应用RAPD技术研究不同种山羊草叶绿体DNA的遗传变异

采用随机扩增多态性ＤＮＡ（ＲＡＰＤ）技术研究了山羊草属ｃｐＤＮＡ的遗传变异性。用改良的“高盐低ｐＨ法”提取了１５种山羊草材料和一种二粒小麦的叶绿体ＤＮＡ,并建立了重复性较好的ＲＡＰＤ标准分析条件。在此标准条件下,检测了１６个材料叶绿体ＤＮＡ的多态性。经１０个引物扩增,在得到的６４个片段中,有６个片段是１６个材料共有的,多态性达９０％。聚类分析结果显示,关系较近的有小亚山羊草和欧山羊草,粘果山羊草和     

棉花线粒体和线粒体DNA的分离与纯化

本文详细报道了一个针对棉花特点的线粒体以及线粒体ＤＮＡ的抽提方法，即先用蔗糖Ｓｔｅｐ递度和ＤＮａｓｅＩ体外消化分离纯化线粒体，然后氯仿抽提纯化ＤＮＡ。实验过程中采用ＰＶＰ排除棉酚和丹宁的干扰，用ＤＩＥＣＡ抑制酚氧化酶活性，并对许多实验细节作了较大改进。得到的ＤＮＡ可满足酶切、ＰＣＲ等分子生物学分析。     

棉花叶绿体70S核糖体S7蛋白基因的克隆和序列分析

叶绿体基因组具有高度保守性，来源于不同植物的叶绿体基因同源性很高。应用ＰＣＲ技术，从棉花叶绿体基因组扩增并克隆了长度为１．０ｋｂ的叶绿体ＤＮＡ片段。该片段含有叶绿体核本小亚基Ｓ７蛋白基因ｒｐｓ７。采用限制性内切酶消化连接的策略构建亚克隆，并测定了该片段的含部核苷酸序列。结果表明，棉花叶绿体ｒｐａｓ７基因与烟草、不稻的相庆基因同源性高，分别为９７．９％和８９．５％，３非编码区的同源性分别为９６．３％     

苜蓿组培中叶绿体DNA和线粒体DNA的变异

用差速离心法自继代培养5个月的愈伤组织中分离叶绿体和线粒体。用饱和酚、氯仿和氯化铯梯度离心的方法纯化二种细胞器的DNA。同时，用同样的方法自供体植物叶中分离和纯化叶绿体和线粒体DNA。采用了6种限制性内切酶、凝胶电泳和Southern印迹杂交的方法，比较了供体植物和愈伤组织中叶绿体DNA和线粒体DNA酶切谱带的变异情况。     

棉花叶绿体基因组的研究进展

近年来,随着分子生物学技术的应用与发展,对棉花叶绿体基因组也有了新认识。本文概述了棉花叶绿体基因组的研究进展,从棉花叶绿体基因组的图谱、功能基因的克隆及研究、叶绿体RNA编辑以及叶绿体转化等方面进行了介绍和评述,并对其在基础研究与应用研究中的前景进行了展望。     

改良CTAB法快速提取棉花DNA

针对棉花（Ｇｏｓｓｐｉｕｍ）富含棉酚、多糖等其它次生干扰物质这一特点而设计的一种快速分离和纯化棉花总ＤＮＡ（ｇＤＮＡ）的方法。该方法是对ＣＴＡＢ（ｃｅｔｙｌｔｒｉｍｅｔｈｙｌａｍｍｏｎｉｕｍｂｒｏｍｉｄｅ）法的改良，它在ＣＴＡＢ法的基础上，首先用ＤＩＥＣＡ（ｄｉｅｔｈｙｌｄｉｔｈｉｏｃａｒｂａｍｉｃａｃｉｄ）抑制酚氧化酶的活性，然后用活性炭和ＰＶＰ４０（ｐｏｌｙｖｉｎｙｌｐｙｒｒｏｌｉｄｏｎｅ）排除棉酚等其它次生干扰物质。试验证明，该方法比较简便、快速、经济、有效，得到的ｇＤＮＡ完全可以满足ＰＣＲ等分子生物学分析。     

雷蒙德氏棉叶绿体基因组Fosmid文库构建

 采用高盐、低pH值法提取雷蒙德氏棉叶绿体DNA;通过物理剪切法获得随机断裂的DNA片段;剪切片段末端、补平修饰后与pCC1FOS载体连接;用噬菌体包装蛋白包装重组DNA,侵染大肠杆菌EPI300,构建了雷蒙德氏棉叶绿体基因组文库。对于叶绿体DNA剪切,以1 mL注射器中等速度吸打18次为最佳参数。叶绿体基因组Fosmid文库滴度为1×10⁴ cfu·mL^-1,插入片段大小平均为38 kb,最终筛选出39个克隆用于后续研究,覆盖叶绿体基因组9.2倍。以叶绿体特异标记筛选出能够覆盖雷蒙德氏棉叶绿体全基因组的6个克隆：F66,F46,F28,F8,F55和F3,为基因组结构和功能基因分析提供了良好的基础。     

光调控下的叶绿体变化

不同的环境都会在叶绿体的形成与发育中起作用，环境对叶绿体产生的改变直接影响光合作用的水平与特性，光是影响叶绿体生长发育最重要的环境因素之一，研究光调控下叶绿体的变化对如何改善作物叶绿体发育条件，促进叶绿体的发育，有效的利用光能，提高农业生产上作物的光合作用,提供理论上的依据。在此，综述和讨论了叶绿体的形态、结构和发育过程，以及叶绿体的内部物质和超微结构在连续光照、脉冲光照、间歇光照及不同光源条件下的变化。     

亚洲栽培稻的核DNA、线粒体DNA和叶绿体DNA籼粳分化的比较研究

本研究对来自１２个国家的７４份亚洲栽培稻进行了核ＤＮＡ,线粒体ＤＮＡ的ＲＦＬＰ分析和叶绿体ＤＮＡ的ＯＲＦ１００的ＰＣＲ分析结果表明参试的７４个栽培稻品种,除ＣＲ１４７的线粒体基因组的类型较为特殊外,其它７３个品种的细胞核基因组,线粒体基因组,叶绿体基因组均可分为籼粳两种类型,可见籼粳分化是栽培稻核ＤＮＡ,ｍｅｔ ＤＮＡ和ｃｐＤＮＡ遗传分化的主流,同一品种３个ＤＮＡ的籼粳属性即有一致的,又有不一致的     

玉米(Zea mays L.)中单14号及亲本叶绿体的组分和功能比较

玉米中单１４号的叶绿体在叶绿素含量、光能吸收、电子传递速率、激发能分配和色素蛋白复合体组成等方面均优于两亲本。其叶绿素ａ、ｂ和总含量提高了３１％￣３６％；对光能吸收强度较大；光还原活力超过双亲２５．５％；分配到ＰＳＩ的激发能比亲本多１４７．１％；叶绿素蛋白复合体分析结果说明，其捕光色素蛋白复合体含量高于亲本。因而中单１４号比亲本在叶绿体的光合功能上显示优势。     

Chloroplast DNA analysis of eggplant (Solanum melongena) and related species for their taxonomic affinity

Summary Total chloroplast DNA (cpDNA) from Solanum incanum, a wild relative of eggplant, was used to probe total DNA of Solanum melongena (eggplant). The DNA fragments detected were the same as observed using purified chloroplast DNA. Chloroplast DNAs were also analysed for nine species of Solanum that are cross-compatible with eggplant: S. aethiopicum, S. anguivi, S. gilo, S. incanum, S. indicum, S. integrifolium, S. macrocarpon, S. olivare and S. panduriforme.Restriction fragments generated by eight enzymes were recorded as present or absent, and a matrix for all fragment positions, species and enzymes was used for cluster analysis. In the resulting dendrogram, the species tested formed three distinct groups: (1) S. aethiopicum, S. anguivi, S. gilo, S. indicum, S. integrifolium and S. olivare, (2) S. incanum, S. melongena and S. panduriforme, (3) S. macrocarpon. Six species of the first group belonging to section Oliganthes appears more closely related to the second group members belonging to section Melongena than does S. macrocarpon, which also belongs to section Melongena. Within the second group, S. panduriforme is slightly more like eggplant than is S. incanum.     

Chloroplast DNA diversity in brinjal eggplant (Solanum melongena L.) and related species

Chloroplast DNA (cpDNA) samples of brinjal eggplant (S. melongena) and representative related species including S. incanum sensu lato (or S. campylacanthum sensu stricto), S. lichtensteinii, S. marginatum, S. macrocarpon, S. anguivi and S. aethiopicum and also S. nigrum as an outgroup taxon, were digested by 14 restriction enzymes and analyzed by using electrophoresis and a cpDNA probe. All the species used here were clearly separated in the cpDNA analysis, except the pair S. anguivi and S. aethiopicum. From the dendrogram constructed by the unweighted pair-group method, it is suggested that S. incanum is the closest to S. melongena and the next closest species is S. macrocarpon followed by S. aethiopicum (and S. anguivi), S. lichtensteinii, S. marginatum and finally the outgroup taxon S. nigrum. The tree derived by the neighbour-joining method suggests phyletic relationships that agree with those indicated by crossability and seed coat anatomy, but conflict with conventional classifications based on morphology. In particular, members of sections Oliganthes and Melongena are not separated and no cpDNA variation was found within either of the morphologically diverse cultigens, S. aethiopicum and S. melongena. Paradoxically, the morphologically similar species S. incanum, S. lichtensteinii and S. marginatum have diverged greatly in their cpDNA. The significance of these results is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

The Use of Stomatal Chloroplast Number for Rapid Determination of Ploidy Level in Maize

The usefulness of using the chloroplast number in epidermal guard cells as an indirect ploidy indicator was evaluated on seed-grown and tissue culture-derived maize plants. For seed-grown plants, two maize genotypes (B89 and R75) which had both diploid and tetraploid seeds available were used as experimental materials. The ploidy levels of seed-grown plants were confirmed by flow cytometric analysis of nuclear suspensions from these plants. For regenerated plants, haploid and diploid levels were examined and the ploidy levels of these plants were determined by chromosome counts of cells from root tips. A positive relationship between the chloroplast number and ploidy level was observed for both seed-grown and regenerated plants. The stomatal guard cells of tetraploid plants had nearly double the number of chloroplasts as the diploid plants. Similar results were found from the regenerated plants. The differences in the mean chloroplast number between diploid and tetraploid seed-grown plants and between haploid and diploid regenerated plants were highly significant. The results of this study demonstrate that counting chloroplasts in guard cells can be an efficient means of screening a large number of plants for ploidy levels. In addition, this study suggests the possibilities of using this method for detecting contaminated seed lots by different ploidy seed and for distinguishing plants of different genotypes.     

番茄质体多顺反子定点整合表达载体的构建及其转烟草的研究

根据烟草叶绿体高频同源重组片段的已知序列(GenBank Z00044.1)设计引物,用PCR的方法克隆到1个3.663 kb的番茄叶绿体DNA片段（psbD/trnG）,命名为ctDNA。该片段与GenBank中烟草的相应片段有96.7%同源性, 与本文所用的烟草的相应片段有95.8%同源性。以其为外源多顺反子定点整合介导的同源重组片段,与来自烟草叶绿体的强启动子Prrn和终止子psbA3’,以及甘露聚糖酶基因man、绿荧光蛋白基因gfp、氨基糖苷3’-腺苷酰基转移酶基因aadA构建番茄质体多顺反子定点整合表达载体pLM2(-psbD-Prrn-RBS-man-RBS-gfp-RBS-aadA-psbA3’-trnG-)。将该载体用基因枪轰击烟草叶片,用添加了壮观霉素的选择分化培养基筛选,获得质体转基因烟草3株。用PCR、激光扫描、Western Blot、RFLP等方法检测都证实man、gfp、aadA基因已整合到烟草质体基因组中,且均得到表达。用番茄质体多顺反子定点整合表达载体成功实现在烟草质体中的表达。     

Distribution of Microsatellites from the Complete Sequence of Gossypium hirsutum Chloroplast DNA

The distribution of chloroplast microsatellites in the chloroplast complete sequence of Gossypium hirsutum was analyzed with bioinformatics software. The results showed that the chloroplast microsatellites of G. hirsutum were mainly distributed in the large single-copy region and the small single-copy region, and most microsatellites were complete repeats. Mononucleotide repeat motifs, dinucleotide repeat motifs and trinucleotide repeat motifs accounted for 76.4%, 20.8% and 2.8% of the total, respectively. Repeats A and T were the main base in the mononucleotide motif, accounting for 92.7% of the total. The most common repeat number of (A)_n and (T)_n was 10 and 11, respectively, and the repeat number of the dinucleotide repeat motif was 5 or 6. These results are available for use in developing general primers from chloroplast DNA in Gossypium.     

山豆根基因组DNA高效提取方法的建立与优化

为了探寻优化山豆根DNA提取的方法,本研究以山豆根的叶片为材料,用3种改良的CTAB法提取山豆根DNA,并将提取的DNA经分光光度计和电泳检测,用于cpDNA引物筛选试验,找出最有效的山豆根DNA提取方法。结果显示,高PVP和β-巯基乙醇提取法和样品预处理法可用于提取山豆根基因组DNA。其中,样品预处理法山豆根DNA的效果最好,提取DNA浓度高、质量好,引物筛选结果条带清晰且单一。本研究建立了山豆根高质量的DNA提取方法,为进行分子生物学研究提供支持。     

鲜米糠中蛋白质的提取工艺研究

以新鲜米糠为原料,采用碱法、酸法、盐法分步对米糠蛋白进行复合提取。在提取过程中,以蛋白质提取率为指标,确定料液比、pH值、温度、时间对米糠蛋白提取率的影响,通过正交试验确定这3种方法各自提取蛋白的最佳工艺,最后依次采用碱提、酸提、盐提的最佳工艺条件分步对米糠蛋白进行提取。结果表明,碱法提取新鲜米糠蛋白最佳工艺条件:时间2.5 h,pH值12,温度30℃,料液比1∶11,提取率为34.49%;酸法提取新鲜米糠最佳工艺条件:时间3 h,pH值0.05,温度35℃,料液比1∶11,提取率为25.88%;盐法提取新鲜米糠蛋白最佳工艺条件:NaCl浓度0.6 mol/L,温度35℃,料液比1∶11,提取率为15.66%。复合法提取新鲜米糠蛋白总提取率达60.12%。