猕猴桃属植物叶绿体基因PCR-RFLP分析

分别用2个不同限制性内切酶对猕猴桃属27个种和15个栽培品种的叶绿体基因(rbcL基因和psbA基因)的PCR扩增产物进行酶切分析,共得到25个限制性位点,其中24个具有多态性。确立了该属植物的单元型分布,对该属植物的系统发育方式和部分重要种类的亲缘关系进行了探讨,拓展了可用于该属植物分子系统学研究的遗传信息。     

抗阿特拉津基因PCR检测方法的建立与应用

阿特拉津是一种均三氮苯类除草剂,其作用机理是取代质体醌与叶绿体类囊体膜上的32kDa蛋白的结合,从而阻断光系统Ⅱ的电子传递而使光合作用受阻。32kDa蛋白由叶绿体psbA基因编码,psbA基因的突变使32kDa蛋白的第264位丝氨酸变为苷氨酸或丙氨酸,从而丧失与阿特拉津结合的能力,导致对阿特拉津除草剂的抗性。由于阿特拉津除草剂在大豆产区的广泛使用,选择和培育阿特拉津抗性     

中国大麦叶绿体DNA和核糖体RNA基因限制性片段长度多型性

本文报道了我们对我国不同大麦区80份大麦品种叶绿体DNA和核糖体RNA基因限制性片段长度多型性的研究。结果表明:rDNA间隔序列长度存在丰富的多样性,80份材料中出现了8种长度变异炎型共组成8种表现型。长度变异类型及其表现型在地理分布上存在着明显的区域性。推测这种分布上的地区性与植物对环境的适应性有关。所用的两个叶绿体DNA克隆片段未检测到限制性片段长度多型性,说明栽培大麦叶绿体DNA变异程度低。     

A rapid method to test for chloroplast DNA involvement in atrazine resistance

A point mutation in the chloroplast psbA gene at codon 264 resulting in an animo acid substitution (ser-gly) manifests itself as atrazine resistance in all recognized weed species studied to date. The single base substitution overlaps a highly conserved Mae1 restriction site which is present in susceptible but not in resistant plants. This restriction enzyme, recently commercialized, has been used to show that it is now possible to discriminate rapidly between the two biotypes without the need for DNA sequencing.     

Chloroplast DNA diversity in the genusRubus (Rosaceae) revealed by Southern hybridization

The variability in chloroplast DNA type of 20Rubus genotypes was examined by Southern hybridization. DNA extracted from theRubus accessions was digested with two restriction enzymes (EcoRI and EcoRV) and heterologous chloroplast DNA sequences from barley and pea were used as probes to detectRubus chloroplast DNA sequences on Southern blots ofRubus total DNA. Restriction fragment length polymorphism was detected and a total of 92 restriction fragments were generated by the probe/enzyme combinations examined. Cladistic principles based on the parsimony assumption were used to assemble a phylogenetic tree based on chloroplast restriction fragment length data. The phylogenetic tree grouped the taxonomically defined species and is in general agreement with information based on morphological criteria. However, the Japanese red raspberryR. illecebrosus was shown to have diverged considerably in terms of evolutionary time from other species in subg.Idaeobatus. Furthermore, the molecular approach provides a quantitative estimate of the relationship between species that is difficult to obtain from morphological data. In order to complement the chloroplast DNA information a ribosomal DNA probe was also included in the analysis and provided further information on the phylogenetic relationships withinRubus.     

水稻叶绿体16S启动子克隆改造、载体构建及转化研究

利用PCR方法从水稻叶绿体基因组DNA中分离16S启动子,并在其下游加入rbcL基因SD序列,以增强该启动子的翻译能力;序列分析表明,除加入的SD序列外,扩增片段与水稻(Oryza sativa)叶绿体基因组DNA序列16S启动子相应区域同源性为100％。将16S启动子与bar基因和gfp基因的融合基因连接,以psbA基因的3′序列为终止子,并以烟草叶绿体trnH—psbA和trnK为同源片段构建了烟草叶绿体表达载体pRl6S。用基因枪转化烟草,转化植株经Southern、Northern检测及后代遗传学分析,发现：16S启动子具有启动活性,融合基因已在烟草叶绿体中稳定整合并遵循母系遗传规律。     

Genetic distribution of Bare–1-like retrotransposable elements in the barley genome revealed by sequence-specific amplification polymorphisms (S-SAP)

Retrotransposons are present in high copy number in many plant genomes. They show a considerable degree of sequence heterogeneity and insertional polymorphism, both within and between species. We describe here a polymerase chain reaction (PCR)-based method which exploits this polymorphism for the generation of molecular markers in barley. The method produces amplified fragments containing a Bare–1-like retrotransposon long terminal repeat (LTR) sequence at one end and a flanking host restriction site at the other. The level of polymorphism is higher than that revealed by amplified fragment length polymorphism (AFLP) in barley. Segregation data for 55 fragments, which were polymorphic in a doubled haploid barley population, were analysed alongside an existing framework of some 400 other markers. The markers showed a widespread distribution over the seven linkage groups, which is consistent with the distribution of the Bare–1 class of retrotransposons in the barley genome based on in situ hybridisation data. The potential applicability of this method to the mapping of other multicopy sequences in plants is discussed.     

Construction of Chloroplast Genomic Library of Amaranthus cruentus R104 and Cloning of the Gene for the Large Subunit of Ribulose-1, 5-disphosphate Carboxylase/Oxygenase

Grain amaranth is an annual food and forage crop and C4-plant with characteristics of rich nutrients, high photosynthesis efficiency and strong stress-resistance. Chloroplast DNA (ctDNA) was prepared from Amaranthus cruentus R104. The ctDNA was digested with restriction enzymes Barn HⅠ, EcoRⅠ, BgⅢ, PstⅠ and SaⅡ, and the fragments thus obtained were electrophoresed in 0.7% agarose gel. The Length of the doublestranded ctDNA was measured to be 140 kb or so. BamHI-digested fragments of R104 ctDNA were inserted into pBR322 and a chloroplast genomic library has been constructed. The recombinant clone containing rbcL gene has been identified and selected out from the library. The restriction analysis of the clone showed that the rbcL gene of A. cruentus R104 has the structure similar to that of C4-plant corn. Moreover the reason of unsuccessful cloning of 5.9 kb BamHI fragment containing psbA gene is discussed.     

Minimal Extent of Sequence Homology Required for Homologous Recombination at the psbA Locus in Chlamydomonas reinhardtii Chloroplasts using PCR-generated DNA Fragments

The Del1 mutant of the green alga Chlamydomonas reinhardtii with a defined deletion in the chloroplast encoded psbA gene is unable to grow photoautotrophically. We show here that this mutant can be transformed with PCR-generated psbA fragments of varying length to yield photosynthetically growing colonies. PCR fragments need not be purified but can be directly precipitated from the amplification reaction onto tungsten particles, allowing fast and efficient mutagenesis experiments. Flanking regions bordering the deletion breakpoints have been systematically shortened from both sides. The shortest fragment giving rise to significant numbers of transformants contains about 50 bp upstream and 120 bp downstream of the deletion breakpoint. This technique greatly simplifies comprehensive structure-function analyses of the D1 protein in Chlamydomonas, but could perhaps be adapted to other chloroplast genes in this or other organisms as well.     

玉米叶绿体表达载体的构建及AsRED基因原核表达

从玉米幼嫩叶片中提取玉米叶绿体基因DNA,通过PCR克隆出叶绿体同源重组片段trnA和trnI、叶绿体特异性启动子Prrn以及终止子psbA.构建玉米叶绿体表达载体pBAIRTARED,含有一个人工操纵子,其中,筛选标记基因aadA和红色荧光蛋白报告基因AsRED处于Prrn启动子和psbA终止子控制.将构建的载体转化大肠杆菌BL21（DE3）,观测到重组细胞呈现红色,表明构建的载体可以用于玉米叶绿体转化以及表达报告基因.     

Preparation of Chloroplast DNA from Barley and Lettuce and Comparison of Restriction Fragments

Chloroplast DNA from three barley cultivars and from one lettuce cultivar was prepared from chloroplasts isolated by Conventional differential centrifugation. Barley chloroplast DNA size was sensibly lower (130 kpb) than lettuce chloroplast DNA (150 kpb). Chloroplast DNAs from the three barley cultivars showed similar restriction fragment patterns after digestion with: BamHI, EcoRI or HindIII. The lettuce chloroplast DNA restriction pattern was very different from the barley chloroplast DNA restriction pattern.     

亚心型扁藻叶绿体psbA基因的克隆及序列分析

psbA基因是叶绿体基因组中一个重要的光调控基因,编码光和系统Ⅱ反应中心的D1蛋白。根据叶绿体基因组序列高度保守的特性,利用菜茵衣藻（Chlamydomonasreinhardtii）psbA基因的保守序列（基因登录号：HQ667991．1）设计引物,采用PCR步移的方法从亚心型扁藻（Platymonassubcordiformis）基因组DNA中克隆到psbA基因全长（基因登录号：KF528742）。序列分析表明,亚心型扁藻psbA基因全长1939bp,编码区长度为1062bp,推导编码353个氨基酸,包括4个赖氨酸残基。有效密码子数显示脚删基因具有明显的密码子偏好性,并且偏好使用以A／T结尾的密码子。相对同义密码子使用度表明25个密码子在编码使用时具有偏好性,其中20个密码子以A／T碱基结尾,占到80％。其终止密码子使用了TAG。     

PCR产物的RFLP分析在黄芪亚族（豆科）系统学研究中的应用初探

用聚合酶链式反应（ＰＣＲ）将豆科黄芪亚族７属９种及甘草亚族１属１种植物叶绿体基因组中ｎｄｈＦ和ｐｓｂＡ基因中一段约３．１ｋｂ的ＤＮＡ扩增出来,并摸索出一最佳的ＰＣＲ条件,使得此条带得以特异性扩增,可直接用于限制性内切酶水解。通过对此扩增片段的限制性片段长度多态性（ＲＦＬＰ）的初步分析,认为利用ＰＣＲ扩增出的ＤＮＡ进行ＲＦＬＰ分析来探讨植物的系统进化问题在中国现行条件下大有潜力,其方法简单易行,结果