碱性土壤基因组DNA的分离纯化和基因文库的构建

直接从广西南宁市凤凰纸业排污沟碱性土壤样品中抽提和分离纯化混合基因组DNA，所获得DNA的产量为每克土壤样品10～30pg。采用限制性内切酶EcoR1酶处理后，构建了以pGEM-3Zf( )为载体的DNA部分文库。文库的容量为23650个转化子。外源片段DNA平均大小为3．2kb。建库效率为每克环境样品获得6000个左右的含1～15kb外源随机插入片段的克隆。通过DNA序列测定和同源性比较，对从文库随机词取的16个转化子序列进行分析，发现13个外源插入片段包含序列尚未确定的DNA片段。     

用水稻基因芯片筛选小麦耐旱相关基因

为了解小麦在干旱逆境条件下的基因转录规律,采用PEG6000对耐旱小麦(Triticum aestivum)品种旱选10号进行拟旱处理,分别提取0、1、6和24h植株的总RNA,经反转录荧光标记制备cDNA探针,并将其与含有6万Oligo的水稻全基因组芯片进行杂交,扫描采集数据后并进行结果分析。在1、6和24h样品中分别检测到差异表达基因166、207和328个,随着处理时间的延长,差异表达基因数目增加。对差异基因进行功能分类,能量代谢途径相关基因在1、6和24h差异表达基因总数中所占比例分别为4.2%、8.2%和16.8%,其中大部分为光合作用相关基因,并且主要表现为上调,但Psbr和Rubisco编码基因的转录水平为下调,暗示它们在耐旱反应中发挥着一定作用。     

不同鳞翅目昆虫细胞色素P450基因(CYPs)的比较基因组学分析

细胞色素P450(cytochrome P450,CYP)涉及许多昆虫生理功能,包括对信号分子的代谢、宿主植物的适应性及杀虫剂的抗性等.鳞翅目(Lepidoptera)昆虫的种类仅次于鞘翅目(Coleoptera),包含了大量的农业害虫.根据鳞翅目烟草天蛾(Manduca sexta L.)的基因组数据,开展CYPs的全基因组学分析,在该基因组中发现了110个可能的CYPs,可分为29个家族,其中有大量CYPs以串联重复形式排列.将分析所得序列与蝶类昆虫黑脉金斑蝶(Danaus plexippus L.)和模式昆虫家蚕(Bombyx mori L.)的CYPs进行比较基因组学分析,结果表明,在CYP2与线粒体集团(clan)中存在12对CYPs直向同源基因,而CYP3与CYP4集团的CYPs呈现种属特异性扩增趋势.通过比较分析鳞翅目昆虫与其他非鳞翅目昆虫的CYPs发现,鳞翅目昆虫之间不仅存在更多的直向同源基因对,而且鉴别出的直向同源基因群也更多,但出现新的家族与亚家族数量相对较少.不同鳞翅目昆虫基因组中的CYPs存在相当广泛的线性保守现象.这些不同昆虫间的比较基因组学分析将促进鳞翅目昆虫CYPs的研究,也有助于害虫治理新靶标的选择.     

欧文氏杆菌CXJZ95-198基因组文库的构建

采用改良鸟枪法构建了草本纤维精制高效菌种Erwinia carotovora CXJZ95-198的基因组文库，结合透明圈法，筛选到了8个甘露聚糖酶基因阳性克隆，并采用PCR方法对它们进行了分析鉴定，结果表明它们含有同一个甘露聚糖酶基因。     

Genomic, protein and antigenic variability of mycoplasma bovis

Restriction endonuclease analysis (REA) with three enzymes SmaI, PstI, BamHI- was used to identify 13 different genomic groups among 37 Mycoplasma bovis strains. One genomic group was comprised of 14 strains. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) patterns for one strain chosen from each genomic group and an international reference strain PG45 were all similar. Antigenic variability in M. bovis species was investigated by immunoblotting, using serum from a calf that had been naturally infected with M. bovis and three M. bovis-specific monoclonal antibodies — mAbs N₂, I₂ and 5D7. Twenty M. Bovis field strains were tested, comprising one from each genomic group, six from the same genomic group and the reference strain. Antigenic profiles obtained with calf serum differed markedly one from the other, the heterogeneity being equally great among the strains belonging to the same genomic group as those coming from different groups. A stable antigen common to 164 out of 168 strains was detected by mAb N₂, whilst with mAbs I₂ and 5D7, two different membrane antigenic systems were demonstrated that were strikingly variable. These variations in expression occurred not only from one strain to another, but also within the same lineage of clones from a single cell.     

玉米基因组DNA的快速高效提取(简报)

采用改良的十六烷基三乙基溴化铵(CTAB)法,以玉米Zea mays的黄化苗叶片为材料提取玉米基因组DNA,提取的DNA经0．8％的琼脂糖凝胶电泳检测,结果表明,此法具有快速、高效和DNA质量好、纯度高等优点。     

禾本科植物内生真菌研究13:禾本科植物内生真菌的分离鉴定及基因组DNA的快速提取

调查我国7省12个不同地区的禾本科植物内生真菌,经分离、培养后鉴定得到2种Epichlo属和4种Neotyphodium属内生真菌。采用改良的QS(improved quick and safe,IQS)法进行禾本科植物内生真菌基因组DNA的提取,并通过PCR进行验证。以生长在平板上的菌丝为起始材料,仪器要求简单、操作简便,快速安全、省时省力,整个提取过程大约40 min。该方法和常规SDS法相比,时间缩短到2周,为原来的1/4。以IQS法提取的DNA为模板,检测禾本科植物内生真菌中的NRPS基因,并在NCBI上进行比对分析,发现与已有的NRPS基因高度相似。这种快速提取法所获得的基因组DNA质量高,可用于后续PCR、基因克隆及其他高通量测试,特别适合获取大量真菌菌株基因组DNA时使用。     

Genomic selection strategies in dairy cattle: Strong positive interaction between use of genotypic information and intensive use of young bulls on genetic gain

We tested the following hypotheses: (i) breeding schemes with genomic selection are superior to breeding schemes without genomic selection regarding annual genetic gain of the aggregate genotype (ΔG(AG) ), annual genetic gain of the functional traits and rate of inbreeding per generation (ΔF), (ii) a positive interaction exists between the use of genotypic information and a short generation interval on ΔG(AG) and (iii) the inclusion of an indicator trait in the selection index will only result in a negligible increase in ΔG(AG) if genotypic information about the breeding goal trait is known. We examined four breeding schemes with or without genomic selection and with or without intensive use of young bulls using pseudo-genomic stochastic simulations. The breeding goal consisted of a milk production trait and a functional trait. The two breeding schemes with genomic selection resulted in higher ΔG(AG) , greater contributions of the functional trait to ΔG(AG) and lower ΔF than the two breeding schemes without genomic selection. Thus, the use of genotypic information may lead to more sustainable breeding schemes. In addition, a short generation interval increases the effect of using genotypic information on ΔG(AG) . Hence, a breeding scheme with genomic selection and with intensive use of young bulls (a turbo scheme) seems to offer the greatest potential. The third hypothesis was disproved as inclusion of genomically enhanced breeding values (GEBV) for an indicator trait in the selection index increased ΔG(AG) in the turbo scheme. Moreover, it increased the contribution of the functional trait to ΔG(AG) , and it decreased ΔF. Thus, indicator traits may still be profitable to use even when GEBV for the breeding goal traits are available.     

Evaluation of a multi-line broiler chicken population using a single-step genomic evaluation procedure

Effects on prediction of analysing a multi-line chicken population as one line were evaluated. Body weight records were provided by Cobb-Vantress for two lines of broiler chickens. Phenotypic records for 183 695 and 164 149 broilers and genotypic records for 3195 and 3001 broilers were available for each line. Lines were combined to create a multi-line population and analysed using a single-step procedure combining the additive relationship matrix and the genomic relationship matrix (G). G was scaled using allele frequencies from each line, the multi-line population, or 0.5. When allele frequencies were calculated from each line, distributions of diagonal elements were bimodal. When allele frequencies were calculated from the multi-line population, the distribution of diagonal elements had one peak. When allele frequency 0.5 was used, the distribution was bimodal. Genomic estimated breeding values (GEBVs) were predicted using each allele frequency. GEBVs differed with allele frequency but had ≥ 0.99 correlations with GEBVs predicted with correct allele frequencies. Means of each line and differences in mean between the lines differed based on allele frequencies. Assumed allele frequencies have little impact on ranking within line but larger impact on ranking across lines. G may be used to evaluate multiple populations simultaneously but must be adjusted to obtain properly scaled estimates when population structure is unknown.     

山苍子AFLP反应体系的建立及其引物筛选

通过对山苍子幼嫩叶片、顶芽、花蕾3种组织的DNA提取效果分析和对影响酶切及选择性扩增效果的4个主要因素(酶切时间、Mg²⁺浓度、dNTPs浓度、引物浓度)的比较研究,建立了适合于山苍子AFLP分析的技术体系。结果表明,山苍子的顶芽是较好的DNA提取材料;山苍子基因组DNA经5 U EcoR I和5 U Mse I酶切1 h即可完全酶切;最佳的选择性扩增体系为20 μL反应体系中含有1.0 U rTaq聚合酶、2.0 μL 10×PCR缓冲液、1.8 μL 25 mmol·L^-1MgCl₂、1.4 μL 2.5 mmol·L^-1dNTP、100 ng·μL^-1引物各1.0 μL。使用该反应体系获得了清晰、稳定的DNA指纹图谱,并筛选出10对多态性较好的AFLP引物组合,为利用AFLP标记技术进一步开展山苍子种群遗传结构、遗传分化等研究奠定了基础。