基于多杀菌素胁迫的桔小实蝇蛋白质组学初步研究

发布时间：2018-06-16 22:30

本文选题：桔小实蝇 + 多杀菌素　；参考：《海南大学》2015年硕士论文

【摘要】：桔小实蝇Bactrocera dorsalis (Hendel)是我国华南地区的重要果蔬害虫,可为害柑桔、芒果、番石榴、杨桃等250多种果实,被许多国家和地区列为检疫性害虫。多杀菌素是在刺糖多胞菌(Saccharopolyspora spinosa)发酵液中提取的一种大环内酯类无公害高效生物杀虫剂,既作用于烟碱型乙酰胆碱受体(nicotinic acetylcholine receptor, nAChR),也作用于γ-氨基丁酸受体(GABAR),能有效控制鳞翅目、双翅目和缨翅目等多种害虫。目前,多杀菌素是生产上有效防治桔小实蝇的主要杀虫剂之但已有报道表明桔小实蝇对多杀菌素产生了一定的抗药性风险。因此,为了合理使用多杀菌素防治桔小实蝇发生与危害,延长多杀菌素的使用寿命,本研究将昆虫毒理学、生物信息学与蛋白质组学相结合,初步开展了基于多杀菌素胁迫的桔小实蝇蛋白质组学研究,建立了桔小实蝇幼虫总蛋白的双向电泳条件,分离和鉴定了多杀菌素胁迫前后的桔小实蝇差异表达蛋白,为深入探讨多杀菌素的抗性形成机制研究奠定了基础。主要结果如下： 1.建立并优化了桔小实蝇幼虫总蛋白的双向电泳条件,确定使用24cm pH4-7的IPG胶条对BPP法提取的桔小实蝇幼虫总蛋白进行双向电泳,采用考马斯亮蓝法对双向电泳凝胶进行染色为最适桔小实蝇蛋白质组学研究的方法,为开展基于多杀菌素胁迫的桔小实蝇蛋白质组学初步研究工作奠定了基础。比较了BPP法和3种TCA-丙酮法提取桔小实蝇幼虫总蛋白的效果,结果表明：BPP法效果最好,可以获得1187±42个蛋白点,且分离得到的蛋白点分辨率更高。比较了13、24cm pH4-7的IPG胶条分离蛋白质的效果,发现使用24cm胶条分离得到1129±46个蛋白点,较13cm胶条增加了483个蛋白点。比较考马斯亮蓝染色与硝酸银染色的效果发现二者染色效果相当,但考马斯亮蓝染色背景较硝酸银染色干净清晰。质谱鉴定的BPP胶中10个特异的蛋白点以Metazoa (Animals)为检索物种可成功检索9个,以Drosophila (fruit flies)为检索物种可成功检索6个,且以Metazoa (Animals)为检索物种可获得更全面的信息。 2.分离和鉴定了5mg/mL多杀菌素短时间胁迫前后的桔小实蝇差异表达蛋白,发现所鉴定的蛋白主要参与物质能量代谢、免疫反应、解毒功能、细胞骨架生成等,为深入探讨桔小实蝇对低浓度多杀菌素胁迫的应答机制提供了基础信息支撑。采用浸渍法处理桔小实蝇3龄老熟幼虫,浸泡时间10s,浸泡后置于土壤中。丙酮处理作为对照组(CK),置于土壤5min后取样,5mg/mL多杀菌素处理作为处理组,分别置于土壤5min、2h后取样。获得丰度变化在2倍以上的58个差异蛋白点并进行了质谱鉴定,共鉴定出44个,其中23个上调表达,21个下调表达。通过生物信息学分析,发现所鉴定的蛋白主要参与物质能量代谢、免疫反应、解毒功能、细胞骨架生成等,其中物质能量代谢相关蛋白所占比例最多,达到43%。 3.分离和鉴定了10mg/mL多杀菌素长时间胁迫前后的桔小实蝇差异表达蛋白,发现所鉴定的蛋白包括物质能量代谢相关蛋白、防御或免疫相关蛋白、神经系统发展相关蛋白、细胞骨架蛋白及其它,为进一步探讨桔小实蝇对高浓度多杀菌素胁迫的应答机制提供了基础信息支撑。采用浸渍法处理桔小实蝇3龄老熟幼虫,浸泡时间10s,浸泡后置于土壤中。丙酮处理作为对照组(CK),置于土壤6h后取样,10mg/mL多杀菌素处理作为处理组,分别置于土壤6h、12h后取样。获得了54个表达量变化2倍以上的差异蛋白点,成功鉴定了42个蛋白点,其中上调蛋白点17个,下调蛋白点25个。鉴定的蛋白经生物信息学分析后按功能分为5类：(1)物质能量代谢相关蛋白,占67%；(2)防御或免疫相关蛋白,占2%；(3)神经系统发展相关蛋白,占7%；(4)细胞骨架蛋白,占2%；(5)其它,占22%。
[Abstract]:Bactrocera ( Hendel ) , an important fruit and vegetable pest in South China , has been classified as quarantine pests in many countries and regions .

In order to prevent and control the occurrence and harm of Bactrocera citrinum and prolong the service life of polybactericidal agent , it has been reported that the two - dimensional electrophoresis condition of the total protein of Bactrocera Bactrocera is established , the two - dimensional electrophoresis condition of the total protein of Bactrocera Bactrocera is established , and the basis of the study is laid for further exploring the mechanism of the resistance formation mechanism of polybactericidal agent . The main results are as follows :

1 . Two - dimensional electrophoresis conditions were established and optimized for the total protein of Bactrocera citrinata larvae . Two - dimensional electrophoresis was carried out on the total protein extracted from BPP by IPG strips of 24 cm pH 4 - 7 .

The effects of BPP method and three TCA - acetone methods on the total protein of fly larvae were compared . The results showed that the results showed that the best results of BPP were 1187 卤 42 protein spots and higher resolution .

2 . Isolation and identification of the differentially expressed proteins of Bactrocera citrinae before and after a short time stress of 5mg / mL , found that the identified proteins mainly involved in substance energy metabolism , immune response , detoxification function , cytoskeletal formation and so on .

Three - instar larvae of orange fly were treated with immersion method for 10 seconds , soaked and placed in soil . After 5 min of soil acetone treatment was taken as control group ( CK ) , the samples were sampled after 5 min of soil , and then sampled at 5 mg / mL for 2 hours .

3 . Isolate and identify the differentially expressed proteins of Bactrocera citrinae before and after long - term stress of 10 mg / mL , and found that the identified proteins include substance energy metabolism - related proteins , defense or immune - related proteins , nervous system development - related proteins , cytoskeletal proteins , and other proteins .

The 3 - year old larvae of orange fly were treated by impregnation . The soaking time was 10 seconds , soaked and placed in the soil . After 6 hours of soil , the samples were sampled after 6 hours of soil and 10 mg / mL multi - bactericide was taken as the treatment group .
( 2 ) defense or immune related protein , accounting for 2 % ;
( 3 ) nervous system development related protein , accounting for 7 % ;
( 4 ) cytoskeletal proteins , accounting for 2 % ;
( 5 ) Other , accounting for 22 % .
【学位授予单位】：海南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433

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