长足大竹象消化道不同分段菌群异质性及竹木质纤维素降解能力

【目的】长足大竹象Cyrtotrachelus buqueti消化道共生菌群参与了竹纤维素的降解。本研究旨在揭示长足大竹象幼虫消化道不同分段共生菌群异质性及木质纤维素的降解能力。【方法】通过对16S rRNA测序对长足大竹象幼虫消化道分段口器(YB)、前肠(YFG)、中肠(YMG)和后肠(YHG)进行菌群组成分析及功能预测;分析各消化道分段核心属细菌基因组中碳水化合物活性酶(carbohydrate active enzyme, CAZy)基因,预测木质纤维素降解能力;应用口器混合菌(MPJ)、前肠混合菌(FJ)、中肠混合菌(MJ)和后肠混合菌(HJ)悬液体外降解竹笋粉,检测共生菌群的木质纤维素的降解能力。【结果】长足大竹象幼虫消化道菌群多样性分析显示,YFG, YMG和YHG的菌群多样性大于YB,YFG的菌群物种多样性最高,而YB最低。YFG, YMG和YHG样本中厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidetes)和变形菌门(Proteobacteria)相对丰度最高,而YB中变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)和放线菌门(Actinobacteria)相对丰度最高。核心属细菌基因组CAZy基因分析表明长足大竹象幼虫消化道中大多数细菌基因组中存在丰富的CAZy基因,且在拟杆菌属Bacteroides细菌基因组中尤为明显,预示其与木质纤维素的降解密切相关。体外降解竹笋粉实验结果表明,长足大竹象幼虫MPJ, FJ, MJ和HJ对竹笋粉纤维素的降解率分别为21.7%, 39.9%, 44.2%和21.0%,对半纤维素降解率分别为72.7%, 52.3%, 65.7%和61.5%,对木质素降解率分别为20.5%, 41.3%, 39.9%和37.9%。【结论】长足大竹象幼虫的消化道菌群的异质性可以影响木质纤维素降解能力,因而这些菌群可以作为分离高效木质纤维素降解菌的重要来源之一。本研究为竹生物质的工业转化利用提供部分参考信息。     

琥珀蚕孵化酶基因AaHE的克隆及启动子活性分析

【目的】琥珀蚕Antheraea assama具有典型的野蚕特征,蚕卵孵化不齐,严重影响琥珀蚕的室内规模化饲养。本研究旨在探究对琥珀蚕卵孵化起关键作用的孵化酶(hatching enzyme)基因及其启动子序列特征,为进一步选择合适的抑制剂或促进剂调节琥珀蚕卵的孵化奠定基础。【方法】采用RACE技术克隆琥珀蚕孵化酶基因的cDNA全长序列,对基因序列进行生物信息学分析;采用qRT-PCR检测琥珀蚕孵化酶基因在琥珀蚕不同发育天数卵中及5龄第3和4天幼虫不同组织(丝腺、马氏管、头、中肠、脂肪体、表皮、血液、精巢和卵巢)中的表达情况;采用染色体步移克隆琥珀蚕孵化酶基因的启动子序列,构建昆虫细胞重组表达载体转染家蚕Bombyx mori BmN细胞,检测琥珀蚕孵化酶基因启动子活性。【结果】获得了琥珀蚕孵化酶基因AaHE(GenBank登录号： KT336227.1)全长cDNA序列,长993 bp,编码294个氨基酸,预测蛋白质分子质量为33.7 kD,理论等电点为5.17。AaHE氨基酸序列含有一个信号肽和一个ZnMc结构域,AaHE是一种含有HExxH锌结合位点的锌依赖性蛋白水解酶,该类酶既是肽酶,同时又是一种消化酶。AaHE在琥珀蚕孵化前的卵及5龄幼虫中肠中特异性高表达,分别与AaHE的肽酶和消化酶的属性相吻合。AaHE启动子核心区存在多个转录因子结合位点,这可能与转录因子参与调节AaHE的表达有关。启动子活性分析表明,AaHE启动子在家蚕BmN细胞中能够启动EGFP基因的表达,具有明显的启动子活性。【结论】AaHE是锌依赖性蛋白水解酶,其启动子核心区存在多个转录因子结合位点。本研究为选择合适的抑制剂或促进剂调节琥珀蚕卵的孵化率提供了参考。     

大豆蚜Kazal型丝氨酸蛋白酶抑制剂基因AgKaSPI 对蜡蚧刺束梗孢菌侵染的表达响应

【目的】探究Kazal型丝氨酸蛋白酶抑制剂KaSPI在大豆蚜Aphis glycines的生长发育、消化和免疫防御等过程中的作用。【方法】基于大豆蚜转录组数据PCR克隆大豆蚜Kazal型丝氨酸蛋白酶抑制剂基因cDNA序列;qRT-PCR分别检测AgKaSPI在大豆蚜1-4龄若虫和成虫以及蜡蚧刺束梗孢菌Akanthomyces     

The role of alanine synthesis and nitrate-induced nitric oxide production during hypoxia stress in Cucurbita pepo nectaries

Floral nectar is a sugary solution produced by nectaries to attract and reward pollinators. Nectar metabolites, such as sugars, are synthesized within the nectary during secretion from both pre-stored and direct phloem-derived precursors. In addition to sugars, nectars contain nitrogenous compounds such as amino acids; however, little is known about the role(s) of nitrogen (N) compounds in nectary function. In this study, we investigated N metabolism in Cucurbita pepo (squash) floral nectaries in order to understand how various N-containing compounds are produced and determine the role of N metabolism in nectar secretion. The expression and activity of key enzymes involved in primary N assimilation, including nitrate reductase (NR) and alanine aminotransferase (AlaAT), were induced during secretion in C. pepo nectaries. Alanine (Ala) accumulated to about 35% of total amino acids in nectaries and nectar during peak secretion; however, alteration of vascular nitrate supply had no impact on Ala accumulation during secretion, suggesting that nectar(y) amino acids are produced by precursors other than nitrate. In addition, nitric oxide (NO) is produced from nitrate and nitrite, at least partially by NR, in nectaries and nectar. Hypoxia-related processes are induced in nectaries during secretion, including lactic acid and ethanolic fermentation. Finally, treatments that alter nitrate supply affect levels of hypoxic metabolites, nectar volume and nectar sugar composition. The induction of N metabolism in C. pepo nectaries thus plays an important role in the synthesis and secretion of nectar sugar.     

甲烷生物利用及嗜甲烷菌的工程改造

作为来源广泛、储量丰富的有机碳一气体,甲烷被认为是下一代工业生物技术中最具潜力的碳原料之一。嗜甲烷菌能够利用其体内的甲烷单加氧化酶,将甲烷作为唯一的碳源和能源进行生长和代谢,这为温室气体减排及其开发利用提供了新的策略。目前,嗜甲烷菌生物催化体系的相关研究已开展多年,随着系统生物学和合成生物学的快速发展,利用代谢工程合理改造嗜甲烷菌代谢途径以提高甲烷转化效率,已经实现了生物转化甲烷制备多种大宗化学品和生物燃料。本文详细讨论并介绍了嗜甲烷菌催化氧化甲烷的相关代谢途径、高效细胞工厂构建及部分化学品生物合成的最新研究进展,并对甲烷生物转化未来的发展方向和面临的技术挑战进行了讨论和展望。     

嫁接诱导大豆低镉富集性状及遗传稳定性

通过野外小区实验,从8个大豆品种中筛选出2个低镉富集品种作为砧木,2个高镉富集品种作为接穗植物,研究嫁接当代以及接穗子代镉富集性状的变化,探明嫁接诱导镉富集性状变异的机制及其遗传稳定性。结果表明:大豆的镉富集性状表现出显著的品种间差异,以低镉富集品种(铁丰29和东鲜1号)作砧木,可以使接穗大豆植株(青仁黑1号和中黄38)地上部分镉含量降低50%~70%;高通量测序和qRT-PCR分析显示,嫁接诱导了相关硫代谢基因的差异表达,与自根植株相比,嫁接植株的APX2、PAPRa、SAM1a和ATPs基因表达水平均显著下调,分别达72.53%、67.62%、42.37%和75.78%,APK4表达量则显著上调2.83倍;以阻控效果最好的青仁黑1号-东鲜1号为例,接穗子代遗传了接穗当代的低镉富集性状,对照亲本,子代籽粒镉含量的降低率仍有30%~50%。研究证明:嫁接可以通过调控含硫化合物的合成与代谢,影响作物地上部分的Cd富集性状,且这种性状可以在子代中稳定遗传。     

模拟降水量变化对荒漠草原土壤酶活性的影响及其相关因素分析

为明确荒漠草原土壤酶活性对降水格局改变的响应机制, 该研究基于宁夏荒漠草原降水量不同梯度变化(减少50%、减少30%、自然降水、增加30%和增加50%)的野外试验(2014年开始试验), 于2016年5-7月采样, 测定分析不同降水梯度2年后对土壤酶活性的影响, 并分析酶活性与植物生物量、微生物生物量C∶N∶P生态化学计量特征以及土壤理化性质的关系。结果表明: (1)与自然降水量相比, 减少30%降水量对3种土壤酶活性均无显著影响, 减少50%降水量显著降低了土壤蔗糖酶活性(^P < 0.05); 增加降水量显著提高了土壤蔗糖酶和磷酸酶活性(^P < 0.05), 但对脲酶活性无显著影响。(2)减少降水量对植物生物量影响较小(尤其减少30%降水量), 但不同程度地降低了微生物生物量C、N、P, 提高了微生物生物量C∶N和C∶P; 增加降水量则不同程度提高了植物生物量及微生物生物量C、N、P。(3)土壤蔗糖酶和磷酸酶活性随植物及微生物生物量增加而增加; 对土壤酶活性影响显著的土壤因子包括: 含水量、NO₃^- N、NH₄⁺ N、C∶P、有机C、全N、C∶N和pH (P < 0.05)。研究认为, 减少降水量(尤其是减少30%降水量)对土壤酶活性影响较小, 增加降水量促进了植物的生长、刺激微生物活性, 进而提高了土壤酶活性, 但随着植物生物量增加, 土壤有机C输入增多, 磷酸酶活性相应增强并促进了有机P的矿化, 导致土壤微生物P限制增加。     

Enzymatic characterization of ancestral/group-IV clade xyloglucan endotransglycosylase/hydrolase enzymes reveals broad substrate specificities

Xyloglucan endotransglycosylase/hydrolase (XTH) enzymes play important roles in cell wall remodelling. Although previous studies have shown a pathway of evolution for XTH genes from bacterial licheninases, through plant endoglucanases (EG16), the order of development within the phylogenetic clades of true XTHs is yet to be elucidated. In addition, recent studies have revealed interesting and potentially useful patterns of transglycosylation beyond the standard xyloglucan–xyloglucan donor/acceptor substrate activities. To study evolutionary relationships and to search for enzymes with useful broad substrate specificities, genes from the ‘ancestral’ XTH clade of two monocots, Brachypodium distachyon and Triticum aestivum, and two eudicots, Arabidopsis thaliana and Populus tremula, were investigated. Specific activities of the heterologously produced enzymes showed remarkably broad substrate specificities. All the enzymes studied had high activity with the cellulose analogue HEC (hydroxyethyl cellulose) as well as with mixed-link β-glucan as donor substrates, when compared with the standard xyloglucan. Even more surprising was the wide range of acceptor substrates that these enzymes were able to catalyse reactions with, opening a broad range of possible roles for these enzymes, both within plants and in industrial, pharmaceutical and medical fields. Genome screening and expression analyses unexpectedly revealed that genes from this clade were found only in angiosperm genomes and were predominantly or solely expressed in reproductive tissues. We therefore posit that this phylogenetic group is significantly different and should be renamed as the group-IV clade.