家蚕蛾触角蛋白的双向电泳分析
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摘要
触角是昆虫间通讯的主要器官,在外部形态、嗅觉和味觉感受机制、昆虫间通讯及发生发育方面表现出一定的雌雄性别差异,目前有关这些差异的分子生理基础并不全面。为探讨这种差异的分子基础,本研究以家蚕蛾作为模式昆虫,运用蛋白质组学技术探究雌雄家蚕蛾触角蛋白的表达谱特征,获得雌雄蛾触角的差异和特异蛋白,采用质谱分析技术鉴定了这些蛋白。在此基础上采用实时荧光定量PCR方法,研究相关特征蛋白的基因转录水平表达变动,获得的研究结果如下:
1家蚕蛾触角蛋白双向电泳表达图谱的研究
1.1家蚕蛾触角蛋白双向电泳图谱建立
采用双向电泳技术获得了家蚕蛾触角总蛋白以及雌雄蛾触角蛋白的电泳图谱。采用Image Master 6.0软件分析电泳图谱,在家蚕蛾触角中检测到约550个点,主要集中在分子量14～70 kD、等电点4～8之间。从显色时间为4min的雌雄蛾触角电泳图谱中分别检测到419和489个蛋白点。其中雌雄匹配蛋白点有326对,匹配率为71.81%。
通过改变显色时间获得了蛋白点数目更多、更清晰的电泳图谱。采用Image Master 6.0软件对显色时间为8 min时获得的电泳图谱进行分析,从雌雄蛾触角电泳图谱中分别检测到693和782个蛋白点。其中雌雄匹配蛋白点有513对,匹配率为69.56%。雌雄间表达差异点有23个,雌雄分别所特有的特异蛋白点分别为4和7个。1.2家蚕雌雄蛾触角中差异和特异蛋白质谱鉴定
采用MALDI-TOF/MS方法对雌雄家蚕蛾触角中差异和特异蛋白进行了鉴定。经数据库检索,共鉴定出9种蛋白:成虫原基生长因子(imaginal disk growth factor)、表皮蛋白RR-1基序15(cuticular protein RR-1 motif 15)、硫醇过氧化还原酶(thiol peroxiredoxin)、空泡ATP酶B亚基(vacuolar ATPase B subunit)、gasp前体(gasp precursor)、成熟的30K脂蛋白(mature 30K lipoprotein)、类异翅盘蛋白(abnormal wing disc-like protein)、转酮醇酶(transketolase)以及一种家蚕性信息素结合蛋白片段的液态结构(Chain A, Solution Structure Of The Bombyx Mori Pheromone-Binding ProteinFragment),其中前六种在雄触角中的表达量要高于雌触角中,类异翅盘蛋白和转酮醇酶在雄触角中的表达量高于雌触角。家蚕性信息素结合蛋白片段的液态结构在雌雄触角都中存在但是在各自的电泳图谱中的位置却不一致。这些蛋白分别涉及不同的代谢途径。
2触角相关基因Bmidgf、Bmtpx以及BmvatB的表达定量分析
采用实时荧光定量PCR方法,对触角相关基因Bmidgf、Bmtpx和BmvatB在5龄第3天的家蚕幼虫各组织以及不同时期成虫触角组织中的转录水平进行检测,并采用家蚕Actin3内参照基因进行归一化处理。结果表明Bmidgf、Bmtpx和BmvatB在不同组织中均表现出一定的转录水平的差异。在不同时期的触角组织中,Bmidgf和BmvatB的转录活性无明显差异,Bmtpx则表现出较为明显的发育阶段差异性。根据以上实验结果推测,以上不同组织和时相的基因表达差异可能与不同时期的触角功能差异相关。
本研究分析了不同性别家蚕蛾触角蛋白的表达谱,通过质谱鉴定得到了成虫原基生长因子、类异翅盘蛋白和家蚕性信息素结合蛋白片段的液态结构等10种差异和特异蛋白。这些蛋白涉及不同的代谢机制,可能与家蚕蛾触角的发生发育和雌雄蛾间的通讯机制相关。上述研究丰富了家蚕蛋白质组学的研究,为进一步研究触角形态和功能差异提供了一定的基础信息。
Antenna is the major communication organ among insects. Antenna have some male and female gender differences in external shape, smell and taste feeling mechanism, communication among insects and development. Currently, the molecular and physiological basis about these differences is not comprehensive. To explore the molecular basis of these differences, we used silk moth as a model insect, and the proteomics technologies were performed to research the protein expression profile characteristics of antenna of male and female silk moth by proteomic techniques, and obtained different and specific proteins between male and female antenna. And then using mass spectrometry techniques, we identified these proteins. On top of it, the characteristics of protein in the level of gene transcription characteristic was studied by quantitative real time PCR(qPCR) method. The results obtained are showned as follows:
1 Research of protein expression profile of antenna protein of silk moth
1.1 The establishment of patterns of antenna protein of silk moth by two dimensional polyacrylamide gel electrophoresis
We obtained expression profiles of total antenna protein and male and female antenna protein. The Image Master 6.0 software was used to analyze the two-dimensional gel electrophoresis profiles. Approximately 550 spots were detected in the antenna of silk moth, most of which were arranged from 14 to 70 kD with pI value(4～8). 419 and 489 spots were detected in the two-dimensional gel electrophoresis profile of female and male antenna at 4 minutes of color-display time, respectively, and the matched protein spots were 326. The matching rate was 71.81%.
We obtained profiles which were clearer and have more protein spots by chananging color-display time. The Image Master 6.0 software was used to analyze the new two-dimensional gel electrophoresis profiles. 693 and 782 spots were detected in the two-dimensional gel electrophoresis profile of female and male antenna, respectively, and the matched protein spots were 513. The matching rate was69.56%. There were23 spots which have different gray value but distributed in both gel electrophoresis profile of female and male antenna. Meanwhile, there were 4 and 7 special spots which detected in gel electrophoresis profile of female and male respectively.
1.2 The spectrum identification of different and specific protein in male and female antenna
The different and specific protein spots were identified by MALDI-TOF MS. Through database, we obtained nine proteins: imaginal disk growth factor, cuticular protein RR-1 motif 15, thiol peroxiredoxin, vacuolar ATPase B subunit, gasp precursor, mature 30K lipoprotein, abnormal wing disc-like protein, transketolase and chain A, solution structure of the Bombyx Mori pheromone-binding protein fragment. Imaginal disk growth factor, cuticular protein RR-1 motif 15, thiol peroxiredoxin, vacuolar ATPase B subunit, gasp precursor and mature 30K lipoprotein have higher expression levels in male antenna than in female antenna. Abnormal wing disc-like protein and transketolase have higher expression levels in female antenna than in male antenna. Chain A exist in all male and female antenna, but its position in the profile of male antenna was inconsistent with that in the profile of female antenna. These proteins are involved in different metabolic pathways respectively.
2 Quantitative analysis of transcription levels of Bmidgf, Bmtpx and BmvatB
The transcription levels in each tissue of Bombyx mori of day 3 of 5th instar and in antenna of different periods of Bmidgf, Bmtpx and BmvatB were studied by quantitative real time PCR(qPCR) method. The detection results were normalized by using a Bombyx mori internal reference gene namely Actin3. The results indicated that Bmidgf, Bmtpx and BmvatB have different transcription levels in different tissues of Bombyx mori of day 3 of 5th instar. The transcription levels of Bmidgf and BmvatB have no obvious differences in antenna of different periods. Bmtpx have different transcription levels in antenna of different periods. It is thus concluded that the result was probably involved in the different function of antenna in different periods.
We have analyzed the protein expression profiles of male and female antenna protein of silk moth. Through mass spectrometry we obtained ten kinds of protein which were expressed differentially and specificly between female and male antenna, including imaginal disk growth factor, abnormal wing disc-like protein and chain A etc. These proteins are involved in different metabolize mechanism, and probably related with the development of antenna of silk moth and the communication mechanism between male and female silk moth. The results not only enriched the proteomics research of Bombyx mori, but provided some basic informations for further study of morphological and functional differences between male and female antenna.
1家蚕蛾触角蛋白双向电泳表达图谱的研究
1.1家蚕蛾触角蛋白双向电泳图谱建立
采用双向电泳技术获得了家蚕蛾触角总蛋白以及雌雄蛾触角蛋白的电泳图谱。采用Image Master 6.0软件分析电泳图谱,在家蚕蛾触角中检测到约550个点,主要集中在分子量14～70 kD、等电点4～8之间。从显色时间为4min的雌雄蛾触角电泳图谱中分别检测到419和489个蛋白点。其中雌雄匹配蛋白点有326对,匹配率为71.81%。
通过改变显色时间获得了蛋白点数目更多、更清晰的电泳图谱。采用Image Master 6.0软件对显色时间为8 min时获得的电泳图谱进行分析,从雌雄蛾触角电泳图谱中分别检测到693和782个蛋白点。其中雌雄匹配蛋白点有513对,匹配率为69.56%。雌雄间表达差异点有23个,雌雄分别所特有的特异蛋白点分别为4和7个。1.2家蚕雌雄蛾触角中差异和特异蛋白质谱鉴定
采用MALDI-TOF/MS方法对雌雄家蚕蛾触角中差异和特异蛋白进行了鉴定。经数据库检索,共鉴定出9种蛋白:成虫原基生长因子(imaginal disk growth factor)、表皮蛋白RR-1基序15(cuticular protein RR-1 motif 15)、硫醇过氧化还原酶(thiol peroxiredoxin)、空泡ATP酶B亚基(vacuolar ATPase B subunit)、gasp前体(gasp precursor)、成熟的30K脂蛋白(mature 30K lipoprotein)、类异翅盘蛋白(abnormal wing disc-like protein)、转酮醇酶(transketolase)以及一种家蚕性信息素结合蛋白片段的液态结构(Chain A, Solution Structure Of The Bombyx Mori Pheromone-Binding ProteinFragment),其中前六种在雄触角中的表达量要高于雌触角中,类异翅盘蛋白和转酮醇酶在雄触角中的表达量高于雌触角。家蚕性信息素结合蛋白片段的液态结构在雌雄触角都中存在但是在各自的电泳图谱中的位置却不一致。这些蛋白分别涉及不同的代谢途径。
2触角相关基因Bmidgf、Bmtpx以及BmvatB的表达定量分析
采用实时荧光定量PCR方法,对触角相关基因Bmidgf、Bmtpx和BmvatB在5龄第3天的家蚕幼虫各组织以及不同时期成虫触角组织中的转录水平进行检测,并采用家蚕Actin3内参照基因进行归一化处理。结果表明Bmidgf、Bmtpx和BmvatB在不同组织中均表现出一定的转录水平的差异。在不同时期的触角组织中,Bmidgf和BmvatB的转录活性无明显差异,Bmtpx则表现出较为明显的发育阶段差异性。根据以上实验结果推测,以上不同组织和时相的基因表达差异可能与不同时期的触角功能差异相关。
本研究分析了不同性别家蚕蛾触角蛋白的表达谱,通过质谱鉴定得到了成虫原基生长因子、类异翅盘蛋白和家蚕性信息素结合蛋白片段的液态结构等10种差异和特异蛋白。这些蛋白涉及不同的代谢机制,可能与家蚕蛾触角的发生发育和雌雄蛾间的通讯机制相关。上述研究丰富了家蚕蛋白质组学的研究,为进一步研究触角形态和功能差异提供了一定的基础信息。
Antenna is the major communication organ among insects. Antenna have some male and female gender differences in external shape, smell and taste feeling mechanism, communication among insects and development. Currently, the molecular and physiological basis about these differences is not comprehensive. To explore the molecular basis of these differences, we used silk moth as a model insect, and the proteomics technologies were performed to research the protein expression profile characteristics of antenna of male and female silk moth by proteomic techniques, and obtained different and specific proteins between male and female antenna. And then using mass spectrometry techniques, we identified these proteins. On top of it, the characteristics of protein in the level of gene transcription characteristic was studied by quantitative real time PCR(qPCR) method. The results obtained are showned as follows:
1 Research of protein expression profile of antenna protein of silk moth
1.1 The establishment of patterns of antenna protein of silk moth by two dimensional polyacrylamide gel electrophoresis
We obtained expression profiles of total antenna protein and male and female antenna protein. The Image Master 6.0 software was used to analyze the two-dimensional gel electrophoresis profiles. Approximately 550 spots were detected in the antenna of silk moth, most of which were arranged from 14 to 70 kD with pI value(4～8). 419 and 489 spots were detected in the two-dimensional gel electrophoresis profile of female and male antenna at 4 minutes of color-display time, respectively, and the matched protein spots were 326. The matching rate was 71.81%.
We obtained profiles which were clearer and have more protein spots by chananging color-display time. The Image Master 6.0 software was used to analyze the new two-dimensional gel electrophoresis profiles. 693 and 782 spots were detected in the two-dimensional gel electrophoresis profile of female and male antenna, respectively, and the matched protein spots were 513. The matching rate was69.56%. There were23 spots which have different gray value but distributed in both gel electrophoresis profile of female and male antenna. Meanwhile, there were 4 and 7 special spots which detected in gel electrophoresis profile of female and male respectively.
1.2 The spectrum identification of different and specific protein in male and female antenna
The different and specific protein spots were identified by MALDI-TOF MS. Through database, we obtained nine proteins: imaginal disk growth factor, cuticular protein RR-1 motif 15, thiol peroxiredoxin, vacuolar ATPase B subunit, gasp precursor, mature 30K lipoprotein, abnormal wing disc-like protein, transketolase and chain A, solution structure of the Bombyx Mori pheromone-binding protein fragment. Imaginal disk growth factor, cuticular protein RR-1 motif 15, thiol peroxiredoxin, vacuolar ATPase B subunit, gasp precursor and mature 30K lipoprotein have higher expression levels in male antenna than in female antenna. Abnormal wing disc-like protein and transketolase have higher expression levels in female antenna than in male antenna. Chain A exist in all male and female antenna, but its position in the profile of male antenna was inconsistent with that in the profile of female antenna. These proteins are involved in different metabolic pathways respectively.
2 Quantitative analysis of transcription levels of Bmidgf, Bmtpx and BmvatB
The transcription levels in each tissue of Bombyx mori of day 3 of 5th instar and in antenna of different periods of Bmidgf, Bmtpx and BmvatB were studied by quantitative real time PCR(qPCR) method. The detection results were normalized by using a Bombyx mori internal reference gene namely Actin3. The results indicated that Bmidgf, Bmtpx and BmvatB have different transcription levels in different tissues of Bombyx mori of day 3 of 5th instar. The transcription levels of Bmidgf and BmvatB have no obvious differences in antenna of different periods. Bmtpx have different transcription levels in antenna of different periods. It is thus concluded that the result was probably involved in the different function of antenna in different periods.
We have analyzed the protein expression profiles of male and female antenna protein of silk moth. Through mass spectrometry we obtained ten kinds of protein which were expressed differentially and specificly between female and male antenna, including imaginal disk growth factor, abnormal wing disc-like protein and chain A etc. These proteins are involved in different metabolize mechanism, and probably related with the development of antenna of silk moth and the communication mechanism between male and female silk moth. The results not only enriched the proteomics research of Bombyx mori, but provided some basic informations for further study of morphological and functional differences between male and female antenna.
引文
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