基于RNA-Seq技术的鲮转录组分析

为满足标记辅助育种的要求,通过454测序平台首次开展了鲮Cirrhina molitorella全鱼转录组深度测序,并用 Newbler 等软件进行数据精细分析。结果表明：共获得了1297479条 reads,总碱基数为486586191 bp,组装后得到19962条contigs,平均长度为1269 bp, N50为1509 bp。基因功能注释研究共获取了10577个特异蛋白,根据特异蛋白注释结果进行GO分析,有7314条contigs有GO注释,包含5381个特异蛋白;采用GO 功能分类工具可将已注释转录物序列划分为分子功能、生物途径和细胞成分3类,为下一步开展生长等性状相关基因功能验证研究提供丰富的序列资源;共鉴定出5931个具有完整的ORF的全长cDNA序列,并且鉴定出2438个微卫星和5014个SNP位点。本研究中,还建立了鲮转录组数据库和网站,方便同行随时调取数据,这为深入开展鲮分子标记辅助的遗传育种、种群遗传学和资源评估等研究提供了丰富的标记资源。     

草鱼生长激素cDNA基因的克隆及序列分析

通过反转录PCR技术，从草鱼脑垂体的总RNA中获得一特异cDNA片段，经克隆后测序，结果表明，该片段长633bp，与报道的草鱼生长激素基因相比，有10个碱基的差异，而推导的蛋白质仅1个氨基酸残基的差异，推测为草鱼生长激素cDNA基因。     

生物质炭对连作参地土壤肥力及微生物特性的影响

为分析生物质炭用于老参地土壤修复的可行性,本研究采用Biolog、高通量测序等技术研究了3种生物质炭对连作参地人参品质、土壤肥力、微生物群落结构与功能多样性变化的影响。结果显示,增施不同生物质炭均有助于两年生重茬人参生物量及总皂苷含量的积累。结合生物质炭对土壤性质的影响研究结果发现,不同生物质炭对土壤有效磷、有机碳含量具有稳定的提升效果,增幅分别为47.04%~237.73%、8.09%~38.71%。增施生物质炭促进了土壤微生物的代谢活性,增加了土壤对聚合物类、酚酸类、氨基酸类、羧酸类碳源的利用效率,其中以酚酸类物质为底物的功能微生物种群数量显著增加。高通量测序结果显示,老参地土壤细菌物种数、丰度与多样性均呈下降趋势,细菌优势门类减少,单个门类优势度上升。生物质炭处理下土壤中细菌丰度与多样性增加,调控了变形菌门、厚壁菌门、疣微菌门、芽单胞菌门的优势度,使其数量变化趋势趋向于新林土。上述分析表明,重茬人参生物量和品质的提高与生物质炭提升土壤中人参生长的关键肥力指标,促进微生物代谢,调控细菌群落结构变化趋势有关。综上,生物质炭对老参地土壤表现出良好的修复、改良作用,适量增施生物质炭有利于老参地土壤质量的调节与恢复。     

基于简化基因组测序技术的甘薯HRM分子标记开发及其应用

目前甘薯中针对SNP位点的分子标记开发及应用的报道较少。为开发甘薯特异SNP分子标记,本研究利用简化基因组测序技术对甘薯种质材料进行测序,筛选稳定的SNP位点,将其开发为基于HRM技术的分子标记,并在甘薯种质材料中进行验证。通过对23个甘薯种质材料简化基因组测序数据的分析,共发现835 756个SNP多态性位点,筛选其中的3 650个含有SNP多态性位点的高质量测序片段,成功设计了134对引物;初步验证发现,22对(16.42%)引物没有扩增产物,15对(11.19%)引物扩增产物2个以上,36对(26.87%)引物的扩增产物没有差异。61对(45.52%)引物在8个样本中的扩增特异性好,而且样本之间有差异。最后筛选34对扩增多态性丰富的引物对52份甘薯种质材料进行扫描,发现材料间多态性介于27.27%~90.91%之间,平均多态性达到59.35%。聚类分析表明,参试52份甘薯种质材料之间的差异较小,多数材料与骨干亲本南瑞苕、徐薯18之间关系较近,国外引进甘薯材料和地方品种差异较大。建议在今后甘薯育种中尽量选用地方品种和国外甘薯品种,从而更好地拓宽甘薯遗传背景。本研究初步建立了基于简化基因组技术和HRM技术开发甘薯SNP分子标记的新思路,为今后甘薯SNP分子标记开发提供了参考。同时本研究开发的甘薯分子标记,丰富了甘薯分子标记类型,为甘薯研究者开展快速的分子标记研究提供了支持。     

Genomic characterization of multidrug-resistant extraintestinal pathogenic Escherichia coli isolated from grain culture soils

Multidrug-resistant (MDR) Escherichia coli, mainly extraintestinal pathogenic E. coli (ExPEC), has been widely reported in infections worldwide. In agricultural soils, manure is a hotspot for the dissemination of antimicrobial resistance genes (ARGs) and pathogenic bacteria; however, MDR bacteria have also been reported in soils with no history of manure use. In addition, cross-resistance and co-resistance have been described as responsible for the metal-driven selection of bacteria resistant to antimicrobials. Therefore, the aim of this study was to analyze three MDR E. coli isolates obtained from Brazilian grain culture soil samples with no history of manure use by whole-genome sequencing. The MDR E. coli isolates were recovered from soils from corn and coffee fields, and presented resistance to β-lactams, quinolones, aminoglycosides, tetracyclines, sulphonamides, and dihydrofolate reductase inhibitor. Resistome analysis showed ARGs to several antimicrobials (i.e., β-lactams, tetracyclines, aminoglycosides, sulphonamides, trimethoprim, phenicols, fosfomycin, and macrolides) as well as several metal resistance genes and antibacterial biocide resistance genes. In addition, known mutations in quinolone-resistance-determining regions of GyrA (Ser83Leu and Asp87Asn), ParE (Ser458Thr), and ParC (Ser80Ile) were also detected. Virulome analysis showed the presence of virulence genes (lpfA, mcmA, gad, mchF, iroN, cma, and iss) associated with ExPEC. Multidrug-resistant ExPEC isolates were assigned to phylogenetic group B1. The presence of MDR B1-ExPEC in soil samples shows the ability of these isolates to survive in soils. This study reports for the first time some sequence types (i.e., ST345, ST448, and ST1146) of MDR E. coli in Brazilian soils. Therefore, these findings contribute to the monitoring of antimicrobial resistance and surveillance studies based on whole-genome sequencing worldwide.     

不同植被对煤矿复垦区域土壤真菌多样性和酶活性的影响

为了评价不同植被对煤矿废弃地复垦区土壤生态修复的效果,本研究开展了桃树、杉树、李树等植被对复垦区土壤酶活性和真菌种群多样性的影响分析。结果表明：（1）不同植被下,脲酶、酸性磷酸酶和转化酶的活性差异显著。脲酶在对照中最高,桃树最低;酸性磷酸酶活性对照最高,杉树（0~20、40~60 cm）和桃树（20~40 cm）土壤中最低,且一般情况下酶活性随土层加深而降低,具有表聚性,但不同植被和不同土层的转化酶活性的变化表现不同;该区域土壤的pH随土层加深增高,最高为5.67,均呈酸性,除对照外,均表现出一定程度的差异性。（2）不同植被下,所有土壤样品的真菌种群优势门均为子囊菌门（Ascomycota）、担子菌门（Basidiomycota）、毛霉菌门（Mucoromycota）,季也蒙酵母属（Meyerozyma）和芽枝霉属（Cladosporium）为所有土壤样品都具有的优势属,而孢霉属（Mortierella）仅在柳树土壤中不是优势属,青霉菌属（Penicillium）、绿核菌属（Ustilaginoidea）是李树土壤中的优势属;盘菌属（Tricharina）是杉树土壤中的优势属;刺毛四枝孢（Tetracladium）是对照土壤中的优势属。通过综合非度量多维尺度分析（Non-metric multidimensional scaling, NMDS）表明,不同植被的土壤真菌群落组成差异较大,柳树0~20 cm、杉树20~40 cm、对照40~60 cm土壤中的真菌数量和多样性较丰富,其中桃树土壤中的真菌丰富度和多样性相对较低。本研究结果可以为其他煤矿复垦区域土壤生态修复研究提供借鉴。     

基于中麻黄萌发种子转录组的黄酮类化合物合成途径基因的挖掘

应用新一代高通量测序技术(Illumina Solexa),对中麻黄(Ephedra intermedin)同萌期的种子进行转录组测序,数据重头(denovo)组装后,共获得52 007条Unigene序列,总长为25 043 596 bp。COG功能注释、GO分类及KEGG代谢通路分析后,获得了64 751个GO功能注释,17 701个COG功能注释以及16 942个KEGG注释,并从KEGG通路中找到参与黄酮类化合物合成途径的关键基因片段283个,其中包含了查尔酮合酶基因、细胞色素相关基因和花青素还原酶等基因。中麻黄转录组测序工作的完成,极大地扩充了麻黄的基因资源,为麻黄属植物分子系统进化分析、抗性和药用功能基因的发掘与利用、遗传改良等研究奠定基础。     

不同封育年限下石灰岩山地植被 细根生物量及其动态

利用土钻法对不同封育年限下石灰岩山地植被细根生物量进行对比研究。结果表明院封育5 年林分细根 生物量为6.6287 t/hm2,封育10 年林分细根生物量为2.3063 t/hm2,封育20 年林分细根生物量为3.1316 t/hm2,封育 30 年林分细根生物量为2.8316 t/hm2;不同封育年限下细根生物量均随着土层深度的增加而降低,0~10 cm 土层的细 根生物量明显高于其他两个土层;不同封育年限细根生物量年动态变化均呈现双峰型。     

Dwarf mistletoe effects on soil basidiomycete community structure, soil fungal functional diversity, and soil enzyme function: Implications for climate change

We used a combination of molecular, culture and biochemical methods to test the hypothesis that severe infection of pine by dwarf mistletoe (genus Arceuthobium) has significant effects on structure and function of soil fungal communities, and on carbon cycling in soils. PCR and DNA sequencing of the basidiomycete communities in paired blocks of uninfected and infected trees revealed: (1) that the top, organic soil layer in this system is inhabited almost exclusively by ectomycorrhizal fungi; (2) no difference in species richness (6 species core⁻¹ in both) or Shannon-Wiener evenness (0.740 and 0.747 in uninfected and infected blocks respectively), however Shannon-Wiener diversity was significantly greater in infected blocks (1.19 vs 1.94 in uninfected and infected blocks respectively, P < 0.05); (3) significant differences in basidiomycete species composition, with nearly complete absence of two system co-dominant Russula species in infected blocks, and replacement of one co-dominant Piloderma species with another in infected plots, indicating physiological variability within the genus. Soil fungal physiological diversity measured using the Fungilog system was significantly greater in terms of both number of carbon substrates used by culturable soil fungi (both ascomycetes and basidiomycetes) in infected blocks, and the rate at which these substrates were used. Soil enzyme assays revealed greater laccase, peroxidase, and cellulase activities in soils associated with infected trees. Thus, event cascades associated with severe dwarf mistletoe infection not only significantly affected soil fungal species composition and increased species diversity, but also impacted on carbon-related function and functional diversity. Given the geographic range of this pathogen, and forecasts that epidemics of this disease will increase in range in severity with global climate change, these effects have the potential to significantly impact local and global carbon budgets.