毛尖紫萼藓类泛素蛋白基因的克隆与表达分析

为研究类泛素蛋白基因在苔藓植物中的生物学功能,从毛尖紫萼藓干旱cDNA文库中获得了类泛素蛋白基因cDNA全长序列,命名为Gp-UBL5。该基因cDNA全长471 bp,包含1个222 bp的完整开放阅读框,编码73个氨基酸组成的蛋白。生物信息学分析表明,Gp-UBL5基因编码蛋白分子质量为8.525 kDa,等电点为7.84,为稳定型蛋白。进化分析显示,该编码蛋白与二穗短柄草、大麦等类泛素蛋白具有较高一致性,达96%~99%。实时荧光定量PCR结果表明,Gp-UBL5基因在复水和快速干旱的各个时期均有表达,推测该基因可能在毛尖紫萼藓的抗旱机制中发挥作用。     

Development of a two‐step,non‐probed multiplex real‐time PCR for surveilling Vibrio anguillarum in seawater

Vibrio anguillarum is an aggressive and halophilic bacterial pathogen most commonly originating from seawater. Vibrio anguillarum presence in fisheries and aquaculture facilities causes significant morbidity and mortality among aquaculture species primarily from haemorrhaging of the body and skin of the infected fish that eventually leads to death, collectively recognized as the disease vibriosis. This study served to develop a non‐probe, multiplex real‐time PCR assay to rapidly detect V. anguillarum presence in seawater. Specific primers targeting genes vah1, empA and rpoN of V. anguillarum were selected for multiplex reaction among 11 different primer sets and the extension step was eliminated. Primer concentration, denaturation time as well as annealing time and temperature of DNA amplification were optimized, thus reducing reaction duration. The two‐step, non‐probed multiplex real‐time PCR set forth by this study detects as little as 3 CFU mL^?1 of V. anguillarum presence in sea water, without enrichment cultivation, in 70 min with molecular precision and includes melting curve confirmation.     

共生菌群在熊蜂生长发育过程中的动态变化

【目的】了解人工饲养的兰州熊蜂（Bombus lantschouensis）肠道内共生菌群的组成,探明主要肠道共生菌群在熊蜂生长发育过程中的变化规律,为进一步开展熊蜂共生菌功能的研究奠定基础。【方法】以兰州熊蜂肠道总DNA为模板,使用细菌通用的774F和1391R引物进行PCR扩增,构建细菌16S rDNA文库,挑取单克隆菌落测序,测得序列去除chimera后,以序列相似性97%为标准,划分分类操作单元（operational taxonomic units,OTU）,采用BLASTn进行序列同源性分析,确定细菌种类,分析熊蜂肠道菌群的组成。根据克隆文库测得的Gilliamella apicola和Snodgrassella alvi细菌16S rDNA序列设计特异性引物。用G. apicola和S. alvi的特异性引物进行PCR扩增,构建重组子质粒,构建好的质粒经浓度测定后,10倍梯度稀释成5个浓度梯度,进行荧光定量PCR检测,绘制标准曲线。以兰州熊蜂的卵、幼虫、蛹以及0、5、10、15、20日龄的工蜂肠道DNA为模板,采用熊蜂β-actin为内参基因,对样品中的共生菌G. apicola和S. alvi进行荧光定量PCR分析,并比较不同日龄、不同虫态每微升肠道基因组DNA样品中检测到的细菌16S rDNA基因的拷贝数,分析共生菌数量在熊蜂生长发育过程中的变化。不同日龄、不同虫态之间相对表达量的显著性差异用软件SPSS19.0的单因素ANOVA方差分析进行统计。【结果】从文库中随机挑选213个单克隆进行测序,经过Chimeras分析后,共得到202个有效序列,这些序列共划分为16个OTU。测得的序列与登录的相应细菌16S rDNA序列的相似性在93%-99%。在克隆文库测得的细菌16S rDNA序列中,G. apicola占45%、S. alvi占30%、Bifidobacterium占10%、Fructobacillus fructose占5%、Lactobacillus占2%、Flavobacterium aciduliphilum占2%,其他细菌占6%。其中G. apicola和S. alvi为兰州熊蜂肠道内的主要共生菌,qPCR结果表明两种共生菌在不同日龄、不同虫态的熊蜂肠道内都能检测到,两种细菌的数量在熊蜂发育过程中的变化趋势相似,即先增加后减少,最后达到稳定状态。G. apicola和S. alvi在熊蜂的卵、幼虫和蛹中数量都较少,在5日龄时的数量达到峰值,显著高于其他日龄,之后又逐渐减少,在15日龄后趋于稳定,第15、20日龄之间差异不显著。【结论】人工饲养的兰州熊蜂肠道中主要有4个种属的常见共生菌：G. apicola、S. alvi、F. fructosus和Bifidobacterium,其中G. apicola和S. alvi是其体内的优势菌。G. apicola和S. alvi在熊蜂中都具有水平和垂直传播的特性,两种共生菌在熊蜂的卵、幼虫和蛹中都检测到,但数量较少,工蜂出房后细菌大量增殖并在出房15 d左右形成稳定的共生菌群。熊蜂生长发育过程中体内共生菌数量的变化可能与这两种细菌对熊蜂的作用有关。     

Alternative dietary protein sources for Atlantic salmon (Salmo salar L.) effect on intestinal microbiota,intestinal and liver histology and growth

The present study has addressed the issue of dietary alterations on gut microbiota in fish by investigating modulation of the allochthonous and autochthonous bacterial gut community of Atlantic salmon following feeding with pea protein concentrate, soy protein concentrate, extracted sunflower, poultry by‐product and feather meal. The results revealed that total allochthonous and total autochthonous bacteria isolated from the distal intestine (DI) were affected by dietary modulations. Total autochthonous bacteria in proximal intestine remained unaffected. Corynebacteriaceae dominated the allochthonous bacteria and Lactobacillaceae dominated the autochthonous bacteria community in proximal and DI indicating that the most abundant groups are not shifted by alternative proteins. Some variations were observed between the bacterial communities as result of dietary manipulations. Alternative protein sources resulted in no severe signs of enteritis. Vacuolization of the liver was unaffected by diet. Furthermore, addition of plant materials significantly affected production of acetic acid. Weight of pyloric‐, proximal‐ and DI was influenced by diet, especially feather meal. Growth performance was significantly affected by inclusion of feather meal to the diet. In conclusion the results showed that pea protein concentrate and poultry by‐products are suitable protein replacements for Atlantic salmon and that soy protein concentrate is a potential candidate.     

罗非鱼催乳素Ⅰ基因的组织分布及盐胁迫对其表达的影响

采用同源克隆方法从橙色莫桑比克罗非鱼（Oreochromis mossambicus）、荷那龙罗非鱼（O.hornorum）及其正反交子代垂体中克隆了催乳素Ⅰ基因（PRLⅠ）,并对其核苷酸序列和推导的氨基酸序列进行了比对分析。结果显示,4种罗非鱼的PRLⅠ基因序列长度均为798 bp,ORF长639 bp,共编码212个氨基酸;橙色莫桑比克罗非鱼和荷那龙罗非鱼仅在31号位点存在一个氨基酸残基差异。用q PCR方法分析橙色莫桑比克罗非鱼和荷那龙罗非鱼PRLⅠ基因在不同组织或器官中的表达,结果表明罗非鱼PRLⅠ基因在垂体中的表达量最高,在其他组织中的表达量较低;盐胁迫后4种罗非鱼垂体中的PRLⅠmRNA水平均显著降低,而在其他组织中的表达变化不大。由此可知PRLⅠ基因主要在垂体表达,参与罗非鱼的渗透压调节。     

Comparison of Arbuscular Mycorrhizal Fungal Community in Roots and Rhizosphere of Invasive Cenchrus incertus and Native Plant in Inner Mongolia,China

Plant invasions could significantly alter arbuscular mycorrhizal (AM) fungal communities, but the effect may vary with plant species and local environments. Identifying changes in the AM fungal community due to plant invasion could improve our understanding of the invasion processes. Here, we examined the AM fungal community composition both in roots and rhizo-sphere soils of the invasive plant Cenchrus incertus and the dominant native plant Setaria viridis in a typical steppe in Inner Mongolia by using terminal restriction fragment length polymorphism analyses (T-RFLP). The results showed that AM fungal abundance in the rhizosphere soils of C. incertus was significantly lower than that of S. viridis. The AM fungal community com-position in the rhizosphere soils of the two plant species also largely differed. In general, AM fungal community structures in roots corresponded very wel to that in rhizosphere soils for both plant species. The dominant AM fungal type both in invasive and native plants was T-RFLP 524bp, which represents Glomus sp. (Virtual taxa 109 and 287). Three specific T-RF types (280, 190 and 141bp) were significantly more abundant in C. incertus, representing three clusters in Glomus which also named as VT (virtual taxa) 287, 64 and 214, Rhizophagus intraradices (VT 113) and Diversispora sp. (VT 60). While the specific T-RF types, 189 and 279bp, for S. viridis, only existed in Glomus cluster 1 (VT 156), were significantly less abundant in C. incertus. These results indicated that AM fungi might play an important role in the invasion process of C. incertus, which stil remains to be fur-ther investigated.     

花绒寄甲Prx6基因的确定和表达分析

【目的】获得花绒寄甲过氧化物还原酶6基因(Prx6),分析其在花绒寄甲发育和衰老过程中相对表达量的变化,以进一步揭示花绒寄甲衰老的分子机制。【方法】基于花绒寄甲成虫转录组数据库获得Prx6基因,并对其进行生物信息学分析。利用实时荧光定量PCR(RT-qPCR)技术,分析Prx6在花绒寄甲发育和衰老阶段的表达量。【结果】从花绒寄甲成虫转录组数据库中获取了2个Prx6基因,分别命名为Prx6-1和Prx6-2,其开放阅读框长度依次为654和672bp,分别编码218和224个氨基酸。多序列比对和系统发育分析表明,这2个基因均属于1-Cys家族,并且与鞘翅目的赤拟谷盗Prx6-1和Prx6-2的同源性最高,分别为71%和82%。发育阶段的定量结果表明,Prx6-1在2龄幼虫、蛹和初孵成虫体内的表达量较高,在6龄幼虫体内的表达量最低;Prx6-2在2龄和4龄幼虫体内表达量较高,在6龄幼虫体内表达量最低。在成虫衰老过程中,2个Prx6基因表达量整体上呈现出随羽化后时间的延长而增加的趋势。【结论】花绒寄甲Prx6基因影响其发育,特别是在2龄和6龄幼虫期。在花绒寄甲衰老过程中,2个Prx6基因的表达量随其羽化后时间的延长而升高。     

冬凌草HMGS基因的克隆与表达分析

羟甲基戊二酰辅酶A合酶（3-Hydroxy-3-methylglutaryl-coenzyme A synthase,HMGS）是甲羟戊酸途径（MVA）中的第一个催化酶。根据冬凌草转录组数据库中HMGS基因序列设计特异引物,采用RT-PCR技术克隆冬凌草IrHMGS基因cDNA全长,并对其序列进行生物信息学分析,通过荧光定量PCR的方法分析其组织表达特性。IrHMGS基因cDNA全长1 382bp,编码460个氨基酸,序列分析结果表明该基因编码的氨基酸序列含有羟甲基戊二酰辅酶A合成酶N末端、C末端等保守结构域。荧光定量PCR表明,IrHMGS在冬凌草组培苗叶和根中的表达量显著高于在组培苗花、茎和愈伤组织中的表达量。本研究为后续深入研究IrHMGS在冬凌草二萜类物质合成途径中的功能奠定了基础。     

苦瓜McAPX2基因及其启动子的克隆与表达分析

基于苦瓜叶片均一化文库克隆得到McAPX2基因的cDNA全长序列(Gen Bank登录号:KJ722767.1),采用染色体步移法获得该基因的启动子序列,实时荧光定量PCR技术检测该基因在不同器官中的表达量及低温胁迫对McAPX2表达量的影响。结果表明:McAPX2基因全长1 086 bp,开放阅读框747 bp,编码249个氨基酸;McAPX2属于过氧化物酶家族ClassⅠ的成员,其脂肪族氨基酸指数为83.90,是一个亲水性蛋白,可推测定位于细胞质中;与甜瓜(NP_001284378.1)、黄瓜(ACO90193.1)、南瓜(AHF27424.1)、苦瓜(AGJ72851.1)同源性较高,分别为95%,94%,94%和93%;q RT-PCR结果显示,在苦瓜的根、茎、叶、雌花和幼果等组织器官中McAPX2的表达量存在显著差异,其中根的表达量最大,而在叶片中的表达量最低;在低温胁迫下,随着胁迫时间的延长,McAPX2表达量逐渐上调,处理12 h时达到最大,随后下降,说明该基因的表达与苦瓜耐低温胁迫相关。分离得到McAPX2基因上游调控序列1 267 bp,其启动子含有与GA、ABA、CTK、乙烯等激素和病原菌等生物胁迫以及热激、干旱、低温、光等非生物胁迫的相关元件。