基于酿酒酵母的多片段质粒的构建

为避免现有的多片段质粒构建技术的弊端,提高多片段质粒构建效率,以构建树干毕赤酵母Agglutinin-like基因的敲除载体为例,利用酿酒酵母活体细胞重组系统,一次性将多个外源DNA片段和线性化质粒重组,形成环状质粒。通过引物设计在相互连接的DNA片段之间引入50bp重叠序列,用PCR扩增DNA片段后,与线性化质粒一起转化酿酒酵母,再用PCR和测序等方法鉴定阳性酵母菌落中质粒的正确性。通过本方法构建的多片段质粒正确率高、耗时短。

基于全局节点和多片段的格栅命名实体识别

现有命名实体识别模型对标注数据量要求较高,基于主动学习的命名实体识别模型需要人工分词造成标注代价大。针对上述问题,提出一种结合全局节点和多片段的格栅命名实体识别模型。将Transformer的全连接结构替换为全局节点和多片段结构,每个节点仅与构造的上下文向量进行注意力计算,全局和片段节点分别获取全局和局部信息,从而降低对标注数据的需求量。对Flat-Lattice结构进行改进,解决现有主动学习策略需要分词的问题,从而在保证模型性能的情况下降低数据标注代价。在MSRA、OntoNotes 5.0、Weibo、PeopleDaily这4个数据集上的实验结果表明,与FLAT模型相比,所提模型达到对应F1阈值所需的标注数据量分别降低了39.90%、2.17%、34.60%和35.67%。

面向多片段答案的抽取式阅读理解模型

随着搜索技术的发展,抽取式阅读理解已经成为搜索引擎中重要的组成部分.给定问题和文本,抽取式阅读理解任务要求从文本中定位出问题的答案.已有工作仅考虑答案片段由文本中的一个片段组成的情况,因此把该问题建模为输入问题和文本,预测出两个文本中的位置索引去指示答案的起始和结束位置.然而现实应用中存在大量问题其答案往往由文本中一个或多个片段组成,想要回答该问题需要从文本中定位出若干的文本片段,而不再是单一片段.已有的阅读理解模型研究主要关注在模型底层结构的设计,对于多片段答案的情况未予考虑,导致已有模型无法从文本中抽取多个答案片段去回答问题.本文提出面向多片段答案的抽取式阅读理解模型BERTBoundary,该模型采用预训练的BERT作为底层结构进行文本和问题的理解.BERT通过自我注意力机制和前向神经网络对文本和问题进行编码表示,同时利用在大规模无监督语料上进行BERT模型参数的预训练达到更强的文本理解.利用新颖的边界序列标注方式去建模一段文本中多个答案片段,模型对答案的起始位置和结束位置分别进行序列标注,对每个词进行二分类,判断其是否是答案的起始位置或者结束位置,并通过简单有效的序列标注方式进行答案片段的解码.BERT-Boundary结合了BERT的文本理解能力和边界序列标注的多片段建模能力.我们在构造的大规模多片段答案的阅读理解数据集上进行详尽地实验和分析,实验结果表明,BERT-Boundary的性能比基线方法取得一致的提升.我们进一步在不同答案片段长度和答案片段数量上比较我们的模型和基线方法,实验数据表明,我们的方法比基线方法取得一致的提升.我们的代码公开发布在https://github.com/lixinsu/multi_span.

多片段Le Fort I型截骨矫治严重牙颌面畸形

目的:采用多片段Le Fort I型截骨术矫治严重牙颌面畸形,并为克服术后骨段固位不良之缺点自制一种腭侧固定夹板。方法:以该手术方法联合双侧下颌升支矢状劈开术（BSSRO）矫治上颌宽度不足伴上下颌其它严重畸形的患者19例。采用单纯多片段Le FortI型截骨6例,联合BSSRO13例。19例中上颌两片段Le FortI型截骨12例,3片段7例。结果:术后随访16例,平均22.6m,66间距离平均扩宽7.3mm,33间平均扩宽3.9mm。无严重并发症及明显复发,咬合关系满意。结论:应用良好的腭侧固定夹板,采用多片段 Le FortI截骨联合BSSRO可一次满意矫治严重双颌畸形。

一步法快速构建多片段连接的同源臂载体

目的：建立一种简便、高效，可一步完成多个片段连接，从而构建含同源臂的载体的方法。方法：按照酶切后可产生前后片段相匹配的粘性末端接头的原则设计PCR引物，在目的片段两端均引入BsaⅠ酶切位点。以G160基因为例，PCR扩增打靶用左右同源臂片段、示踪基因CMV-EGFP片段、载体骨架pMD19－T等4个片段，纯化后一起加入一个反应管中，并加入BsaⅠ限制性内切酶和T7DNA连接酶及相应缓冲液，进行酶切、酶连接共10～50个循环反应，一步构建含同源臂载体的质粒；产物经高温处理后，直接转化感受态细胞，并进行重组子PCR鉴定；对pMD19－T载体进行优化，突变载体上的BsaⅠ酶切位点，把示踪基因CMV-EGFP片段引入pHSG298－T载体，再选择不同的G160基因同源臂片段组合对构建系统进行验证。结果：重组质粒酶切和PCR结果表明，应用一步法可成功连接多个片段来构建含同源臂及示踪基因的克隆载体；用优化后的pMD19－T－O载体体系，在2d内即完成了6种各含4个片段的载体的构建。结论：多个基因片段一步无缝连接的方法简便、易行、可靠，不仅可快速构建某类载体系统，还可对基因进行精确的点突变，该系统可用于快速构建基因打靶载体。

多片段酶切/连接反应结合同源重组的DNA组装方法

为了寻找一种准确高效的多片段组装及多点突变方案,将目前常用的多片段组装方法进行了整合与优化,并以果糖-1,6-二磷酸酶1(fructose-1,6-diphosphatase 1,FBP1)基因4位点突变为例进行验证。通过引入突变位点和Bsa I识别序列连接含有突变位点的片段,经过酶切/连接反应后扩增组装成功的目的片段,并引入与线性化载体两端同源的序列,最后进行片段与线性化载体的重组并转化大肠杆菌。经筛选与测序,得到4位点突变重组质粒。该方案弥补了常规的Gibson组装和Golden Gate组装的部分缺点,是一种高效进行多片段组装和多点突变的实验方案。

利用同序异尾限制性内切酶快速克隆多基因片段的新方法

介绍一种可以快速进行多基因片段克隆的新方法——同序异尾限制性内切酶(isoschizomer-heterotailrestrictionendonuclease,IHRE)一步克隆法,该方法可以一步完成多达6个DNA片段的连接,具有简单快速、节省试剂、成功率高、不引入非目的基因序列等很多优点.应用该方法已经成功完成对人肠激酶轻链多基因克隆、HSV多表位DNA疫苗的构建等.

鱼类环境DNA metabarcoding片段的近缘物种识别差异

已知的鱼类环境DNA(eDNA)metabarcoding片段均未被针对性考察其对近缘物种的适用性,实际使用过程中存在“物种丢失”风险。为筛选出物种识别率最高的片段,本研究比较了15个主流片段对106属(共935种)鱼类的识别差异。研究结果如下:(1)蛋白质编码基因(COI,片段15)的物种识别率最高,但其引物通用性最差;片段09、片段11、片段07、片段03、片段12的引物序列总平均遗传距离也较大,均存在eDNA低效扩增的风险;(2)片段长度影响物种识别率,核糖体基因中片段05、片段06、片段01、片段02及片段13的物种识别率较高;(3)非度量多维尺度分析(NMDS)显示,不同基因、同一基因不同片段的识别结果存在较大差异,应考虑多片段、多基因组合应用;片段01与片段02、片段05与片段06等在NMDS图上距离较近,存在相互替代性;(4)物种类群影响识别结果,eDNA研究仍需要进一步开发高识别率片段。综合物种识别率、引物通用性、NMDS分析等多方面因素,本研究推荐2×150 bp测序平台使用片段01(Mi Fish-U)、2×250 bp测序平台使用片段05(Ac12S),辅以片段13(Vert-16S-eDNA)等进行近缘鱼类多样性调查。本研究旨在为提高鱼类eDNA调查结果准确性提供一定技术支撑。

中国猪瘟C-株(脾淋毒)全长cDNA的分子克隆

应用RT PCR、NestedPCR和Half nestedPCR技术从实验感染兔脾组织的总RNA中得到了覆盖猪瘟病毒(CSFV)C 株全基因组的7个cDNA重叠片段,分别克隆于pMD18 T或pGEM TEasy载体后进行测序。运用T A克隆和多片段一步连接法,将cDNA重叠片段F1、F2、F3、F4与F5、F6、F7分别克隆到pGEM 5Zf(+)载体后,得到两个半长cDNA亚克隆重组质粒pGEM 5Zf(+)/F1 4和pGEM 5Zf(+)/F5 7,再将两个半长cDNA重叠片段连接并克隆到pGEM 5Zf(+)载体,构建出了CSFVC 株全长cDNA重组质粒pGEM 5Zf(+)/F1 7。全长cDNA的成功构建为进一步研究CSFV分子生物学提供了良好的工具。

高校运用有限摄编媒体资源实现多机位摄制高质量教学影片探析

摄制高质量的教学影片是各高校追求的重要目标,部分题材的影片摄制要实现高质量,必须以多台摄影机、多景别、多视角拍摄来实现,然而,部分高校由于经费的原因,不具备这样的条件,这是个矛盾。本文以摄、编、导等多个环节较为详细的阐述了运用有限的摄编媒体资源来实现多机位(15机位)摄制高质量教学影片的思路、方法与技巧,并以实例佐证,以求与同行们切磋。

多机位编辑在视频公开课节目制作中的应用

视频公开课节目制作要求使用多机位进行拍摄制作,传统多机位制作多是用现场切换进行一次性录制制作,这种制作方式有前期工作量大,后期编辑不方便等缺点,本文结合具体案例,介绍了视频公开课多机位制作的流程,以及苹果公司后期制作软件Final Cut Pro X的多机位编辑技术。

Bioleaching of Pb-Zn-Sn chalcopyrite concentrate in tank bioreactor and microbial community succession analysis

The variation of microbial community structure was investigated for the tank bioleaching process of Pb-Zn-Sn chalcopyrite concentrate in the presence of mixed moderately thermophilic bacteria. The parameters, such as pH value, solution potential and concentrations of metal ions, were determined by the method of polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） to analyze the succession of microbial community. The results showed that a final copper extraction rate of 85.6% could be obtained after tank bioleaching for 30 d. The Acidithiobacillus caldus was the dominant population with abundance of about 73.80%in the initial stage, then Sulfobacillus thermosulfidooxidans dominated from the 18th day to the end of bioleaching, while the abundance of Leptospirillum ferriphilum changed slightly. A higher solution potential within a certain range and appropriate concentration of ferric ions were essential for this tank bioleaching of chalcopyrite.

Discussion on AFLP Molecular Markers in Piper methysticum and Pepper

The aim of the research was to discuss the genetic relationships between Piper methysticum, Pepper and other wild species in Pepper genus. DNA was extracted from leaves which belonged to 28 germplasms including 6 materials of P. methysticum, 21 maerials of cultivated and wild Pepper, 1 material of Peperomia pellucida belonged to different genus. Premiers with good band-type and high polymorphism and resolution were selected from 64 pairs of primers for AFLP amplification and the clustering analysis was conducted with MVSP3.13f software. 191 bands were amplified by 4 pairs of premiers, 189 of which had polymorphism, being 98.6%. 28 germplasms were classified into 6 different groups at the genetic similarity coefficient of 0.52 by silver staining AFLP, in which 6 materials of Piper methysticum were clustered into a single group, indicating that P. methysticum belonged to Pepper family of Pepper genus but were distantly related to the others. The research provided the basis for selecting rootstocks for P. methysticum graft, molecular identification of P. methysticum and the fingerprint construction of P. methysticum.

Studies on the relationship between the point mutation of ras oncogenes and the prognosis of patients with gastric cancer

AIM To study the relationship between the point mutation of ras oncogenes and the prognosis of patients with gastric cancer.

Location and Analysis of Introgressed Segments in the Parthenogenetic Progenies of Zea mays×Z. diploperennis by GISH

用来自玉米 (ZeamaysL .)与二倍体多年生类玉米 (Z .diploperennisIltis,DoebleyandGuzm偄ｎ)杂交的孤雌生殖后代同一抗病个体的 4个株系进行了基因组原位杂交 ,用改进的杂交技术获得了近 10 0 %的检出率 ,每一检出片段在同源染色体两成员和每两个姊妹染色单体上均有清晰的信号。

Development of a SCAR marker for male gametophyte of Gracilariopsis lemaneiformis based on AFLP technique

The red alga Gracilariopsis lemaneiformis(Bory) is an economically valuable macroalgae. As a means to identify the sex of immature Gracilariopsis lemaneiformis, the amplifi ed fragment length polymorphism(AFLP) technique was used to search for possible sex- or phase-related markers in male gametophytes, female gametophytes, and tetrasporophytes, respectively. Seven AFLP selective amplifi cation primers were used in this study. The primer combination E-TG/M-CCA detected a specifi c band linked to male gametophytes. The DNA fragment was recovered and a 402-bp fragment was sequenced. However, no DNA sequence match was found in public databases. Sequence characterized amplifi ed region(SCAR) primers were designed from the sequence to test the repeatability of the relationship to the sex, using 69 male gametophytes, 139 female gametophytes, and 47 tetrasporophytes. The test results demonstrate a good linkage and repeatability of the SCAR marker to sex. The SCAR primers developed in this study could reduce the time required for sex identifi cation of Gracilariopsis lemaneiformis by four to six months. This can reduce both the time investment and number of specimens required in breeding experiments.

COX-2 polymorphisms-765G→C and-1195A→G and colorectal cancer risk

AIM： TO determine the possible modulating effect of the COX-2 polymorphisms, -765G→C and -1195A→G, on the risk of colorectal cancer （CRC） in a Dutch population. METHODS： This case-control study includes 326 patients with CRC and 369 age- and gender-matched controls. Genotypes of the COX-2 polymorphisms -7dEG→C and -1195A→G were determined by polymerase chain reaction-based restriction fragment length polymorphism. COX-2 genotypes and haplotypes were analyzed and odds ratios with 95% confidence intervals were estimated by logistic regression. RESULTS： The -765GG genotype was associated with an increased risk of developing CRC （OR, 1.45; 95% CI, 1.03-2.04）. No significant difference was observed in the genotype distribution of the -1195A→G polymorphism between patients and controls. The GG/AC haplotype was present significantly less often in patients than in controls （OR 0.44; 95% CI, 0.22-0.85）. When the AC, AG and GG haplotypes were investigated separately, the AC haplotype showed a tendency to be less frequent in patients than in controls （OR（AG/AC） 0.78; 95% CI, 0.57-1.06）. CONCLUSION： The -765GG genotype is associated with an increased risk of developing CRC and the G6/ AC haplotype seems to protect against CRC. These findings suggest a modulating role for the COX-2 polymorphisms -765G→C and -1195A→G in the development of CRC in a Dutch population.

Genetic alterations of hepatocellular carcinoma by random amplified polymorphic DNA analysis and cloning sequencing of tumor differential DNA fragment

AIM: To study the genetic alterations and their association with clinicopathological characteristics of hepatocellular carcinoma (HCC), and to find the tumor related DNA fragments.METHODS: DNA isolated from tumors and corresponding noncancerous liver tissues of 56 HCC patients was amplified by random amplified polymorphic DNA (RAPD)with 10 random 10-mer arbitrary primers. The RAPD bands showing obvious differences in tumor tissue DNA corresponding to that of normal tissue were separated,purified, cloned and sequenced. DNA sequences were analyzed and compared with GenBank data.RESULTS: A total of 56 cases of HCC were demonstrated to have genetic alterations, which were detected by at least one primer. The detestability of genetic alterations ranged from 20% to 70% in each case, and 17.9% to 50% in each primer. Serum HBV infection, tumor size,histological grade, tumor capsule, as well as tumor intrahepatic metastasis, might be correlated with genetic alterations on certain primers. A band with a higher intensity of 480 bp or so amplified fragments in tumor DNA relative to normal DNA could be seen in 27 of 56 tumor samples using primer 4. Sequence analysis of these fragments showed 91% homology with Homo sapiens double homeobox protein DUX10 gene.CONCLUSION: Genetic alterations are a frequent event in HCC, and tumor related DNA fragments have been found in this study, which may be associated with hepatocarcinogenesis. RAPD is an effective method for the identification and analysis of genetic alterations in HCC, and may provide new information for further evaluating the molecular mechanism of hepatocarcinogenesis.

N-Acetyltransferase 2 genetic polymorphisms and risk of colorectal cancer

AIM:To investigate the possible association between meat intake,cigarette smoking and N-acetyltransferase 2 (NAT2) genetic polymorphisms on colorectal cancer (CRC) risk.METHODS:Patients with CRC were matched for gender and age to healthy controls.Meat intake and cigarette smoking were assessed using a specific frequency questionnaire.DNA was extracted from peripheral blood and the genotypes of the polymorphism were assessed by polymerase chain reaction-restriction fragment length polymorphism.Five NAT2 alleles were studied (WT,M1,M2,M3 and M4) using specific digestion enzymes.RESULTS:A total of 147 patients with colorectal cancer (76 women and 90 men with colon cancer) and 212 controls were studied.The mean age of the two groups was 62 years.More than half the subjects (59.8% in the case group and 51.9% in the control group) were NAT2 slow acetylators.The odds ratio for colorectal cancer was 1.38 (95% CI:0.90-2.12) in slow acetylators.Although the number of women was small (n=76 in the case group),the cancer risk was found to be lower in intermediate (W/Mx) acetylators [odds ratio (OR):0.55,95% confidence interval (95% CI):0.29-1.02].This difference was not observed in men (OR:0.56,95% CI:0.16-2.00).Among NAT2 fast acetylators (W/W or W/Mx),meat consumption more than 3 times a week increased the risk of colorectal cancer (OR:2.05,95% CI:1.01-4.16).In contrast,cigarette smoking increased the risk of CRC among slow acetylators (OR:1.97,95% CI:1.02-3.79).CONCLUSION:The risk of CRC was higher among fast acetylators who reported a higher meat intake.Slow NAT2 acetylation was associated with an increased risk of CRC.

Preliminary Analysis of Population Genetic Diversity of Cultivated Laminaria japonica Sporophyte via AFLP Technique

The amplified fragment length polymorphic DNA (AFLP) technique was adopted to estimate the population genetic polymorphism among 30 sporophytes of Laminaria japonica collected from a cultivating farm in Rongcheng,China.Three methods were used for genomic DNA extraction from Laminaria japonica sporophyte and only the products obtained using the improved genomic DNA extraction kit method proved qualified for AFLP analysis.The parameters of the method were optimized.Samples of forty milligrams and the cell lysis time of 120 min were suggested to replace the parameters recommended by the manufacturer.Thirty individuals of Laminaria japonica from the same cultivating site were investigated using one pair of selective primers.A total of 21 loci were obtained and 17 of them were polymorphic.The mean percent age of polymorphic loci of this population was 80.95%.The Nei's gene diversity (H) within this population was 0.3028 and the average Shannon's Information index (I) was 0.4498.A genetic distance matrix among different individuals was constructed as well.Through this study,an applicable AFLP genetic analysis working system for Laminaria japonica sporophyte was established.The results of this research also revealed a high level of genetic diversity within the studied population.