家蚕近等基因系感染浓核病毒(BmDNV-Z)前后中肠组织蛋白的差异表达分析

Differential Expression of Midgut Proteins of Silkworm Near-isogenic Line Before and After Densovirus Infection

家蚕浓核病毒(BmDNV)是危害家蚕的主要病毒之一。为鉴定与家蚕抗病毒相关的蛋白质,以对BmDNV完全不感染的家蚕品种兰10(L10)为供体亲本,将其抗性基因导入家蚕敏感品种菁松(JS)中,构建抗性近等基因系NIL(BC6F2)。对JS及其近等基因系NIL分别用家蚕浓核病毒镇江株(BmDNV-Z)添毒,二者分别表现为高度敏感和完全不感染。对JS和NIL在病毒感染前后中肠组织蛋白双向电泳(2D-PAGE)图谱中的差异蛋白点进行串联质谱(MALDI-TOF-TOF)分析,共鉴定了41个差异表达的蛋白点,其中有35个蛋白点可能与病毒的诱导相关,有6个蛋白点可能与家蚕的组成抗性相关。鉴定的差异表达蛋白包括糖酵解酶类、能量代谢酶类、参与基因表达的蛋白质以及参与其它细胞功能的蛋白质等。选取10个差异表达蛋白点进行基因表达定量PCR分析,进一步确证了这些蛋白质在JS及其近等基因系NIL之间以及在病毒诱导前后的差异表达。例如:精氨酸激酶在NIL和JS中均可被诱导表达;V-ATP合成酶及热激蛋白HSP70只在NIL中被特异性诱导表达;烯醇化酶在NIL中的表达水平显著高于JS,且不能被病毒感染所诱导,是一个典型的组成抗性相关蛋白。鉴定出的41个差异表达蛋白点有可能参与了家蚕对BmDNV-Z的抗性。

Bombyx mori densovirus （BmDNV） is one of the major virulent pathogens to domesticated silkworm. To identify the antivirus-related proteins in silkworm, an antivirus near-isogenic line （NIL）, BC6F2, was constructed by using silkworm variety Lan 10 （LIO） with complete resistance to BmDNV as donor parent, and used to transfer the resistance genes into sensitive silkworm variety Jingsong （JS）. JS and its near-isogenic line NiL showed high sensitivity and complete resistance to BmDNV resloectivelv after infection by was carried out to analyze the differentially expressed midgu＇（ proteins present in the two dimensional electrop-horetogram （2D-PAGE） before and after virus in- fection. Altogether 41 differential protein spots were iden- tified, among which 35 protein spots are probably virus induction-related proteins, and 6 protein spots may be re- lated to constitutive resistance in silkworm. These identi-fled proteins included glycolytic enzyme, energy metabolism enzyme and the proteins involved in gene expression and other cellular functions. Quantitative PCR analysis to gene expression of 10 selected differentially expressed protein spots further confirmed the differential expression between JS and NIL before and after viral infection. For instance, arginine ki- nase was induced both in JS and NIL, while vacuolar ATP synthase and heat shock 70 kD protein were specifically ir^- duced in NIL, The expression level of enolase in NIL was significantly higher than that in JS. Enolase is a typical constitutive resistance protein, and could not be induced by virus challenge. The 41 identified differentially expressed proteins may be involved in the resistance of silkworm to BmDNV-Z.