成与含量的变化。寄生蛹脂肪体可溶性蛋白及芳基贮存蛋白的组成与未寄生蛹相比没有明显变化,但含量均低于未寄生蛹。寄生蛹血淋巴中可溶性蛋白的组成与未寄生蛹相比差异不大,其含量低于未寄生蛹。与脂肪体不同,寄生后12 h时寄生蛹血淋巴中芳基贮存蛋白亚基的免疫印迹结果出现2条免疫条带,这表明其可能先于未寄生蛹发生降解,而芳基贮存蛋白的含量与未寄生蛹相比差异多不显著。
Two cDNA sequences of hexameric storage protein,SpeAry and SpeLSP-2,were cloned from Boettcherisca peregrine and their gene expression patterns were revealed. Then the effects of parasitization by Nasonia vitripennis(Hymenoptera:Pteromalidae)on the expressional and transcriptional levels were investigated.The results are as follows:
1 Cloning the cDNAs of SPs subunit in B.peregrine and sequence analysis
By using the homology searching software of GenBank,full length of the cloned Speary cDNA was found to be 2,534 bp with 2,346 b.p of open reading frame.The molecular weight of the SpeAry deduced by amino acid sequence was 95.42 kDa.The calculated isoelectric point was 5.01.Utilizing online software,SignalP,the signal peptide was consisted of 15 amino acids at the 3'- end excluding the poly(A)-tail.The deduced primary structure indicated a high content of aromatic amino acids up to 23.8%.Full length of the cloned SpeLSP-2 cDNA was 2,235 bp with 2,106 bp of ORF.We also found a polyadenylation signal peptide with 19 amino acid at the poly-A initiation site.The putative protein encoded by the SpeLSP-2 gene was with a molecular weight of 82.96 kDa, an isoelectric point of 5.94.The primary structure with a content of about 11.1%aromatic amino acids which was less than 15%,showed a strong homology with other LSP-2 gene sequences.
Clustal W and phylogenetic tree analysis showed that S.peregrine arylphorin was most closely related with CviAry in amino acid sequence and should belong to the same cluster. Furthermore,SpeLSP-2 was most closely related to CviLSP-2.According to the taxonomy that if the evolutionary relationship was nearer and the conservative were higher.SPs of S.peregrine and other insect storage proteins had relatively high similarity,from which we can conclude that they were highly conservative.
2 The transcriptional dynamics of the two SPs from B.peregrine with instar and its changes after parasitization.
By pre-experiment,we determined the conditions for Semi-quantitative RT-PCR of the fat body genes of B.peregrine.The results of showed the transcriptional levels of SpeAry and SpeSP2 had strong correlation with instar.The transcription level increased gradually from the first to the third instar,then decreased significantly in prepupae,and disappeared in late pupae and adult.The transcription trend of SpeSP2 was similar with that of SpeAry,with exception that there was no transcription in the first instar.This revealed that transcription of SpeSP2 was later than that of SpeAry.In addition,there was no transcritption observed in the fat body of either parasitized or non-parasitized pupae, from which we can conclude that parasitization had no influence on the transcription.
3 Effects of parasitization by Nasonia vitripennis on the compositions and contents of soluble proteins and arylphorin in its host B.peregrin pupae
In this article,the changes in compositions and contents of soluble proteins and Arylphorin,in fat body and hemolymph or B.peregrine pupae parasitized by N.vitripennis are investigated.The composition of soluble proteins and Arylphorin in fat body and hemolymph of parasitized pupae are similar to those of non-parasitized pupae.In contrast, their contents in both fat body and hemolymph of parasitized pupae are significantly lower than those of non-parasitized ones.Similarly,the composition of soluble proteins in hemolymph of parasitized pupae is likely to that of non-parasitized ones,while their contents are lower.In contrast to fat body,12 h after parasitization the hemolymph of parasitized pupae possess two bands reacting with the polyclonal antiserum against arylphorin in the western-blotting membrane.It is implicated that the Arylphorin in hemolymph might be broken down,which is earlier in parasitized than in non-parasitized pupae.The difference in the content of arylphorin in hemolymph is not significant between parasitized and non-parasitized pupae,except 12h after parasitization.
引文
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