家蠶蛾觸角蛋白的雙向電泳分析.pdf

1、 II家蠶蛾觸角蛋白的雙向電泳分析 中文摘要 觸角是昆蟲間通訊的主要器官，在外部形態(tài)、嗅覺和味覺感受機制、昆蟲間通訊及發(fā)生發(fā)育方面表現(xiàn)出一定的雌雄性別差異， 目前有關(guān)這些差異的分子生理基礎(chǔ)并不全面。為探討這種差異的分子基礎(chǔ)，本研究以家蠶蛾作為模式昆蟲，運用蛋白質(zhì)組學技術(shù)探究雌雄家蠶蛾觸角蛋白的表達譜特征， 獲得雌雄蛾觸角的差異和特異蛋白， 采用質(zhì)譜分析技術(shù)鑒定了這些蛋白。在此基礎(chǔ)上采用實時熒光定量PCR方法，研究相關(guān)特征蛋白的基因轉(zhuǎn)錄水

2、平表達變動，獲得的研究結(jié)果如下： 1 家蠶蛾觸角蛋白雙向電泳表達圖譜的研究 1.1家蠶蛾觸角蛋白雙向電泳圖譜建立 采用雙向電泳技術(shù)獲得了家蠶蛾觸角總蛋白以及雌雄蛾觸角蛋白的電泳圖譜。 采用 Image Master 6.0 軟件分析電泳圖譜，在家蠶蛾觸角中檢測到約 550 個點，主要集中在分子量 14～70 kD、等電點 4～8 之間。從顯色時間為 4min 的雌雄蛾觸角電泳圖譜中分別檢測到 419 和 489 個蛋白點。其中雌雄匹配蛋

3、白點有 326 對，匹配率為71.81%。 通過改變顯色時間獲得了蛋白點數(shù)目更多、更清晰的電泳圖譜。采用 Image Master 6.0 軟件對顯色時間為 8 min 時獲得的電泳圖譜進行分析，從雌雄蛾觸角電泳圖譜中分別檢測到 693 和 782 個蛋白點。其中雌雄匹配蛋白點有 513 對，匹配率為69.56%。 雌雄間表達差異點有 23 個， 雌雄分別所特有的特異蛋白點分別為 4 和 7 個。1.2 家蠶雌雄蛾觸角中差異和特異蛋白質(zhì)

4、譜鑒定 采用 MALDI-TOF/MS 方法對雌雄家蠶蛾觸角中差異和特異蛋白進行了鑒定。經(jīng)數(shù)據(jù)庫檢索，共鑒定出 9 種蛋白：成蟲原基生長因子(imaginal disk growth factor)、表皮蛋白 RR-1 基序 15(cuticular protein RR-1 motif 15)、硫醇過氧化還原酶(thiol peroxiredoxin)、空泡 ATP 酶 B 亞基(vacuolar ATPase B subunit)、

5、gasp 前體(gasp precursor)、 成熟的 30K 脂蛋白(mature 30K lipoprotein) 、 類異翅盤蛋白(abnormal wing disc-like protein)、轉(zhuǎn)酮醇酶(transketolase)以及一種家蠶性信息素結(jié)合蛋白片段的液態(tài)結(jié)構(gòu)(Chain A, Solution Structure Of The Bombyx Mori Pheromone-Binding Protein IVA

6、nalysis of antenna protein of silk moth by two -dimensional electrophoresis Abstract Antenna is the major communication organ among insects. Antenna have some male and female gender differences in external shape, smell a

7、nd 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,

8、 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 obtaine

9、d 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 transcriptio

10、n 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 pat

11、terns 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

12、 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 wer

13、e 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 obtaine

14、d 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 d