拟南芥bre突变体分析及小麦ver203F基因表达模式研究

第一部分 茎尖分生组织的中央部位是由干细胞组成，维持干细胞的动态平衡对于植物器官启动显得尤为重要。在拟南芥花序分生组织和花分生组织中分别有WUS/CLV和(WUS+LFY)/AG两个负反馈调节环控制着这一平衡，保证了花序分生组织的非决定性和花分生组织的决定性。 本文用T-DNA激活标签技术得到功能增强突变体bre，序列分析表明BRE就是干细胞特征基因WUSCHEL。定量RT-PCR表明在突变体的茎节间WUS大量表达，证实在WUS基因的调控区插入的4个35S增强子使WUS表达增强。突变体bre生长发育缓慢，茎弯曲，花器官数目改变，心皮发育加快。尤为突出的是在茎皮层直接分化出花分生组织。这些表型可能与WUS的表达增强有关。这说明WUS功能是多效的，除了在分生组织中决定干细胞命运、促进CLV和AG的表达外，还可能与生长素极性运输以及花分生组织特征基因的表达有关。 第二部分 春化作用是促进植物从营养生长向生殖生长转变的有效途径之一，作者所在实验室曾经分离到多个小麦春化相关基因。为研究春化期间它们在不同组织或不同细胞内的表达模式，首先建立并完善了RNA组织原位杂交系统。以春化相关基因ver203F为模板，采用地高辛为标记分子，借助体外转录制备RNA探针，分析了小麦胚芽和幼苗中ver203F的表达模式。结果表明：春化前和脱春化的胚芽中不表达ver203F，春化处理14天以上的胚芽幼叶表达ver203F,而且主要定位在叶的边缘分生组织细胞内，叶维管束和表皮组织未检测到转录物，胚芽鞘和茎尖分生组织中不表达。幼苗的侧芽中的幼叶有相同的表达模式。茉莉酸可以诱导ver203F的表达。．说明春化时的低温信号可能由幼叶接收，并有可能涉及到茉莉酸介导的信号转导途径。

LPA3蛋白参与调控PSII组装的功能研究

PSII是一个叶绿体类囊体膜的蛋白复合体，它由20多个蛋白亚基组成，这些蛋白由核基因和叶绿体基因共同编码。由于PSII结构的复杂性，PSII的组装是多步骤的，并得到辅因子和调控蛋白的协助。但我们对参与调控步骤的蛋白因子还了解不多。鉴于叶绿体有限的编码能力，推测参与叶绿体组装调控的因子主要是由核基因来编码的。辨定这些核编码的叶绿体蛋白并深入研究其作用的分子机制将有助于我们了解PSII生物发生的分子机理。为此，我们利用叶绿素荧光成像系统对pER16 T-DNA插入突变体库进行了筛选，并对高荧光突变体lpa3进行了研究。主要研究结果如下： 突变体lpa3生长较为缓慢，叶色黄绿，叶绿素含量低。在突变体中，最大荧光量子产率Fv/Fm降低到0.514，表明PSII光合功能受到了损伤。突变体lpa3叶绿素荧光慢诱导曲线的正常下降表明PSII后的电子传递正常。突变体P700氧化还原动力学与野生型一致，则进一步表明PSI在突变体中是具有功能的。77K发射荧光光谱显示PSII的特征峰在突变体中较高，而PSI的特征荧光峰没有变化，则进一步显示突变体lpa3是一个PSII突变体。 通过Tail-PCR，发现突变体中T-DNA插入导致基因lpa3缺失表达，并且lpa3基因的互补可以使突变体的性状得到恢复。该基因表达的蛋白LPA3含有一个跨膜区域，是一个类囊体膜蛋白。该蛋白不是PSII的蛋白组成成分，可能与自身或者其它的蛋白组成一个复合体而起作用。 在突变体lpa3中PSII蛋白质尤其是核心蛋白D1、D2，含量下降。并且突变体中PSII蛋白复合体含量下降。但是突变体中PSII基因的表达并没有在转录水平受到调节，并且蛋白D1和D2的翻译起始也没有受到影响。体外标记实验表明，PSII中D1蛋白的合成明显降低，而其它蛋白的合成没有改变。进一步实验表明，突变体中PSII的组装效率降低。推测D1蛋白由于不能有效组装而反馈调节自身的合成。 酵母双杂交实验表明LPA3蛋白可以与D1蛋白相互作用，并且也与参与PSII组装的蛋白Alb3相互作用。因此，LPA3蛋白可能和蛋白Alb3形成一个复合体，该复合体与D1蛋白直接相互作用而参与调控PSII的组装。

拟南芥AST基因的精细作图

拟南芥ast (anthocyanin spotted testa) 突变体是由碳离子束诱导产生的与花青苷生物合成有关的突变体，受单隐性核基因控制。由于花青苷的异常积累，突变体未成熟种子的种皮呈现紫红色的斑点;野生型植株幼嫩的种皮没有花青苷的异常积累，呈淡绿色。初步作图分析表明，AST基因定位于拟南芥第I号染色体上，并且位于SSLP分子标记nga280和CAPS分子标记PAB5之间。 AST基因与SSLP分子标记nga280紧密连锁，遗传距离为3.2cM;与CAPS分子标记PAB5相距较远，遗传距离为21.1cM。 采用DDRT-PCR的策略，分析野生型与突变型植株未成熟角果中基因表达的差异。通过调整DDRT-PCR中总RNA、锚定引物、随机引物、cDNA和dNTP等关键试剂的用量，优化了适用于银染检测的DDRT-PCR方法。PCR扩增产物经6%变性聚丙烯酰胺凝胶垂直电泳分离后，银染能检测到多而清晰的条带。泳道中的条带数最少为40个，最多达80个，平均为60个，条带大小分布在100bp-900bp范围，银染的灵敏度为5pg/mm2。此方法操作简便快速，灵敏度高，重复性好。采用这个改良的的方法，分析了拟南芥野生型和ast突变型植株未成熟角果中16,000个cDNA扩增产物条带，从中筛选出28个差异条带。二次PCR扩增后，进一步筛选出10个差异表达的cDNA条带，其中6个是野生型特异表达的，4个是突变型特异表达的。对这10个差异片段进行测序。BLASTN分析表明，这10个差异表达的cDNA片段与数据库中花青苷生物合成途径中的结构基因和调节基因序列没有同源性，表明用DDRT-PCR的方法克隆特定的AST基因有一定的局限性。 利用图位克隆（map-based cloning）的策略，对拟南芥AST 基因进行克隆。根据拟南芥数据库中的SNPs (simple nucleotide polymophisms) 序列和插入/缺失多态性（insertion/deletion polymorphisms）序列，设计了一系列分子标记。利用这些分子标记，对600个F2代有突变表型的植株进行重组子筛选，完成了对拟南芥AST基因的精细作图，成功地将AST 基因定位到BAC克隆T13M11上。初步确定该BAC克隆中的基因T13M11.8 可能是AST基因。该基因的DNA序列长1432bp，含有6个外显子和5个内含子，编码的蛋白与花青苷生物合成途径中的二氢黄酮醇4-还原酶有较高的同源性。功能互补实验正在进行当中。

Construction, characterization and chromosomal mapping of bacterial artificial chromosome (BAC) library of Yunnan snub-nosed monkey (Rhinopithecus bieti)

We constructed a high redundancy bacterial artificial chromosome library of a seriously endangered Old World Monkey, the Yunnan snub-nosed monkey (Rhinopithecus bieti) from China. This library contains a total of 136 320 BAC clones. The average insert size of BAC clones was estimated to be 148 kb. The percentage of small inserts (50-100 kb) is 2.74%, and only 2.67% non-recombinant clones were observed. Assuming a similar genome size with closely related primate species, the Yunnan snub-nosed monkey BAC library has at least six times the genome coverage. By end sequencing of randomly selected BAC clones, we generated 201 sequence tags for the library. A total of 139 end-sequenced BAC clones were mapped onto the chromosomes of Yunnan snub-nosed monkey by fluorescence in-situ hybridization, demonstrating a high degree of synteny conservation between humans and Yunnan snub-nosed monkeys. Blast search against human genome showed a good correlation between the number of hit clones and the size of the chromosomes, an indication of unbiased chromosomal distribution of the BAC library. This library and the mapped BAC clones will serve as a valuable resource in comparative genomics studies and large-scale genome sequencing of nonhuman primates. The DNA sequence data reported in this paper were deposited in GenBank and assigned the accession number CG891489-CG891703.

Site-directed gene integration in transgenic zebrafish mediated by cre recombinase using a combination of mutant Lox sites

With current gene-transfer techniques in fish, insertion of DNA into the genome occurs randomly and in many instances at multiple sites. Associated position effects, copy number differences, and multiple gene interactions make gene expression experiments difficult to interpret and fish phenotype less predictable. To meet different fish engineering needs, we describe here a gene targeting model in zebrafish. At first, four target zebrafish lines, each harboring a single genomic lox71 target site, were generated by zebrafish transgenesis. The zygotes of transgenic zebrafish lines were coinjected with capped Cre mRNA and a knockin vector pZklox66RFP. Site-specific integration event happened from one target zebrafish line. In this line two integrant zebrafish were obtained from more than 80,000 targeted embryos (integrating efficiency about 10(-4) to 10(-5)) and confirmed to have a sole copy of the integrating DNA at the target genome site. Genomic polymerase chain reaction analysis and DNA sequencing verified the correct gene target events where lox71 and lox66 have accurately recombined into double mutant lox72 and wild-type loxP. Each integrant zebrafish chosen for analysis harbored the transgene rfp at the designated egfp concatenates. Although the Cre-mediated recombination is site specific, it is dependent on a randomly placed target site. That is, a genomic target cannot be preselected for integration based solely on its sequence. Conclusively, an rfp reporter gene was successfully inserted into the egfp target locus of zebrafish genome by Cre-lox-mediated recombination. This site-directed knockin system using the lox71/lox66 combination should be a promising gene-targeting platform serving various purposes in fish genetic engineering.

Selection and ultrastructural observation of a high-CO2-requiring mutant of cyanobacterium Synechococcus sp PCC7942

A high-CO2-requiring mutant of Synechococcus sp. PCC7942 las been isolated after chemical mutagenesis of ethyl methane sulphonate (EMS). It was able to grow at 4% CO2, but not under ambient CO2. The initial screening of the mutant showed that the genetic reversion rate was about 10(-7) and death occurred 2 -3 days after being transferred from 4% CO2 to the ambient air. Its photosynthetic dependence on external dissolved inorganic carbon was higher than that of the wild type cells, but its carbonic anhydrase activity was comparatively low. In the ultrastructural level, various types of aberrant carboxysomes appeared in the mutant cells: rod-shaped carboxysomes, irregular carboxysomes and the "empty-inclusion carboxysomes" with increasing number of glycogen granules surrounding the thylakoids. All these alterations indicated that the mutant was defective in utilizing the external CO2. The induction of carboxysomes by lower levels of CO2 and the biogenesis of carboxysomes are herein discussed.

Comparative analysis of astaxanthin and its esters in the mutant E1 of Haematococcus pluvialis and other green algae by HPLC with a C30 column

A gradient reversed-phase high-performance liquid chromatography (HPLC) method using a C30 column was developed for the simultaneous determination of astaxanthin, astaxanthin monoesters and astaxanthin diesters in the green algae Chlorococcum sp., Chlorella zofingiensis, Haematococcus pluvialis and the mutant E1, which was obtained from the mutagenesis of H. pluvialis by exposure to UV-irradiation and ethyl methanesulphonate (EMS) with subsequent screening using nicotine. The results showed that the contents of total astaxanthins including free astaxanthin and astaxanthin esters ranged from 1.4 to 30.9 mg/g dry biomass in these green algae. The lower total astaxanthin levels (< 2 mg/g dry biomass) were detected in the green algae Chlorococcum sp. and C. zofingiensis. The higher total astaxanthin levels (> 16 mg/g dry biomass) were found in the green alga H. pluvialis and its mutant E1. It is notable that the mutant E1 is found to have considerably higher amounts of total astaxanthin (30.9 mg/g) as compared to the wild strain of H. pluvialis (16.1 mg/g). This indicates that UV-irradiation and EMS compound mutagenesis with subsequent screening using nicotine is an effective method for breeding of a high-producing astaxanthin strain of H. pluvialis. In addition, the green alga C. zofingiensis had a remarkably higher percentage of astaxanthin diesters (76.3% of total astaxanthins) and a remarkably lower percentage of astaxanthin monoesters (18.0% of total astaxanthins) in comparison with H. pluvialis (35.5% for diesters and 60.9% for monoesters), the mutant E1 (49.1% and 48.1%) and Chlorococcum sp. (18.0% and 58.6%).

Isolation and mapping of telomeric pentanucleotide (TAACC)(n) repeats of the pacific whiteleg shrimp, Penaeus vannamei, using fluorescence in situ hybridization

To develop genetic and physical maps for shrimp, accurate information on the actual number of chromosomes and a large number of genetic markers is needed. Previous reports have shown two different chromosome numbers for the Pacific whiteleg shrimp, Penaeus vannamei, the most important penaeid shrimp species cultured in the Western hemisphere. Preliminary results obtained by direct sequencing of clones from a Sau3A-digested genomic library of P. vannamei ovary identified a large number of (TAACC/GGTTA)-containing SSRs. The objectives of this study were to (1) examine the frequency of (TAACC)(n) repeats in 662 P. vannamei genomic clones that were directly sequenced, and perform homology searches of these clones, (2) confirm the number of chromosomes in testis of P. vannamei, and (3) localize the TAACC repeats in P. vannamei chromosome spreads using fluorescence in situ hybridization (FISH). Results for objective I showed that 395 out of the 662 clones sequenced contained single or multiple SSRs with three or more repeat motifs, 199 of which contained variable tandem repeats of the pentanucleotide (TAACC/GGTTA),, with 3 to 14 copies per sequence. The frequency of (TAACC)n repeats in P. vannamei is 4.68 kb for SSRs with five or more repeat motifs. Sequence comparisons using the BLASTN nonredundant and expressed sequence tag (EST) databases indicated that most of the TAACC-containing clones were similar to either the core pentanucleotide repeat in PVPENTREP locus (GenBank accession no. X82619) or portions of 28S rRNA. Transposable elements (transposase for Tn1000 and reverse transcriptase family members), hypothetical or unnamed protein products, and genes of known function such as 18S and 28S rRNAs, heat shock protein 70, and thrombospondin were identified in non-TAACC-containing clones. For objective 2, the meiotic chromosome number of P. vannamei was confirmed as N = 44. For objective 3, four FISH probes (P1 to P4) containing different numbers of TAACC repeats produced positive signals on telomeres of P. vannamei chromosomes. A few chromosomes had positive signals interstitially. Probe signal strength and chromosome coverage differed in the general order of P1 > P2 > P3 > P4, which correlated with the length of TAACC repeats within the probes: 83, 66, 35, and 30 bp, respectively, suggesting that the TAACC repeats, and not the flanking sequences, produced the TAACC signals at chromosome ends and TAACC is likely the telomere sequence for P. vannamei.

Genetic mapping of laminaria japonica and l. longissima using amplified fragment length polymorphism markers in a "two-way pseudo-testcross" strategy

With a "two-way pseudo-testcross" mapping strategy, we applied the amplified fragment length polymorphism (AFLP) markers to construct two moderate density genetic linkage maps for Laminaria. The linkage maps were generated from the 60 progenies of the F, cross family (Laminaria longissima Aresch. x L. japonica Miyabe) with twenty pairs of primer combinations. Of the 333 polymorphic loci scored in 60 progenies, 173 segregated in a 1:1 ratio, corresponding to DNA polymorphisms heterozygous in a single parent, and the other 58 loci existing in both parents followed a 3:1 Mendelian segregation ratio. Among the loci with 1:1 segregating ratios, 79 loci were ordered in 14 linkage groups (648.6 cM) of the paternal map, and 72 loci were ordered in 14 linkage groups (601.9 cM) of the maternal map. The average density of loci was approximately 1 per 8 cM. To investigate the homologies between two parental maps, we used 58 loci segregated 3:1 for further analysis, and deduced one homologous linkage group. The linkage data developed in these maps will be useful for detecting loci-controlling commercially important traits for Laminaria.

Two-Point Closure Strategy in the Mapping Closure Approximation Approach

A two-point closure strategy in mapping closure approximation (MCA) approach is developed for the evolution of the probability density function (PDF) of a scalar advected by stochastic velocity fields. The MCA approach is based on multipoint statistics. We formulate a MCA modeled system using the one-point PDFs and two-point correlations. The MCA models can describe both the evolution of the PDF shape and the rate at which the PDF evolves.