高含固率木质纤维素厌氧发酵物总DNA的4种提取方法比较

不同的样本特性和提取方法对获得微生物总DNA的质量有重要影响。文章基于高含固率木质纤维素厌氧发酵物腐殖酸、酚类物质含量高、质地均一性差、微生物浓度低的特点,研究了4种方法提取不同高含固率粪秸厌氧发酵物中微生物总DNA的效果。结果表明,常规的十二烷基磺酸钠法(sodium dodecyl sulfate,SDS)、十二烷基磺酸钠和溴化十六烷基三甲铵结合法(sodium dodecyl sulfate and cetyltrimethyl ammonium bromide,SDS-CTAB)和商业的粪便试剂盒法提取的DNA质量均较差,SDS法和试剂盒法未能获得聚合酶链式反应(polymerase chain reaction,PCR)扩增目的条带,SDS-CTAB法得到的条带较模糊;改进SDS-CTAB法获得的DNA杂质少、纯度高,具有较好的稳定性,A260/A280和A260/A230值分别为1.74~1.86和1.65~1.86,每克样品的DNA浓度在50 ng·μL^-1以上,电泳条带单一齐整、清晰明亮,PCR扩增的目的条带清晰度高,适宜后续分子生物学技术的分析。林格氏液洗脱、聚乙烯吡咯烷酮-40(Polyvinyl Pyrrolidone-40,PVP-40)洗涤液除杂以及裂解液和多种酶联合破壁是改进SDS-CTAB法获得该类专一性样本高质量微生物总DNA的关键步骤。     

甘蔗热带种金属硫蛋白家族基因的克隆及响应重金属胁迫的表达分析

金属硫蛋白是一类富含巯基的低分子量蛋白,在植物的重金属解毒及细胞氧化还原调控等方面起重要的作用。本研究以甘蔗热带种Badila组培苗为材料,分别测定了其在CdCl2、ZnSO4和CuCl2水溶液培养条件下地上部和地下部的重金属含量,结果显示其对上述3种重金属有较强的耐受与富集能力。继而克隆了ScMT1(登录号为KJ504373)、ScMT2-1-5(登录号为MH191346)和ScMT3(登录号为KJ5043704)3个金属硫蛋白家族基因,它们分别属于植物MT亚家族中的MT1、MT2和MT3型基因。ScMT1含有1个内含子和2个外显子,开放阅读框(Open Reading Frame,ORF)长228 bp,编码75个氨基酸;ScMT2-1-5含有2个内含子和3个外显子,ORF长246 bp,编码81个氨基酸;ScMT3含有1个内含子和2个外显子,ORF长198 bp,编码65个氨基酸。RT-qPCR显示,Cd2+胁迫下,在甘蔗地上部和地下部,ScMT2-1-5均连续显著上调表达,而ScMT1的上调应答出现延迟。ScMT3在地上部的上调应答出现延迟,在地下部呈“扬-抑”趋势,提示甘蔗响应Cd^2+胁迫过程中ScMT2-1-5起更积极的作用,ScMT1参与胁迫后期的分子响应,而ScMT3不起主导作用。Cu^2+胁迫下,地上部ScMT1连续显著上调表达,ScMT2-1-5和ScMT3呈总体上调的表达趋势;地下部,ScMT1和ScMT2-1-5的上调表答均出现延迟,仅在胁迫后期显著上调表达,而ScMT3仅在胁迫前期显著上调表达。该结果提示了ScMT1、ScMT2-1-5和ScMT3在Cu2+胁迫响应过程中的协作关系,三者共同参与了地上部的胁迫响应,其中ScMT1起更积极的作用;此外三者还先后参与了地下部对Cu^2+胁迫的分子响应。Zn^2+胁迫下,ScMT1和ScMT3分别仅在地上部和地下部显著上调表达;ScMT2-1-5在地上部和地下部均呈“扬-抑”的应答趋势;提示了在甘蔗响应Cd^2+胁迫应答过程中ScMT1和ScMT3分别在地上部和地下部起主要作用,ScMT2-1-5参与了胁迫前期的分子响应。ScMT1、ScMT2-1-5和ScMT3在甘蔗不同组织中及在重金属(Cd^2+、Zn^2+或Cu^2+)不同累积水平下呈现出相似或互补的应答特性,提示上述甘蔗MT家族不同成员在重金属解毒及细胞氧化还原调控等方面产生了功能分化,且三者在应对过量Cd^2+、Zn^2+或Cu^2+对甘蔗组织造成伤害的过程中存在时空上的协同作用。该研究为深入理解多倍体植物甘蔗中MT家族各成员基因在重金属耐受过程中的协同作用机制奠定了基础。     

食源性单核细胞增生李斯特菌lmoF2365_0037基因克隆及序列分析

为了对食源性单核细胞增生李斯特菌LM5567的lmoF23650037基因进行克隆和序列分析。试验利用PCR方法对lmoF23650037基因进行扩增,连接pMD19-T载体进行克隆,筛选阳性菌株进行测序比对分析。结果显示:扩增获得的lmoF23650037基因序列长为548 bp,克隆得到lmoF23650037基因的阳性转化子;生物信息学分析表明:lmoF23650037蛋白属于跨膜蛋白,无信号肽序列;二级结构中无规则卷曲和延伸链比例较大,分别是46.31%和32.89%;序列比对结果表明,LM5567株lmoF23650037基因核苷酸序列与02-6680株(4b,奶酪,加拿大)、10-0811株(1/2b,螃蟹,加拿大)、10-0809株(4b,粪便,加拿大)、81-0558株(4b,脑脊髓液、加拿大)、02-1103株、02-1792(4b,奶酪,加拿大)、81-0861株(4b,卷心菜,加拿大)相似性均为100%;LM5567 lmoF23650037基因编码的氨基酸序列与上述菌株相似性均为93.3%。本试验成功克隆了lmoF23650037基因,为进一步探究lmoF23650037基因的功能奠定了基础。     

文冠果实时荧光定量PCR内参基因的筛选

为筛选适用于文冠果(Xanthoceras sorbifolium Bunge)性别分化过程研究和不同器官的内参基因,本研究以文冠果性别分化关键期的顶芽和侧芽、根、茎、叶、种仁、雌能花和雄能花为材料,通过实时荧光定量PCR(qRT-PCR)鉴定7个候选内参基因(Actin,UBQ,EF-1α,TUB,eIF-4α,18S rRNA和GAPDH)在各样品中的表达量,并评价7个基因的表达稳定性。结果表明,7个候选内参基因在不同样品中的表达量存在明显差异,各基因在雌能花中的表达均显著高于其他材料,在雄能花中的表达处于较低水平;适用于文冠果性别分化过程的最优内参基因为UBQ和eIF-4α,适用于不同器官的最优内参基因为EF-1α和GAPDH。研究结果将对今后进行文冠果性别分化过程研究和不同器官中的基因表达分析提供参考。     

Quantitative trait loci mapping for yield-related traits under low and high planting densities in maize (Zea mays)

Average maize yield per hectare has increased significantly because of the improvement in high-density tolerance, but little attention has been paid to the genetic mechanism of grain yield response to high planting density. Here, we used a population of 301 recombinant inbred lines (RILs) derived from the cross YE478 × 08–641 to detect quantitative trait loci (QTLs) for 16 yield-related traits under two planting densities (57,000 and 114,000 plants per ha) across four environments. These yield-related traits responded differently to high-density stress. A total of 110 QTLs were observed for these traits: 33 QTLs only under low planting density, 50 QTLs under high planting density and 27 QTLs across both densities. Only two major QTLs, qCD6 and qWKEL2-2, were identified across low- and high-density treatments. Seven environmentally stable QTLs were also observed containing qED6, qWKEL3, qRN3-3, qRN7-2, qRN9-2 and qRN10 across both densities, as well as qRN9-1 under low density. In addition, 16 and eight pairs of loci with epistasis interaction (EPI) were detected under low and high planting densities, respectively. Additionally, nine and 17 loci showed QTL × environment interaction (QEI) under low- and high-density conditions, respectively. These interactions are of lesser importance than the main QTL effects. We also observed 26 pleiotropic QTL clusters, and the hotspot region 3.08 concentrated nine QTLs, suggesting its great importance for maize yield. These findings suggested that multiple minor QTLs, loci with EPI and QEI, pleiotropy and the complex network of “crosstalk” among them for yield-related traits were greatly influenced by plant density, which increases our understanding of the genetic mechanism of yield-related traits for high-density tolerance.     

Rapid screening of genetically modified ingredients in soybean and cotton processing by-product and waste using direct qPCR

To develop a simple and fast method for screening genetically modified ingredients from processing by-product and waste, direct quantitative PCR (qPCR) kit-Taqman which omitting multi genomic DNA preparing steps was developed in this study. A total of 18 oil crop processing by-products and wastes including 10 soybean and 8 cotton materials were collected from food processing factories. Compared with 2 commercial direct qPCR kits, conditions of DNA releasing procedure and PCR amplification were optimized. Element screening was performed at the initial step of genetically modified (GM) ingredient testing procedure via direct qPCR. GM event identification was carried out in positive samples by initial screening. Totally 5 screening elements (P–35S, T-NOS, Cp4-epsps, bar and pat) for soybean materials and 6 screening elements (P–35S, T-NOS, NPTII, Cry1Ac, bar and pat) for cotton samples were detected. In GM event identification, MON531 and MON1445 were found in cotton materials. Results were further confirmed by real-time PCR with DNA extraction and purification. The direct qPCR system proposed by this research was convenient for rapid screening and identification of GM ingredients in oil crop primary by-product and waste.     

Molecular prevalence,risk factors assessment and haemato-biochemical alterations in hepatozoonosis in dogs from Punjab,India

Hepatozoonosis caused by Hepatozoon canis is an important tick-borne disease of dogs in tropical and sub-tropical regions throughout the world. In the present study evaluation of blood samples collected from 225 dogs presented at Small Animal Clinics, GADVASU, Ludhiana, Punjab (India) was done for the presence of H. canis by PCR based assay targeting a portion of 18S rRNA gene. Of the total samples subjected to PCR, an amplicon of 666 bp was detected in 13.78% samples whereas, routine blood smear examination revealed gamonts in 5.78% samples. Furthermore, prevalence of H. canis infection was found to be significantly associated with season, being highest in summer and lowest in winter while other risk factors e.g. age, sex and breed showed non-significant association. In terms of various clinico-pathological parameters, significant drop in haemoglobin, total red blood cell count, packed cell volume and lymphocytes were recorded in positive cases whereas the total white blood cell count was non-significantly increased. The haematological alterations in the positive cases were lymphopenia, anaemia, thrombocytopenia, relative neutrophilia, neutrophilic leucocytosis, eosinophilia, monocytosis and lymphocytosis while the biochemical profile revealed hypoproteinemia and increased levels of blood urea nitrogen and creatinine (in positive cases) pointing towards renal failure.     

A genetic linkage map in southern‐by‐spring oat identifies multiple quantitative trait loci for adaptation and rust resistance

We developed 178 recombinant inbred lines from a southern‐by‐spring oat population designated as “TxH.” These lines were genotyped to generate a high‐quality linkage map that resolved 6,902 markers into 21 linkage groups that matched closely with the latest hexaploid oat consensus map. Three major quantitative trait loci (QTLs) affecting heading date were found in locations that are consistent with known QTLs and candidate genes, and two other QTLs affecting heading date were found in novel locations. Five QTLs affecting plant height were found. Both sets of QTLs are responsible for transgressive segregation observed for these two traits. Four QTLs affecting resistance to crown rust, caused by the pathogen Puccinia coronata f. sp. avenae, were identified. Two of these QTLs are consistent with known clusters of rust resistance genes, while two may represent new locations of novel rust resistance genes. A complete set of SNP sequences suitable for generating markers for molecular selection is provided.     

Bulk pollen pollination in maize for efficient construction of introgression populations with high genome coverage

Introgression populations consist of a set of introgression lines or families, constructed by continuous backcrossing to the recurrent parent, while carrying a limited number of chromosome segments from a donor parent in their genomes. Increasing the genome coverage is an important aim when constructing introgression population. In this study, we proposed bulk pollen pollination (BPP) method and used it to increase the genome coverage of a maize introgression population. The results showed that the genome coverage of the introgression population constructed using BPP method reached 100% at BC₃ generation, which accorded with the simulation result. The BPP‐based BC₃F_1:2 population could identify most quantitative trait loci (QTL) detected using the F_2:3 population, especially major QTL. Simulation analysis showed that the genome coverage of introgression population increased with the increase of population size and the number of bulked plants, and decreased with the increase of backcross generation. Our results proved the reliability of the BPP‐based introgression population in increasing genome coverage and detecting QTL, and provided references for constructing high‐coverage introgression populations.