中华蜜蜂表皮蛋白基因的克隆与功能分析
摘要
昆虫的蜕皮和变态是昆虫发育生物学研究的热点之一。昆虫的角质层主要由表皮蛋白和几丁质组成；几丁质由N-乙酰葡萄糖胺聚合而成，结构明确，而表皮蛋白的结构和特征具有多样性，昆虫角质层的性质主要由其中的表皮蛋白性质所决定。表皮蛋白和表皮蛋白基因被认为是研究昆虫蜕皮与变态调控机理的重要模型，受到越来越多的重视。目前关于表皮蛋白家族基因的研究多数集中在果蝇、家蚕等物种，蜜蜂中这类基因的相关研究很少。中华蜜蜂是我国特有的优良土著蜂种，对维护植物资源物种的多样性，促进农业的可持续发展具有重要的意义。研究表皮蛋白家族基因功能可以为深入阐明分析中华蜜蜂的蜕皮发育机制，丰富中华蜜蜂资源保护理论提供科学依据。
本研究以中华蜜蜂（Apis cerana cerana）为材料，利用同源序列，通过RT-PCR和RACE-PCR的方法克隆得到中华蜜蜂两个表皮蛋白新基因，分别命名为AccCPR1和AccCPR2并对它们进行了生物信息学分析、表达特性分析和功能预测。本研究的具体结果如下：
（1）从中华蜜蜂中分离得到首个R&R亚族的表皮蛋白基因，命名为AccCPR1，Genbank登陆号为JQ282798。其cDNA全长为822nt，包括573nt的开放阅读框，编码一个190个氨基酸残基的多肽，预测的分子量和等电点分别为21.3kDa和6.50。生物信息学分析表明，多肽链中包含一个几丁质结合区域，属于典型的R&R-2型表皮蛋白；在几丁质结合区域β-折叠频繁发生（42.5％），而在其他的区域一个β-折叠也没有；AccCPR1与同属于膜翅目的CPRs比属于双翅目或半翅目的CPRs在系统进化关系上更亲密。R＆R保守基序中最保守的序列：G-x8-G-x6-Y/F，几乎在所有的表皮蛋白（CPRs）保持不变，但AccCPR1及其直系同源物对于最保守的R＆R基序的组成序列（G-x8-G-x6-Y/F）是个例外。在其启动子序列中发现5个E74和4个BR-C转录因子结合位点，推测AccCPR1可能参与蜜蜂的蜕皮与变态。
实时荧光定量PCR发现，当蜜蜂体内蜕皮激素的浓度到达高峰开始下降时AccCPR1启动转录，这说明AccCPR1的转录需要一个蜕皮激素的“脉冲”机制。我们采用不同的试验方法验证了这个假设。20E是蜕皮激素的主要成分，当给幼虫饲喂含有不同浓度的20E时，高浓度的蜕皮激素抑制了AccCPR1的表达。将白蛹期的胸部表皮在含有不同浓度的20E的培养基中培养，发现高浓度的蜕皮激素抑制AccCPR1的表达，除去20E可以恢复表达。组织特异性表达与免疫组织化学定位表明AccCPR1在表皮与中肠表达。这些结果表明，AccCPR1是一种典型的R&R-2型表皮蛋白，在蜜蜂的发育中发挥重要作用，并且蜕皮激素的“脉冲”机制对AccCPR1的表达非常重要。
（2）从中华蜜蜂中分离得到第二个R&R亚族的表皮蛋白基因，并命名为AccCPR2，Genbank登陆号为KJ502287。其cDNA全长为1134nt，编码一个234个氨基酸残基的多肽，预测的分子量为26.3kDa，预测的等电点为6.69。多肽链中包含一个几丁质结合区域，R＆R保守基序中最保守的序列：G-x8-G-x6-Y/F，可以在AccCPR2及其直系同源物的几丁质结合区域发现。AccCPR2基因组的第一个内含子在编码5个氨基酸之后中断信号肽，而这是昆虫表皮蛋白家族基因组序列的一个典型特征；在5′端空白区域发现了若干个假定的与发育有关的转录因子结合位点，包括3个假定的BR-C结合位点、5个假定的CdxA结合位点、1个假定的HB结合位点和3个假定的DFD结合位点，说明AccCPR2可能参与蜜蜂的蜕皮与变态。
生物信息学分析表明AccCPR2也属于与几丁质结合的R＆R-2类型表皮蛋白，但是其表达方式与以往在有些昆虫上已经发现的“脉冲”机制有比较大的差异。不同发育时期的表达特性分析表明，在蛹期AccCPR2的表达量随着蜕皮激素浓度的升高而增加，说明AccCPR2的转录需要蜕皮激素持续存在。将预蛹期的表皮在含有不同浓度的20E的培养基中培养，发现随着20E浓度的升高，AccCPR2表达量增加，并进一步确定了20E诱导表皮蛋白表达需要的时间。将刚出房的成年幼蜂暴露于各种应激条件中检测发现，AccCPR2mRNA的表达水平在重金属（HgCl2，CdCl2）处理后上升，而在杀虫剂（溴氰菊酯，百草枯）处理后先上升后下降。AccCPR2基因的抗逆性直接证据来自重组蛋白的活性分析，尽管重金属和杀虫剂处理间基因表达的模式有所不同，但是菌落扩散试验的结果是相似的。异源表达结果表明，AccCPR2可能参与抗逆性应激反应。
本研究结果发现，AccCPR1和AccCPR2同属R＆R-2类型表皮蛋白基因，但在多肽链最保守基序上存在结构差异并且两者在蜕皮激素调控下具有不同表达模式，表明不同表皮蛋白家族基因在中华蜜蜂变态发育机制研究中的多样性。
Insect molting and metamorphosis is one of the hot topics in the field of developmentalbiology. The insect cuticle is mainly composed of cuticular protein and chitin. The structure ofchitin is clear, which is polymerized of N-acetyl glucosamine, while the structure andcharacteristics of cuticular protein is various. The nature of the insect cuticle is mainlydetermined by the nature of the cuticular protein. Cuticular protein and cuticular protein gene,which are considered to be an important model for the study of regulation mechanism ofinsect molting and metamorphosis, have received more and more attention. So far,researches about cuticular protein gene are mainly focus on the model speices, such as D.melanogaster and Bombyx mori, and information in honeybee is relatively limited. TheChinese honeybees which are the excellent indigenous bee species are of great significance tothe maintenance of biological diversity of plant resources in China, and to promote thesustainable development of agriculture. The research of cuticular protein family genes mayprovide scientific basis of elucidating the developmental mechanisms of Chinese honeybeesand protecting the indigenous excellent germplasm resource.
In this research, we selected the Chinese honeybee as the experiment material, and twogenes, AccCPR1, AccCPR2, have been cloned by RT-PCR and RACE-PCR. Then, a seriesof research have been managed on the isolation, sequence and expression profile analysis, andfunctional identification of the two genes. The main results are as follows:
(1) The first cuticle protein R&R gene named AccCPR1was identified from A. ceranacerana. The full-length cDNA (GenBank accession number: JQ282798) has a length of822nt,which contains an open reading frame (ORF) of573nt encoding a190amino acidpolypeptide with a predicted molecular mass of21.3kDa and an isoelectric point of6.50. Theprotein contains a chitin binding region and is a typical cuticle R&R-2protein. The analysisof the secondary structure indicated thatβ-sheets occurred frequently (42.5%) throughout thestructure of the region, whereas there were none in other regions. The phylogenetic treeindicated that AccCPR1has a greater level of sequence identity with Hymenoptera CPRs thanwith Diptera or Hemiptera CPRs. The strict R&R consensus: G-x8-G-x6-(Y/F) was found inthis region. But AccCPR1and its orthologs are exception for the most conservative sequencemotif. Five putative E74binding sites and four BR-C binding sites were predicted in the5'-flanking region, which suggests a potential function in molting and metamorphosis.
RT-qPCR showed that AccCPR1transcript occurred as the ecdysteroid titer decreased after reaching a peak, which suggests AccCPR1expression requires a “pulse” regimen ofecdysteroids. This hypothesis was tested using different experimental strategies.20E is amajor component of ecdysone. When larvae were reared with different concentrations of20Ein their diet, the ecdysteroid peak repressed AccCPR1expression. Exposure of the thoracicintegument of the pupae in vitro to different concentration of20E repressed AccCPR1expression, which recovered after the removal of20E. Tissue-specific expression andimmunohistochemical localization showed AccCPR1expressed in the epidermis and in themidgut. These results suggest that AccCPR1is a typical cuticle R&R-2protein that plays animportant role in development, and an ecdysteroid pulse is critical for high AccCPR1geneexpression.
(2) We cloned and characterized another cuticle protein R&R gene, referred to AccCPR2,from the honeybee (A. cerana cerana). The full-length cDNA (GenBank accession number:KJ502287) has a length of1134nt, which contains an open reading frame (ORF) of705ntencoding a234amino acid polypeptide with a predicted molecular mass of26.3kDa and anisoelectric point of6.90. The protein contains a chitin binding region and the strict R&Rconsensus: G-x8-G-x6-(Y/F) was found in this region. The first intron interrupts the signalpeptide after five coding amino acids in the AccCPR2genomic sequence, which is a typicalcharacteristic of CPR genomic sequences in insects. Three BR-C, five CdxA, one HB andthree DFD binding sites were predicted in the5'-flanking region, suggesting a potentialfunction in molting and metamorphosis.
Bioinformatics analysis indicated that AccCPR2contains a chitin binding region and is atypical cuticle R&R-2protein, while its expression pattern has a relatively large differenceswith "pulse" mechanismin that have been found in some insects. Expression profile of thedifferent developmental stages showed that the expression level of AccCPR2increased withthe increase of ecdysone concentration during the pupal stage, indicating that AccCPR2transcription needs ecdysone persist. Exposure of the integument of the pharte pupae in vitroto different concentration of20E, the expression of AccCPR2was identified with the increaseof20E concentration. Environmental stressors, such as heavy metals and pesticides, alsoinfluenced gene expression. In addition, a disc diffusion assay showed that AccCPR2enhanced the ability of bacterial cells to resist multiple stresses. We infer from our results thatAccCPR2acts in honeybee development and in protecting these insects from abiotic stresses.
The results of this study found that, AccCPR1and AccCPR2belong to R&R-2typecuticle protein genes. There are structural differences of the most conserved motifs betweenthe two polypeptide chains and the two genes are regulated by ecdysone titer with different expression patterns, suggesting different cuticle protein genes in Apis cerana cerana play avariety of roles in metamorphosis mechanisms.
本研究以中华蜜蜂（Apis cerana cerana）为材料，利用同源序列，通过RT-PCR和RACE-PCR的方法克隆得到中华蜜蜂两个表皮蛋白新基因，分别命名为AccCPR1和AccCPR2并对它们进行了生物信息学分析、表达特性分析和功能预测。本研究的具体结果如下：
（1）从中华蜜蜂中分离得到首个R&R亚族的表皮蛋白基因，命名为AccCPR1，Genbank登陆号为JQ282798。其cDNA全长为822nt，包括573nt的开放阅读框，编码一个190个氨基酸残基的多肽，预测的分子量和等电点分别为21.3kDa和6.50。生物信息学分析表明，多肽链中包含一个几丁质结合区域，属于典型的R&R-2型表皮蛋白；在几丁质结合区域β-折叠频繁发生（42.5％），而在其他的区域一个β-折叠也没有；AccCPR1与同属于膜翅目的CPRs比属于双翅目或半翅目的CPRs在系统进化关系上更亲密。R＆R保守基序中最保守的序列：G-x8-G-x6-Y/F，几乎在所有的表皮蛋白（CPRs）保持不变，但AccCPR1及其直系同源物对于最保守的R＆R基序的组成序列（G-x8-G-x6-Y/F）是个例外。在其启动子序列中发现5个E74和4个BR-C转录因子结合位点，推测AccCPR1可能参与蜜蜂的蜕皮与变态。
实时荧光定量PCR发现，当蜜蜂体内蜕皮激素的浓度到达高峰开始下降时AccCPR1启动转录，这说明AccCPR1的转录需要一个蜕皮激素的“脉冲”机制。我们采用不同的试验方法验证了这个假设。20E是蜕皮激素的主要成分，当给幼虫饲喂含有不同浓度的20E时，高浓度的蜕皮激素抑制了AccCPR1的表达。将白蛹期的胸部表皮在含有不同浓度的20E的培养基中培养，发现高浓度的蜕皮激素抑制AccCPR1的表达，除去20E可以恢复表达。组织特异性表达与免疫组织化学定位表明AccCPR1在表皮与中肠表达。这些结果表明，AccCPR1是一种典型的R&R-2型表皮蛋白，在蜜蜂的发育中发挥重要作用，并且蜕皮激素的“脉冲”机制对AccCPR1的表达非常重要。
（2）从中华蜜蜂中分离得到第二个R&R亚族的表皮蛋白基因，并命名为AccCPR2，Genbank登陆号为KJ502287。其cDNA全长为1134nt，编码一个234个氨基酸残基的多肽，预测的分子量为26.3kDa，预测的等电点为6.69。多肽链中包含一个几丁质结合区域，R＆R保守基序中最保守的序列：G-x8-G-x6-Y/F，可以在AccCPR2及其直系同源物的几丁质结合区域发现。AccCPR2基因组的第一个内含子在编码5个氨基酸之后中断信号肽，而这是昆虫表皮蛋白家族基因组序列的一个典型特征；在5′端空白区域发现了若干个假定的与发育有关的转录因子结合位点，包括3个假定的BR-C结合位点、5个假定的CdxA结合位点、1个假定的HB结合位点和3个假定的DFD结合位点，说明AccCPR2可能参与蜜蜂的蜕皮与变态。
生物信息学分析表明AccCPR2也属于与几丁质结合的R＆R-2类型表皮蛋白，但是其表达方式与以往在有些昆虫上已经发现的“脉冲”机制有比较大的差异。不同发育时期的表达特性分析表明，在蛹期AccCPR2的表达量随着蜕皮激素浓度的升高而增加，说明AccCPR2的转录需要蜕皮激素持续存在。将预蛹期的表皮在含有不同浓度的20E的培养基中培养，发现随着20E浓度的升高，AccCPR2表达量增加，并进一步确定了20E诱导表皮蛋白表达需要的时间。将刚出房的成年幼蜂暴露于各种应激条件中检测发现，AccCPR2mRNA的表达水平在重金属（HgCl2，CdCl2）处理后上升，而在杀虫剂（溴氰菊酯，百草枯）处理后先上升后下降。AccCPR2基因的抗逆性直接证据来自重组蛋白的活性分析，尽管重金属和杀虫剂处理间基因表达的模式有所不同，但是菌落扩散试验的结果是相似的。异源表达结果表明，AccCPR2可能参与抗逆性应激反应。
本研究结果发现，AccCPR1和AccCPR2同属R＆R-2类型表皮蛋白基因，但在多肽链最保守基序上存在结构差异并且两者在蜕皮激素调控下具有不同表达模式，表明不同表皮蛋白家族基因在中华蜜蜂变态发育机制研究中的多样性。
Insect molting and metamorphosis is one of the hot topics in the field of developmentalbiology. The insect cuticle is mainly composed of cuticular protein and chitin. The structure ofchitin is clear, which is polymerized of N-acetyl glucosamine, while the structure andcharacteristics of cuticular protein is various. The nature of the insect cuticle is mainlydetermined by the nature of the cuticular protein. Cuticular protein and cuticular protein gene,which are considered to be an important model for the study of regulation mechanism ofinsect molting and metamorphosis, have received more and more attention. So far,researches about cuticular protein gene are mainly focus on the model speices, such as D.melanogaster and Bombyx mori, and information in honeybee is relatively limited. TheChinese honeybees which are the excellent indigenous bee species are of great significance tothe maintenance of biological diversity of plant resources in China, and to promote thesustainable development of agriculture. The research of cuticular protein family genes mayprovide scientific basis of elucidating the developmental mechanisms of Chinese honeybeesand protecting the indigenous excellent germplasm resource.
In this research, we selected the Chinese honeybee as the experiment material, and twogenes, AccCPR1, AccCPR2, have been cloned by RT-PCR and RACE-PCR. Then, a seriesof research have been managed on the isolation, sequence and expression profile analysis, andfunctional identification of the two genes. The main results are as follows:
(1) The first cuticle protein R&R gene named AccCPR1was identified from A. ceranacerana. The full-length cDNA (GenBank accession number: JQ282798) has a length of822nt,which contains an open reading frame (ORF) of573nt encoding a190amino acidpolypeptide with a predicted molecular mass of21.3kDa and an isoelectric point of6.50. Theprotein contains a chitin binding region and is a typical cuticle R&R-2protein. The analysisof the secondary structure indicated thatβ-sheets occurred frequently (42.5%) throughout thestructure of the region, whereas there were none in other regions. The phylogenetic treeindicated that AccCPR1has a greater level of sequence identity with Hymenoptera CPRs thanwith Diptera or Hemiptera CPRs. The strict R&R consensus: G-x8-G-x6-(Y/F) was found inthis region. But AccCPR1and its orthologs are exception for the most conservative sequencemotif. Five putative E74binding sites and four BR-C binding sites were predicted in the5'-flanking region, which suggests a potential function in molting and metamorphosis.
RT-qPCR showed that AccCPR1transcript occurred as the ecdysteroid titer decreased after reaching a peak, which suggests AccCPR1expression requires a “pulse” regimen ofecdysteroids. This hypothesis was tested using different experimental strategies.20E is amajor component of ecdysone. When larvae were reared with different concentrations of20Ein their diet, the ecdysteroid peak repressed AccCPR1expression. Exposure of the thoracicintegument of the pupae in vitro to different concentration of20E repressed AccCPR1expression, which recovered after the removal of20E. Tissue-specific expression andimmunohistochemical localization showed AccCPR1expressed in the epidermis and in themidgut. These results suggest that AccCPR1is a typical cuticle R&R-2protein that plays animportant role in development, and an ecdysteroid pulse is critical for high AccCPR1geneexpression.
(2) We cloned and characterized another cuticle protein R&R gene, referred to AccCPR2,from the honeybee (A. cerana cerana). The full-length cDNA (GenBank accession number:KJ502287) has a length of1134nt, which contains an open reading frame (ORF) of705ntencoding a234amino acid polypeptide with a predicted molecular mass of26.3kDa and anisoelectric point of6.90. The protein contains a chitin binding region and the strict R&Rconsensus: G-x8-G-x6-(Y/F) was found in this region. The first intron interrupts the signalpeptide after five coding amino acids in the AccCPR2genomic sequence, which is a typicalcharacteristic of CPR genomic sequences in insects. Three BR-C, five CdxA, one HB andthree DFD binding sites were predicted in the5'-flanking region, suggesting a potentialfunction in molting and metamorphosis.
Bioinformatics analysis indicated that AccCPR2contains a chitin binding region and is atypical cuticle R&R-2protein, while its expression pattern has a relatively large differenceswith "pulse" mechanismin that have been found in some insects. Expression profile of thedifferent developmental stages showed that the expression level of AccCPR2increased withthe increase of ecdysone concentration during the pupal stage, indicating that AccCPR2transcription needs ecdysone persist. Exposure of the integument of the pharte pupae in vitroto different concentration of20E, the expression of AccCPR2was identified with the increaseof20E concentration. Environmental stressors, such as heavy metals and pesticides, alsoinfluenced gene expression. In addition, a disc diffusion assay showed that AccCPR2enhanced the ability of bacterial cells to resist multiple stresses. We infer from our results thatAccCPR2acts in honeybee development and in protecting these insects from abiotic stresses.
The results of this study found that, AccCPR1and AccCPR2belong to R&R-2typecuticle protein genes. There are structural differences of the most conserved motifs betweenthe two polypeptide chains and the two genes are regulated by ecdysone titer with different expression patterns, suggesting different cuticle protein genes in Apis cerana cerana play avariety of roles in metamorphosis mechanisms.
引文
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