基于基因编辑技术研究特定基因对小鼠肠道微生物的影响

基因编辑是一种能够精确修改基因组的技术,它为基因功能研究提供了强大的工具。利用锌指核酸酶(ZFNs)、转录激活类效应核酸酶(TALENs)、规律成簇的间隔短回文重复序列(CRISPR)等技术,研究人员可以操作目标基因,实现在小鼠模型中敲除、插入或修改特定基因,用以研究其对肠道微生物组成的影响,探索肠道微生物与宿主相互作用的机制,理解这些相互作用对宿主健康的影响。近期的研究发现,小鼠基因编辑对其肠道微生物的影响有了重要进展。特定基因的敲除能够导致微生物组成的改变,进而影响宿主的代谢、免疫和疾病风险。这些研究揭示了肠道微生物与宿主之间复杂的相互作用网络,为理解肠道微生物学和宿主健康的关键因素提供了重要线索。未来,进一步研究小鼠基因组编辑对肠道微生物的影响,将有助于开发新的治疗策略和预防措施,以维护宿主健康。

泛耐药鲍氏不动杆菌流行株全基因组与特定基因分析

目的调查一株泛耐药鲍氏不动杆菌流行株的遗传学背景,为耐药菌的快速诊断提供参考。方法泛耐药鲍氏不动杆菌WA2859分离自2010年3月住院患者痰液标本,作gyrA与parC基因测序、BLASTn比对确认为鲍氏不动杆菌,采用Illumina HiSeq与Ion Torrent PGM两种大规模并行测序仪进行全基因组分析,再进行人工测序补缺口,并进行了特定基因分析(包含β-内酰胺类、氨基糖苷类抗菌药物获得耐药基因,ISaba1-blaADC、ISaba1-blaOXA-23连锁检测)。结果泛耐药鲍氏不动杆菌WA2859株全基因组测序未获完整序列,得到一条推定的染色体序列,长3 887 116bp(内含两个缺口);推定的质粒序列,长260513bp(内含9个缺口);特定基因检出blaTEM、blaADC、blaOXA-23β-内酰胺类抗菌药物获得耐药基因和aac(6′)-Ⅰb、ant(3′)-Ⅰ、ant(2″)-Ⅰ、aph(3′)-Ⅰ氨基糖苷类抗菌药物获得耐药基因,并且ISaba1-blaADC、ISaba1-blaOXA-23连锁检测均阳性。结论特定基因检测与全基因组测序互为补充,对特定基因检测结果作样本聚类分析并可得到菌株亲缘关系结果,可对临床分离的耐药细菌作快速诊断和分子流行病学研究。

结直肠癌肝转移特定基因miRNA芯片甄别

目的:筛选结直肠癌(colorectal cancer,CRC)肝转移miRNA对应的特定基因。方法:收集10例CRC患者肿瘤组织标本,其中同时伴肝转移者5例,无远处器官转移5例。应用miRNA芯片方法对两组样本的miRNA表达差异情况进行研究,并通过实时定量RT-PCR对miRNA的芯片表达差异进行验证,再利用软件预测靶基因。结果:总RNA提取的质量分析显示,每份样品总RNA的A260/A280为2.11～2.15。电泳结果显示,每份样品的总RNA均有清晰的28S和18S条带,RNA完好无降解,质量符合miRNA芯片的质量要求。肝转移组较非转移组筛选出6种CRC表达失调的miRNA,其中上调的为miR-224、miR-1236和miR-622,下调的为miR-155、miR-342-5p和miR-363,miR-224的差异信号值最高(>500)。芯片实验显示,hsa-miR-224在肝转移组呈现显著性上调,P=0.038 2;而hsa-miR-155在肝转移组却呈现显著性下调,P=0.043 2。miR-224的表达倍数在肝转移CRC组织中较非转移组表达上调(Fold change=2.47),P=0.016 6。结论:可调控CRC肝转移的特定基因miR-224的筛选,可作为早期诊断治疗的新手段。

日本用小分子RNA成功抑制特定癌细胞基因

日本学者通过给人体癌细胞注入起干扰作用的小分子RNA(核糖核酸)的方法,成功地抑制了癌细胞中特定基因的表达,这一成果将有助于开发疗效好的抗癌剂。日本产业技术综合研究所近日发表的新闻公报说,取得这一成果的是该研究所的多比良和诚与东京大学的川崎广明两位学者。他们在研究中设计了5种呈短发夹结构的RNA,将它们注入人乳腺癌细胞后,这些RNA会生成能对癌细胞中erbB

复合探针荧光定量PCR方法的建立

为了对特定基因进行实时检测,根据荧光能量转移(FRET)原理,设计及合成了一种新的FRET复合探针,该探针由一条长的荧光杂交探针和短的淬灭探针构成,其中荧光探针5'端接一荧光素分子,3'端接一延伸阻断分子磷酸,淬灭探针3'端连接一个淬灭分子对甲基红,淬灭探针与荧光探针5'端互补,无模板时,该探针杂交形成复合探针,无荧光产生,当有模板时,荧光探针与模板杂交,荧光不能被淬灭,产生的荧光与模板量成正比。根据复合探针的反应原理,研究了该探针的FRET性质及影响因素包括淬灭探针及扩增片段长度、荧光探针与淬灭探针的合适比例及镁离子浓度。实验结果显示淬灭探针及扩增片段长度对复合探针的作用有明显的影响,本实验采用淬灭探针长21个核苷酸,扩增片段长127bp,荧光探针与淬灭探针的合适比例为1∶1,镁离子浓度为3mmol/L,可获得最佳的反应体系;该复合探针合成简单,淬灭彻底,具有良好的准确性与特异性,敏感性达102拷贝,并具有较宽的动力学定量范围,可对102～109拷贝范围内的待检样品进行准确的定量。复合探针技术可应用于病毒感染水平、转基因拷贝数及单核苷酸多态性等检测。

Cre/lox系统介导的位点特异性重组技术及其应用

Ｃｒｅ／ｌｏｘ系统是源于Ｐ１噬菌体的一个ＤＮＡ重组体系，它能导致在特定的ＤＮＡ序列（ｌｏｘＰ位点）处发生定点重组。该系统可以将外源基因定点整合到染色体上或将特定ＤＮＡ片段删除；这种定位重组系统在遗传操作中发挥了重要的作用。

Analysis of Seed-specificity of Silencing fad_2 Gene Expression in Transgenic Rapeseed Line W-4(Brassica napus L.)

This study was to investigate the efficiency and specificity of RNAi silencing on the expression of endogenous fad2 gene in transgenic line W-4. [Method] The relative expression of fad2 gene in seeds at different developmental stages of 7th, 14th, 21st and 28th day after flowering （DAF） as wel as the root, stem, leaf at winter seedling stages of both the transgenic line W-4 and non-transgenic control Westar by real-time fluorescence quantitative PCR. [Results] The results showed the relative expression of fad2 gene was gradual y increasing with the days after flowering in the seeds of the control Westar, while it was found decreasing significantly since the 21st DAF in the seeds of the line W-4. The decline was up to 60% in comparison with the control Westar. However, no significant difference in the relative expression of fad2 gene in other organs like root, stem and leaf was observed between transgenic line W-4 and non-transgenic control Westar. Fatty acid composition analysis showed the oleic acid desaturation parameter（ODP） in seeds of the line W-4 was 0.07 in average, decreased by nearly 75% than control Westar which was 0.24 in average, while no significant difference in the seedling root, stem and leaf was measured between transgenic rapeseed and control. [Conclusion] The results above validated that RNA interference in transgenic rapeseed W-4 is at a seed-specific manner, not interfering with fad2 gene expression in organs such as the root, stem and leaf. The study also found that the period of fad2 gene expres-sion decline was wel coincided with the expression of napin gene, both appeared at the 21st DAF, indicating that the expression of dsRNA of fad2 gene is precisely control ed by the napin promoter.

Uncertainty in Measuring Construct-specific Fragments of Genetically Modified Maize MON863 by Real Time Quantitative PCR

[Objective] The study aimed at evaluating the uncertainty in measuring the construct-specific fragments of genetically modified maize MON863 by real time quantitative PCR.[Method] The content of construct-specific fragments in MON863 samples was determined by real time quantitative PCR,and then the uncertainty of measurement result was evaluated according to the sources of uncertainty like the PCR system,the data processing and the micropipette.[Result] Type A evaluation of uncertainty（uA） in the measurement was 1.7×10^-2;Type B evaluation of uncertainty（uB） was 9.0×10^-4;the combined standard uncertainty（uC） was 1.7×10^-2;the expanded uncertainty（U95） was 0.036 and the finally measured result was 1.08%±0.036.[Conclusion] The main uncertainty of the result measured by real time quantitative PCR came from the randomizing effect in the experimental process.

Morphological Analysis and Gene Mapping of a Rice Dwarf Mutant

[Objective] This study aimed to conduct morphological analysis and gene mapping of a rice dwarf mutant. [Method] A dwarf mutant （Xiaoxiang＇ai） was used as test material for morphological observation. Xiaoxiang＇ai was used as female parent and semi-dwarf material Xiangzao143 was used as male parent to construct populations for genetic analysis. Gene mapping was conducted by using micro- satellite markers. [Result] The average height of Xiaoxiang＇ai was 55 cm; genetic analysis results showed that plant height of F1 individuals was similar with the male parent, while semi-dwarf plants and dwarf plants were observed in F2 populations and the segregation ratio was nearly 3：1, indicating that plant height trait of Xiaoxi- ang＇ai is mainly controlled by one pair of recessive gene located on rice chromo- some 5 and in the upstream of RM249, with a genetic distance of 8.4 cM. [Conclu- sion] rRH is a new dwarf gene in rice.

Recent advances in identification of male specificity determinant and its function in S-RNase-mediated gametophytic self-incompatibility

S-RNase-mediated gametophytic self-incompatibility （GSI） is controlled by a multiallelic S-locus at which two separate genes, the female （pistil） and male （pollen） specificity determinants, are tightly linked. This review described both the identification of pollen specific F-box genes, SLF/SFBs, in Antirrhinum, Petunia and Prunus species and the demonstration of SLF/SFB as pollen determinant together with their functions in GSI response. Recent studies of how the pollen determinant functions in pollination reaction revealed that pollen determinant interacted with S-RNases in a non-allele-specific manner. It targeted all of the non-self S-RNases for ubiquitination through a functional SCF complex and subsequent degradation via 26S proteasome pathway in compatible reaction. It allows pollen tube to reach into the embryo sac and to finish double fertilization. In incompatible response, the intact self S-RNases were left to function as a cytotoxin that degrades self-pollen tube RNA, resulting in the cessation of pollen tube growth.

Analysis of Quantitative Trait Loci for Starch Properties of Rice Based on an RIL Population

Rice (Oryza sativa L.) eating and cooking quality is mainly influenced by its starch properties. Mapping quantitative trait loci (QTL) for starch properties not only helps us understand their genetic basis leading to acceleration of quality improvement, but also helps us find possible genes participating in the synthesis of starch. A recombinant inbred line (RIL) population consisting of 107 lines, derived from an indica (Zaiyeqing 8, ZYQ 8) and a japonica (Jingxi 17, JX 17) rice, was used to investigate the genetic factors affecting starch quality parameters, such as apparent amylose content (AAC), gel consistency (GC), starch pasting viscosity parameters, gel textural properties, gelatinization temperature (GT) and starch retrogradation properties. A total of 44 QTLs covered chromosomes 2-6, 8, 9 and 11 were detected for the 22 traits, with at least one QTL and as many as four QTLs for each individual trait. The results indicated that two major genes were responsible for most starch property traits. The Wx gene that encodes granule bound starch synthase on chromosome 6 was significant for AAC, GC, starch pasting viscosity parameters, gel textural properties and starch retrogradation properties. The alk gene linked with Wx on chromosome 6 was significant for starch gelatinization temperature characteristics. All other QTLs were minor genes. One QTL on chromosome 9 flanked by RZ404 and G295 was significant for gel hardness (HD), gumminess (GUM), chewiness (CHEW), peak temperature of retrogradated starch (RTp), and percentage retrogradation (R%) and all these traits were not tested before.

Purification of a Diatom and Its Identification to Cylindrotheca closterium

A diatom was purified with colony selection and continuous dilution methods. It was identified to Cylindrotheca closterium according to its morphological characteristics and rbc L and 18 s r RNA gene sequences. The alga was not sensitive to ampicillin and neomycin, but sensitive to chloramphenicol which inhibited its growth at concentrations ranging from 50 to 150 μg m L-1. The purified alga was easy to culture and its specific growth rate was 0.207 ± 0.002(d-1). It was resistant to pollution and could be harvested in an easy way. It was relatively high in lipid content(20.08% ± 0.67% of dry weight) and the combined amount of its 16:0 and 16:1(n-7), the most suitable resource of biodiesel, was as high as 64% of the total fatty acids, while the amount of polyunsaturated fatty acids reached 19.96%–20% of the total fatty acids. Thus the purified C. closterium can be cultured as a biodiesel producer or a nutrition supplement producer.

Relationship between nm23H1 genetic instability and clinical pathological characteristics in Chinese digestive system cancer patients

AIM: To study the relationship between nm23H1 gene genetic instability and its clinical pathological characteristics in Chinese digestive system cancer patients. METHODS: Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) was used to analyze the microsatellite instability (MSI) and loss of heterozygosity (LOH). Immunohistochemistry was employed to detect the expression of nm23H1. RESULTS: The MSI was higher in TNM stageⅠ + Ⅱ than in stage Ⅲ + Ⅳ of gastric, colonic and gallbladder carcinomas. The LOH was higher in TNM stage Ⅲ + Ⅳ than in stageⅠ + Ⅱ of gastric, colonic and hepatocellular carcinomas. Lymphatic metastasis was also observed. The expression of nm23H1 protein was lower in TNM stage Ⅲ + Ⅳ than in stageⅠ + Ⅱ of these tumors and in patients with lymphatic metastasis.The nm23H1 protein expression was higher in the LOH negative group than in the LOH positive group.CONCLUSION: MSI and LOH may independently control the biological behaviors of digestive system cancers. MSI could serve as an early biological marker of digestive system cancers. Enhanced expression of nm23H1 protein could efficiently inhibit cancer metastasis and improve its prognosis. LOH mostly appears in late digestive system cancer.

Antagonistic Activity, Antimicrobial Susceptibility and Potential Virulence Factors of Enterococcus faecalis

Enterococcus faecalis isolates （87） were phenotypically and genotypically identified and subsequently subjected to the antagonism test and antimicrobial susceptibility. The lipolitic, hemolytic and DNAse activities were identified along with the genes gelE, cylL, cylS, ccf, cpd and cob that, encode virulence determinants. Thirty seven percent of isolates inhibited Listeria monocytogenes （CERELA）, Listeria innocuous （CERELA）, Staphylococcus aureus （ATCC25932）, Lactococcus lactis （IL1403）, Micrococcus luteus （ATCC 10240） and Enterococcusfaecalis （ATCC29212）. All strains were sensitive to the ampicillin antibiotic, but 47% were resistant to at least one antimicrobial agent and 6% of isolates presented multidrug resistance. Ninety seven percent of isolates contained the gelE gene, but 77% of these isolates showed gelatinase activity. Presence of cylL and cylS genes was observed in 25% of the isolates, but only 5% presented hemolytic activity. None isolates showed lipase and DNAse activities. Eight percent of isolates contained the ccf gene and 2% showed the presence of the cpd and cob genes. The ability to inhibit pathogenic bacteria, low resistance to antibiotics and absence of virulence factors make some of Enterococcusfaecalis strains characterized in the present study promising for exploitation in other applications such as probiotics in aquaculture.

The Study on the Gene Expression of Preimplantation IVF Bovine Embryos

The cattle different stage embryos obtained from in vitro was studied using the technology of single preimplantation embryo mRNA different display:single 8-cell and blastocyst stage embryos were studied using technology of mRNA different display and one different fragment was found. The result suggested that this fragment displayed high homology (99%) to cattle mRNA for ribosomal protein L31. Then to detect the expression of RPL31mRNA in 8 cell and blastocyst stage embryos by real-time quantitative PCR,the result showed the relative amount of 8 cells was 3.2 times of blastocyst's.

Development of a Real-Time PCR Method (Taqman) for Rapid Identification and Quantification of Prorocentrum donghaiense

Prorocentrum donghaiense is a dinoflagellate that is widely distributed in the East China Sea and has become increasingly involved in Harmful Algal Blooms (HABs). Therefore, it is necessary to study this dinoflagellate to monitor HABs. In this study, 13 pairs of primers specific to P. donghaiense (within its internal transcribed spacer (ITS) regions) were designed for SYBR Green I real-time PCR. As the SYBR Green I real-time PCR could not identify P. donghaiense in a specific manner, a Taqman real-time PCR method was developed by designing a set of specific primers and a Taqman probe. A 10-fold serial dilution of recombinant plasmid containing ITS regions of P. donghaiense was prepared as standard samples and the standard curve was established. Additionally, we quantified the genomic DNA in P. donghaiense cells and utilized this DNA to prepare another 10-fold serial dilution of standard sample and accordingly set up the standard curve. The mathematic correlation between the cell number and its corresponding plasmid copy number was also established. In order to test the efficiency of the real-time PCR method, laboratory samples and P. donghaiense HAB field samples were employed for identification and quantitative analysis. As to laboratory samples, as few as 102 cells of P. donghaiense could be quantified precisely utilizing both centrifugation and filtration techniques. The quantification results from field samples by real-time PCR were highly similar to those by light microscopy. In conclusion, the real-time PCR could be applied to identify and quantify P. donghaiense in HABs.

Rapid Identification of Pathogenic Bacteria by means of TwoConservative Gene Loci′ Specific PCR-CE-RFLP

To establish a rapid identification method for common pathogenic bacteria on the basis of molecular biology and to construct a preliminary Polymerase Chain Reaction-Capillary Electrophoresis - Restriction Fragment Length Polymorphism (PCR-CE-RFLP) database of bacteria isolated from clinical specimens frequently, 183 strains collected from clinical samples belonging to 12 genera and 19 species whose biochemical characterizations corresponded to the typical ones were examined. The genomic DNAs were amplified by two pairs of fluorescence labeled primers aiming at 16S rRNA gene and 16S-23S rRNA spacer region gene respectively at the same time. PCR products were then digested by restriction endonuclease HaeⅢ incompletely before taking capillary electrophoresis. The results with the PCR-CE-RFLP patterns of 16S rRNA genes were just alike within some genera, but when it comes to 16S-23S rRNA spacer region genes, each bacterium showed a unique pattern, which can be distinguished from each other easily. It seems that PCR-CE-RFLP patterns of 16S rRNA gene could only be used to classify the bacteria into family level, whereas the data of 16S-23S rRNA spacer region gene could be utilized to identify the whole microorganisms as precisely as the species level. In spite of the data of the spacer region gene alone can be sufficiently to verify the whole bacteria, we insist that the 16S rRNA gene could be of some assistant in case that there should be lots of families of bacteria, in which some similar ones, with the same RFLP data of 16S-23S rRNA spacer region gene, may coexist. This study proves that the utility of PCR-CE-RFLP is a convenient, rapid method to identify pathogenic bacteria, and is also a quick diagnosis measure for application to clinical use.

Detecting Factor Ⅺ Deficiency in Holstein Cattle Using PCR Analysis

[Objective] This study established a method to detect Factor Ⅺ by polymerase chain reaction analysis.[Method]A pair of primers was designed and synthesized according to sequences of FⅪ gene in Holstein calves,published in Genbank. Polymerase chain reaction was used to analyze FⅪ deficiency of 576 Holstein calves in Henan,and the result was verified by DNA sequencing. [Result] We detect 576 cows,which include two carriers and one F Ⅺ deficiency,and the result was consistent with the DNA sequencing. The frequency of the FⅪ mutant allele was 0.3%,the carrier was 0.3%,the prevalence was 0.2%.[Conclusion]A method detecting FⅪ by polymerase chain reaction analysis was established. This method is not only simple and convenient,but also has a high accuracy and low cost,which is more suitable for large-scale FⅪ investigation.