草坪害虫华北大黑鳃金龟幼虫围食膜蛋白Ho-Peritrophin3基因克隆及序列分析

 华北大黑鳃金龟是一种重要的草坪害虫，其幼虫中肠围食膜是害虫生物防治的潜在靶标。本研究利用棉铃虫围食膜蛋白多克隆抗体，从已构建的华北大黑鳃金龟中肠ｃＤＮＡ 表达文库中筛选得到一个编码围食膜蛋白的ｃＤＮＡ 克隆Ｈｏ-Ｐｅｒｉｔｒｏｐｈｉｎ３，其ｃＤＮＡ 长度为１７３７ｂｐ，在ｐｏｌｙＡ 末端上游含有１个多聚腺苷酸信号序列ＡＡＴＡ-ＡＡ，最长开放阅读框（ＯＲＦ）编码５２８个氨基酸，与华北大黑鳃金龟围食膜蛋白Ｈｏ-Ｐｅｒｉｔｒｏｐｈｉｎ２的相似性最高，为６４．９％。结构域分析表明，Ｈｏ-Ｐｅｒｉｔｒｏｐｈｉｎ３只含有较少的Ｏ-糖基化位点和Ｎ-糖基化位点，不含有类粘蛋白结构域，具有５个几丁质结合功能域（ｃｈｉｔｉｎｂｉｎｄｉｎｇｄｏｍａｉｎ，ＣＢＤ），它们均由６个保守的半胱氨酸残基组成，与Ｈｏ-Ｐｅｒｉｔｒｏｐｈｉｎ１、Ｈｏ-Ｐｅｒｉｔｒｏｐｈｉｎ２羧基端的ＣＢＤ 只含有４个半胱氨酸残基明显不同。Ｈｏ-Ｐｅｒｉｔｒｏｐｈｉｎ３的胰蛋白酶和胰凝乳蛋白酶的作用位点主要位于５个ＣＢＤ 内部，受到其保护，不会被降解；与Ｈｏ-Ｐｅｒｉｔｒｏｐｈｉｎ１、Ｈｏ-Ｐｅｒｉｔｒｏｐｈｉｎ２不同的是在第５个ＣＢＤ 下游区域的１０７个氨基酸中，有３６．３％的氨基酸属于胰蛋白酶和胰凝乳蛋白酶的酶切位点。     

Effects of xylan and cottonseed meal on growth performance,digestion, intestinal morphology and the expression of two peritrophin genes of Chinese mitten crab,Eriocheir sinensis

Xylan is one of the main non‐starch polysaccharides (NSPs) in cottonseed meal. This study aimed to evaluate the anti‐nutrition effect of xylan and to better understand cottonseed meal as a feed ingredient for the Chinese mitten crab, Eriocheir sinensis. A diet containing 280 g/kg fishmeal without cottonseed meal or xylan was formulated as a control (FM diet). A second diet (XYL diet) was supplemented with 53.3 g/kg xylan according to its amount in the third diet, the cottonseed meal diet (CSM diet), which was formulated to include 400 g/kg cottonseed meal. The last diet (CSM + XYLase diet) included 400 g/kg xylanase hydrolysed cottonseed meal, in which most xylan was hydrolysed. Diets were isonitrogenous (370 g/kg crude protein) and isoenergetic (17.58 KJ/g) and were randomly fed to 12 tanks of crabs for 8 weeks. Results showed that no difference was detected among groups in growth performance, serum biochemical index, or digestive and metabolism enzyme activity (p > 0.05). The apparent digestibility coefficient (ADC) of dry matter was FM > XYL > CSM + XYLase > CSM (p < 0.05). The ADC of protein in crab fed diet FM and XYL was significantly higher than CSM and CSM + XYLase (p < 0.05). Cottonseed meal decreased the fold height of the hindgut, upregulated the expression of peritrophin genes of Es‐PL44 and Es‐PP1 (p < 0.05), while xylan decreased the fold height of the hindgut and upregulated the expression of Es‐PP1 (p < 0.05). These results indicated that the negative effect of cottonseed meal and xylan on digestibility partly compensated by enhanced absorption of the hindgut innerly and by supplementation of xylanase externally, and cottonseed meal is an attractive alternative protein source in general for Chinese mitten crab.     

凡纳滨对虾围食膜蛋白基因全长cDNA的克隆及序列分析

为研究凡纳滨对虾围食膜蛋白在IHHNV感染虾体过程中的作用,实验采用SMART技术构建了凡纳滨对虾肠道组织的全长cDNA文库,电子拼接获得了对虾围食膜蛋白基因全长cDNA序列,并进行了功能分析。全长cDNA文库的库容达1.05×106pfu/mL,重组率为95%。随机挑选1 600个克隆进行测序,去除载体后得到插入长度≥450 bp的有效序列1 531条,根据同源片段长度不小于100 bp,90%同源性的原则对这些序列归并unigene,得到unigene 399条。从所测克隆中得到一个围食膜蛋白基因,其cDNA序列长度为1 002 bp,编码由303个氨基酸组成的蛋白,基因序列比对发现该序列与斑节对虾卵巢围食膜蛋白1和2、围食膜蛋白前体3,短钩对虾围食膜蛋白1、2,以及中国对虾围食膜蛋白编码序列的同源性较高,均达到80%以上。通过半定量RT-PCR对该基因在不同组织的表达分析结果表明该基因在抗感染对虾传染性皮下及造血组织坏死病毒(IHHNV)的对虾的心脏和胃中高表达,在肝脏、肠和肌肉中表达较低,在鳃中表达最弱,在眼中不表达;该基因在感染IHHNV病毒的对虾的心脏表达明显减弱,在肝脏、眼、肌肉和鳃中表达较低,在肠道和胃中几乎不表达,说明该基因参与对虾抵御病原体侵入过程。     

凡纳滨对虾卵巢类围食膜蛋白基因克隆及其在早期胚胎发育中的表达

围食膜蛋白最早是从昆虫围食膜中解离得到的,在保护昆虫免受外源微生物侵染过程中发挥了重要作用。为了解围食膜蛋白在对虾免疫过程中的作用,实验前期进行了凡纳滨对虾转录组分析,并克隆获得了凡纳滨对虾2条类围食膜蛋白基因LvPT1和LvPT2,其编码的2条氨基酸序列相似性极高,同已报道的其他虾类围食膜蛋白具有较高同源性。利用Real-time PCR方法进行组织表达分析发现,LvPT1和LvPT2与对虾卵巢围食膜蛋白(SOPs)类似,在卵巢中高度表达,利用半定量PCR方法,分析LvPT2在对虾早期发育中的表达情况。结果显示,LvPT2在受精卵时期表达量最高,随着发育进行,表达量逐渐降低,至无节幼体孵出后无明显表达。研究表明,LvPT1和LvPT2与虾SOPs应为同一类围食膜蛋白,可能参与了受精卵细胞的形成,并在保护对虾早期胚胎发育时免受外界病原微生物的侵染中发挥着重要作用。