Single cell proteomics microchip to profile immune function, with applications in stem cell biology, translational disease mechanism study and clinical therapeutics monitoring

In response to infection or tissue dysfunction, immune cells develop into highly heterogeneous repertoires with diverse functions. Capturing the full spectrum of these functions requires analysis of large numbers of effector molecules from single cells. However, currently only 3-5 functional proteins can be measured from single cells. We developed a single cell functional proteomics approach that integrates a microchip platform with multiplex cell purification. This approach can quantitate 20 proteins from >5,000 phenotypically pure single cells simultaneously. With a 1-million fold miniaturization, the system can detect down to ~100 molecules and requires only ~104 cells. Single cell functional proteomic analysis finds broad applications in basic, translational and clinical studies. In the three studies conducted, it yielded critical insights for understanding clinical cancer immunotherapy, inflammatory bowel disease (IBD) mechanism and hematopoietic stem cell (HSC) biology.

To study phenotypically defined cell populations, single cell barcode microchips were coupled with upstream multiplex cell purification based on up to 11 parameters. Statistical algorithms were developed to process and model the high dimensional readouts. This analysis evaluates rare cells and is versatile for various cells and proteins. (1) We conducted an immune monitoring study of a phase 2 cancer cellular immunotherapy clinical trial that used T-cell receptor (TCR) transgenic T cells as major therapeutics to treat metastatic melanoma. We evaluated the functional proteome of 4 antigen-specific, phenotypically defined T cell populations from peripheral blood of 3 patients across 8 time points. (2) Natural killer (NK) cells can play a protective role in chronic inflammation and their surface receptor – killer immunoglobulin-like receptor (KIR) – has been identified as a risk factor of IBD. We compared the functional behavior of NK cells that had differential KIR expressions. These NK cells were retrieved from the blood of 12 patients with different genetic backgrounds. (3) HSCs are the progenitors of immune cells and are thought to have no immediate functional capacity against pathogen. However, recent studies identified expression of Toll-like receptors (TLRs) on HSCs. We studied the functional capacity of HSCs upon TLR activation. The comparison of HSCs from wild-type mice against those from genetics knock-out mouse models elucidates the responding signaling pathway.

In all three cases, we observed profound functional heterogeneity within phenotypically defined cells. Polyfunctional cells that conduct multiple functions also produce those proteins in large amounts. They dominate the immune response. In the cancer immunotherapy, the strong cytotoxic and antitumor functions from transgenic TCR T cells contributed to a ~30% tumor reduction immediately after the therapy. However, this infused immune response disappeared within 2-3 weeks. Later on, some patients gained a second antitumor response, consisted of the emergence of endogenous antitumor cytotoxic T cells and their production of multiple antitumor functions. These patients showed more effective long-term tumor control. In the IBD mechanism study, we noticed that, compared with others, NK cells expressing KIR2DL3 receptor secreted a large array of effector proteins, such as TNF-α, CCLs and CXCLs. The functions from these cells regulated disease-contributing cells and protected host tissues. Their existence correlated with IBD disease susceptibility. In the HSC study, the HSCs exhibited functional capacity by producing TNF-α, IL-6 and GM-CSF. TLR stimulation activated the NF-κB signaling in HSCs. Single cell functional proteome contains rich information that is independent from the genome and transcriptome. In all three cases, functional proteomic evaluation uncovered critical biological insights that would not be resolved otherwise. The integrated single cell functional proteomic analysis constructed a detail kinetic picture of the immune response that took place during the clinical cancer immunotherapy. It revealed concrete functional evidence that connected genetics to IBD disease susceptibility. Further, it provided predictors that correlated with clinical responses and pathogenic outcomes.

Comparative studies of Vulva development in C. briggsae

As evolution progresses, developmental changes occur. Genes lose and gain molecular partners, regulatory sequences, and new functions. As a consequence, tissues evolve alternative methods to develop similar structures, more or less robust. How this occurs is a major question in biology. One method of addressing this question is by examining the developmental and genetic differences between similar species. Several studies of nematodes Pristionchus pacificus and Oscheius CEW1 have revealed various differences in vulval development from the well-studied C. elegans (e.g. gonad induction, competence group specification, and gene function.)

I approached the question of developmental change in a similar manner by using Caenorhabditis briggsae, a close relative of C. elegans. C. briggsae allows the use of transgenic approaches to determine developmental changes between species. We determined subtle changes in the competence group, in 1° cell specification, and vulval lineage.

We also analyzed the let-60 gene in four nematode species. We found conservation in the codon identity and exon-intron boundaries, but lack of an extended 3' untranslated region in Caenorhabditis briggsae.

Plastidic Phosphoglucose Isomerase Is an Important Determinant of Starch Accumulation in Mesophyll Cells, Growth, Photosynthetic Capacity, and Biosynthesis of Plastidic Cytokinins in Arabidopsis

Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. It is involved in glycolysis and in the regeneration of glucose-6-P molecules in the oxidative pentose phosphate pathway (OPPP). In chloroplasts of illuminated mesophyll cells PGI also connects the Calvin-Benson cycle with the starch biosynthetic pathway. In this work we isolated pgi1-3, a mutant totally lacking pPGI activity as a consequence of aberrant intron splicing of the pPGI encoding gene, PGI1. Starch content in pgi1-3 source leaves was ca. 10-15% of that of wild type (WT) leaves, which was similar to that of leaves of pgi1-2, a T-DNA insertion pPGI null mutant. Starch deficiency of pgi1 leaves could be reverted by the introduction of a sex1 null mutation impeding beta-amylolytic starch breakdown. Although previous studies showed that starch granules of pgi1-2 leaves are restricted to both bundle sheath cells adjacent to the mesophyll and stomata guard cells, microscopy analyses carried out in this work revealed the presence of starch granules in the chloroplasts of pgi1-2 and pgi1-3 mesophyll cells. RT-PCR analyses showed high expression levels of plastidic and extra-plastidic beta-amylase encoding genes in pgi1 leaves, which was accompanied by increased beta-amylase activity. Both pgi1-2 and pgi1-3 mutants displayed slow growth and reduced photosynthetic capacity phenotypes even under continuous light conditions. Metabolic analyses revealed that the adenylate energy charge and the NAD(P) H/NAD(P) ratios in pgi1 leaves were lower than those of WT leaves. These analyses also revealed that the content of plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP)-pathway derived cytokinins (CKs) in pgi1 leaves were exceedingly lower than in WT leaves. Noteworthy, exogenous application of CKs largely reverted the low starch content phenotype of pgi1 leaves. The overall data show that pPGI is an important determinant of photosynthesis, energy status, growth and starch accumulation in mesophyll cells likely as a consequence of its involvement in the production of OPPP/glycolysis intermediates necessary for the synthesis of plastidic MEP-pathway derived hormones such as CKs.

Hypoxic and low pH water in the nearshore marine environments of Monterey Bay, California: characterizing a decade of oxygen and pH, and drivers of variability.

A decade-long time series recorded in southern Monterey Bay, California demonstrates that the shallow, near-shore environment (17 m depth) is regularly inundated with pulses of cold, hypoxic and low pH water. During these episodes, oxygen can drop to biologically threatening levels, and pH levels were lower than expected. Weekly water chemistry monitoring revealed that the saturation state of aragonite (the more soluble form of calcium carbonate) was often below saturation and had a moderate positive relationship with pH, however, analytical and human error could be high. Pulses of hypoxia and low pH water with the greatest intensity arise at the onset of the spring upwelling season, and fluctuations are strongly semidurnal (tidal) and diurnal. Arrival of cold, hypoxic water on the inner shelf typically occurs 3 days after the arrival of a strong upwelling event and appears to be driven by upwelling modulated by internal tidal fluctuations. I found no relationship between the timing of low-oxygen events and the diel solar cycle nor with terrestrial nutrient input. These observations are consistent with advection of hypoxic water from the deep, offshore environment where water masses experience a general decline of temperature, oxygen and pH with depth, and inconsistent with biochemical forcing. Comparisons with concurrent temperature and oxygen time series taken ~20 km away at the head of the Monterey Canyon show similar patterns but even more intense hypoxic events due to stronger semidiurnal forcing there. Analysis of the durations of exposure to low oxygen levels establishes a framework for assessing the ecological relevance of these events. Increasing oceanic hypoxia and acidification of both surface and deep waters may increase the number, intensity, duration and spatial extent of future intrusions along the Pacific coast. Evaluation of the resiliency of nearshore ecosystems such as kelp forests, rocky reefs and sandy habitats, will require consideration of these events.

Aplicações de métodos quimiométricos e de técnicas analíticas em amostras alimentícias e farmacêuticas

Esse trabalho compreende dois diferentes estudos de caso: o primeiro foi a respeito de um medicamento para o qual foi desenvolvida uma metodologia para determinar norfloxacino (NOR) por espectrofluorimetria molecular e validação por HPLC. Primeiramente foi desenvolvida uma metodologia por espectrofluorimetria onde foram feitos alguns testes preliminares a fim de estabelecer qual valor de pH iria fornecer a maior intensidade de emissão. Após fixar o pH foi feita a determinação de NOR em padrões aquosos e soluções do medicamento usando calibração univariada. A faixa de concentração trabalhada foi de 0500 μg.L-1. O limite de detecção para o medicamento foi de 6,9 μg.L-1 enquanto que o de quantificação foi de 24,6 μg.L-1. Além dessas, outras figuras de mérito também foram estimadas para desenvolvimento da metodologia e obtiveram resultados muito satisfatórios, como por exemplo, os testes de recuperação no qual a recuperação do analito foi de 99.5 a 103.8%. Para identificação e quantificação do NOR da urina foi necessário diluir a amostra de urina (estudada em dois diferentes níveis de diluição: 500 e 1000 x) e também uso do método da adição de padrão (na mesma faixa de concentração usada para medicamento). Após a aquisição do espectro, todos foram usados para construção do tensor que seria usado no PARAFAC. Foi possível estimar as figuras de mérito como limite de detecção de 11.4 μg.L-1 and 8.4 μg.L-1 (diluição de 500 e 1000 x respectivamente) e limite de quantificação de 34 μg.L-1 e 25.6 μg.L-1 (diluição de 500 x e 1000 x respectivamente). O segundo estudo de caso foi na área alimentícia no qual se usou espectroscopia NIR e FT MIR acopladas a quimiometria para discriminar óleo de soja transgênica e não transgênica. Os espectros dos óleos não mostraram diferença significativa em termos visuais, sendo necessário usar ferramentas quimiométricas capazes de fazer essa distinção. Tanto para espectroscopia NIR quanto FT MIR foi feito o PCA a fim de identificar amostras discrepantes e que influenciariam o modelo de forma negativa. Após efetuar o PCA, foram usadas três diferentes técnicas para discriminar os óleos: SIMCA, SVM-DA e PLS-DA, sendo que para cada técnica foram usados também diferentes pré processamento. No NIR, apenas para um pré processamento se obteve resultados satisfatórios nas três técnicas, enquanto que para FT-MIR ao se usar PLS-DA se obteve 100% de acerto na classificação para todos os pré processamentos

Influence of ocean conditions on the timing of early life history events for blue rockfish (Sebastes mystinus) off California

Settled juvenile blue rockfish (Sebastes mystinus) were collected from two kelp beds approximately 335 km apart off Mendocino in northern California and Monterey in central California. A total of 112 rockfish were collected from both sites over 5 years (1993, 1994, 2001, 2002, and 2003). Total age, settlement date, age at settlement, and birth date were determined from otolith microstructure. Fish off Mendocino settled mostly in June and fish off Monterey settled mostly in May (average difference in settlement=23 days). Although the difference in the timing of settlement followed this same pattern for both areas over the five years, settlement occurred later in 2002 and 2003 than in the prior years of sampling. The difference in the timing of settlement was due primarily to differences in birth dates for the two areas. The time of settlement was positively related to upwelling and negatively related to sea level anomaly for most of the months before settlement. Knowledge of the timing of settlement has implications for design and placement of marine protected areas because protection of nursery grounds is frequently a major objective of these protected areas. The timing of settlement is also an important consideration in the planning of surveys of early recruits because mistimed surveys (caused by latitudinal differences in the timing of settlement) could produce biased estimates.

Development of larval and early juvenile penpoint gunnel (Apodichthys flavidus) (family: Pholidae)

The penpoint gunnel (Apodichthys flavidus) is a member of the perciform family Pholidae. Pholids, commonly referred to as gunnels, are eel-like fishes that inhabit the rocky intertidal and subtidal regions of the northern oceans and are often associated with macroalgae, such as Fucus spp. or kelp (Watson, 1996). Gunnels are ecologically important forage fishes that form part of the diet of birds and commercially important groundfish species (Hobson and Sealy, 1985; NMFS1; Golet et al., 2000). The diet of A. flavidus and other pholids comprises primarily harpactacoid copepods, gammarid amphipods, isopods, and other crustaceans (Cross, 1981). Apodichthys flavidus ranges along the west coast of North America from southern California to the Gulf of Alaska (Mecklenburg et al., 2002). Adult A. flavidus are distinguished from other pholids by their total vertebral counts, the presence of a thick and grooved first anal spine, a preanal length that is approximately 60% standard length (SL), and a dark green to light olive coloration (Yatsu, 1981). It is one of the largest pholids (up to 46 cm) and is important in the live fish trade for both home and public aquaria (Froese and Pauly2).

A computer software package for assessing and managing risks posed by experiments with genetically modified fish and shellfish

Assessment and management of risk is needed for sustainable use of genetically modified aquatic organisms (aquatic GMOs). A computer software package for safely conducting research with genetically modified fish and shellfish is described. By answering a series of questions about the organism and the accessible aquatic ecosystem, a researcher or oversight authority can either identify specific risks or conclude that there is a specific reason for safety of the experiment. Risk assessment protocols with examples involving transgenic coho salmon, triploid grass carp and hybrid tilapia are described. In case a specific risk is identified, the user is led to consider risk management measures, involving culture methods, facilities design and operations management, to minimize the risk. Key features of the software are its user-friendly organization; easy access to explanatory text, literature citations and glossary; and automated completion of a worksheet. Documented completion of the Performance Standards can facilitate approval of a well designed experiment by oversight authorities.

The review of recent advances in fish genetics and biotechnology

Great advances have been, and are being made in our knowledge of the genetics and molecular biology (including genomics, proteomics and structural biology). Global molecular profiling technologies such as microassays using DNA or oligonucleotide chip, and protein and lipid chips are being developed. The application of such biotechnological advances are inevitable in aquaculture in the areas of improvement of aquaculture stocks where many molecular markers such as RFLPs, AFLDs and RAPD are now available for genome analysis, finger printing and genetic linkage mapping. Transgenic technology has been developed in a number of fish species and research is being pursed to produce transgenic fish carrying genes that encode antimicrobial peptides such as lysozyme thereby achieving disease resistance in fish. Also it is a short cut to achieving genetic change for fast growth and other desirable traits like early sexual maturity, temperature tolerance and feed conversion efficiency. KEYWORDS: Fish genetics, transgenesis, monoploidy, diploidy, polyploidy,gynogenesis, androgenesis, cryopreservation.

A interação entre ciência e valores segundo Hugh Lacey: estratégias, controvérsias e a garantia do bem-estar de todos

Este trabalho analisa questões relacionadas a interação entre ciência e os valores de acordo com o modelo de desenvolvimento científico apresentado por Hugh Lacey. É realizado um exame das principais questões relacionadas ao ideal de uma ciência livre de valores, que possui raízes na defesa da autonomia científica feita por Galileu Galilei; o lugar que estes valores (cognitivos e não-cognitivos) ocupam e seu grau de influência dentro da pesquisa científica. Uma comparação entre as noções de paradigma e estratégia propostas por Thomas Kuhn e Hugh Lacey, respectivamente, são apresentadas, bem como as posições de Helen Longino como representante de um fazer científico feminista. Esta dissertação também apresenta o estudo de caso acerca da implantação e uso das sementes transgênicas. Mais do que um simples exemplo, o estudo de caso apresentado por Lacey abre oportunidade para a pluralidade de estratégias na ciência como principal modo de alcançar o bem estar de todos.

Ecology and growth of juvenile California spiny lobster, Panulirus interruptus (Randall)

ABSTRACT TRANSCRIBED FROM ENGLE'S PH.D. ORAL DEFENSE PAMPHLET: The natural history of juvenile California spiny lobster, Panulirus interruptus (Randall), was investigated, with primary emphasis placed on ascertaining juvenile habitats, determining juvenile growth rates and component growth processes, and evaluating ecological and behavioral phenomena associated with juvenile survival and growth. Habitat surveys of island and mainland localities throughout southern and lower California revealed that small, greenish juveniles typically inhabit crevices or temporary burrows in 0-4m deep, wave-swept rocky habitats covered by dense beds of surf grass, Phyllospadix torreyi S. Watson. Phyllospadix beds were more abundant on gradually sloping rocky mainland beaches than on steeply sloping island shores. Phyllospadix abundance was positively correlated with P. interruptus abundance; however, at Santa Catalina Island, the Phyllospadix habitat was not extensive enough to be the sole lobster nursery. In laboratory tests, puerulus larvae and early juveniles chose Phyllospadix over rubble rocks or broad-bladed kelp, but did not consistently prefer Phyllospadix over reticulate algae. Ecology, growth, and behavior of juvenile P. interruptus inhabiting a discrete Phyllospadix habitat at Bird Rock, Santa Catalina Island, were investigated from October 1974 through December 1976 by means of frequent scuba surveys. Pueruli settled from June to November. Peak recruitment occurred from July to September, when seasonal temperatures were maximal. Settled larvae were approximately one year old. Juvenile growth was determined by size-frequency, single molt increment, mark-recapture, and laboratory culture studies. Carapace length vs. wet weight relationships fit standard power curve equations. Bird Rock juveniles grew from 7 to 32mm CL in 10-11 molts and from 32 to 56mm CL in 5-6 molts during their first and second benthic years, respectively. Growth rates were similar for males and females. Juveniles regenerating more than two limbs grew less per molt than intact lobsters. Long-term growth of laboratory-reared juveniles was 20% less than that of field lobsters. Growth component multiple regression analyses demonstrated that molt increment was directly proportional to premolt size and temperature for age 1+ lobsters. Molt frequency was inversely proportional to size and directly proportional to temperature. Temperature affected age 2+ lobsters similarly, but molt increment was independent of size, and molt frequency declined at a different rate. Juvenile growth rates more than doubled during warm water months compared to cold water months, primarily because of increased molt frequency. Based on results from this study and from previous investigations, it is estimated that P. interruptus males and females become sexually mature by ages 4 and 5 years, respectively, and that legai size is reached by 7 or 8 years of age. Juvenile P. interruptus activity patterns and foraging behavior were similar to those of adults, except that juvenile home ranges were proportionally smaller, and small juveniles were apparently not attracted to distant food. Small mollusks, abundant in Phyllospadix habitats, were the major food items. Size-dependent predation by fish and octopus apparently caused the considerable juvenile mortality observed at Bird Rock. Juveniles approaching 2 years of age gathered in mixed size-class aggregations by day and foraged beyond the grass beds at night. In autumn, these juveniles migrated to deeper habitats, coincident with new puerulus settlement in the Phyllospadix beds. Based on strong inferences from the results, it is proposed that size-dependent predation is the most important factor determining the !ife history strategy of juvenile P. interruptus. Life history tactics promoting rapid growth apparently function dually in reducing the period of high vulnerability to predation and decreasing the time required to reach sexual maturity. The Phyllospadix habitat is an excellent lobster nursery because it provides shelter from predators and possesses abundant food resources for sustaining optimum juvenile growth rates in shallow, warm water.

A biogeographic assessment of the Channel Islands National Marine Sanctuary: a review of boundary expansion concepts for NOAA’s National Marine Sanctuary Program

The priority management goal of the National Marine Sanctuaries Program (NMSP) is to protect marine ecosystems and biodiversity. This goal requires an understanding of broad-scale ecological relationships and linkages between marine resources and physical oceanography to support an ecosystem management approach. The Channel Islands National Marine Sanctuary (CINMS) is currently reviewing its management plan and investigating boundary expansion. A management plan study area (henceforth, Study Area) was described that extends from the current boundary north to the mainland, and extends north to Point Sal and south to Point Dume. Six additional boundary concepts were developed that vary in area and include the majority of the Study Area. The NMSP and CINMS partnered with NOAA’s National Centers for Coastal Ocean Science Biogeography Team to conduct a biogeographic assessment to characterize marine resources and oceanographic patterns within and adjacent to the sanctuary. This assessment includes a suite of quantitative spatial and statistical analyses that characterize biological and oceanographic patterns in the marine region from Point Sal to the U.S.-Mexico border. These data were analyzed using an index which evaluates an ecological “cost-benefit” within the proposed boundary concepts and the Study Area. The sanctuary resides in a dynamic setting where two oceanographic regimes meet. Cold northern waters mix with warm southern waters around the Channel Islands creating an area of transition that strongly influences the regions oceanography. In turn, these processes drive the biological distributions within the region. This assessment analyzes bathymetry, benthic substrate, bathymetric life-zones, sea surface temperature, primary production, currents, submerged aquatic vegetation, and kelp in the context of broad-scale patterns and relative to the proposed boundary concepts and the Study Area. Boundary cost-benefit results for these parameters were variable due to their dynamic nature; however, when analyzed in composite the Study Area and Boundary Concept 2 were considered the most favorable. Biological data were collected from numerous resource agencies and university scientists for this assessment. Fish and invertebrate trawl data were used to characterize community structure. Habitat suitability models were developed for 15 species of macroinvertebrates and 11 species of fish that have significant ecological, commercial, or recreational importance in the region and general patterns of ichthyoplankton distribution are described. Six surveys of ship and plane at-sea surveys were used to model marine bird diversity from Point Arena to the U.S.-Mexico border. Additional surveys were utilized to estimate density and colony counts for nine bird species. Critical habitat for western snowy plover and the location of California least tern breeding pairs were also analyzed. At-sea surveys were also used to describe the distribution of 14 species of cetaceans and five species of pinnipeds. Boundary concept cost-benefit indices revealed that Boundary Concept 2 and the Study Area were most favorable for the majority of the species-specific analyses. Boundary Concept 3 was most favorable for bird diversity across the region. Inadequate spatial resolution for fish and invertebrate community data and incompatible sampling effort information for bird and mammal data precluded boundary cost-benefit analysis.

植物生产生物可降解塑料PHB的研究：真养产碱杆菌H16的phbB和phbC的基因克隆、植物表达载体构建和转基因马铃薯植株的获得

利用聚合酶链式反应(PCR)技术从Alcaligenes eutrophus H16染色体DNA中扩增并克隆了调控聚-3-羟基丁酸(poly-3-hydroxy-butyrate，PHB)生物合成的两个关键酶基因：依赖NADPH的乙酰乙酰CoA还原酶基因(phbB)和PHB合成酶基因(phbC)。限制性内切酶图谱和核苷酸序列分析证实了克隆结果，并表明克隆的基因与国外所报道的有很高的同源性。经过基因拼接，构建了块茎特异性表达的高等植物表达载体pPSAGB（嵌合phbB）、pBIBGC(嵌合phbC)和pPSAGCB（嵌合phbB和phbC）。并以试管薯(microtuber)为外植体经Agrobacterium介导转化了虎头、京丰、Bintje、Favorita、高原4号和88-5共6个马铃薯品种，获得49个株系。经PCR检测导入phbB的株系共有44个，对其中30个株系进行DNA dot blot分析，结果表明phbC导入呈阳性的株系有20个。深入的鉴定工作还在进行中。

菠菜硬脂酰ACP去饱和酶基因的克隆及其抗寒性转基因烟草的获得

低温是限制植物分布和生物产量的一个重要环境因子。低温的危害也是农业生产上经常遭受的主要自然灾害之一。改良作物的抗寒性是植物科学研究的一个重要课题。植物基因工程的兴起为此目的提供了有力的手段。 大量的研究证明，低温对植物的伤害，首先是使生物膜发生相变和相分离。因此，保持低温下生物膜功能性的液晶态是抗寒的重要机制。研究表明，生物膜这种具流动性的功能态的保持，是与其组成上的膜脂脂肪酸的不饱和度成正相关的。 已有几个关于通过提高膜脂脂肪酸不饱和度的基因操作而增加植物抗寒性的报道。在众多的植物脂肪酸去饱和酶中，硬脂酰ACP去饱和酶(SAD)是最为关键的酶之一。它催化脂肪酸的第一步去饱和反应：18：0-18：l¨。多不饱和脂肪酸是在1 8：1 中由其他去饱和酶再加入双键而生成的。因此，SAD的活性水平是决定植物膜脂不池和度的一个关键因素。 本研究以酸酚法提取的菠菜总RNA为材料，采用反转录一PCR的方法，克隆得到SAD基因，经定向缺失法获得一套该基因的缺失突变体后,用DNA 自动测序仪和双脱氧链终止法测序。获得的SAD基因序列与Nishlda(1992)等发表的菠菜sADcDNA核苷酸序列比较。两者的编码SAD的ORF都为ll97bp，核苷酸差异仅为8bp。但令人惊奇的是我们克隆到的基因，其5‘端上游还存在-个小的ORF，长30bp．编码10个氨基酸。其他报道的SAD基因中都没有这个ORF。 将克隆到的SAD基因构建成两个植物双元表达载体：正义的pB112-13和反义的pB112-6。用叶盘法转化烟草。DNA点杂交和Southern杂交筛选出转基因植株。抗寒性测定表明：当植株置于6'C40小时，转基因植株和对照的相对电导率比较一致，无明显改变;而在88小时，pB121-6转化植株和对照的相对电导率明显升高，以pB1121-6植株升高更多，但pB1121-13转化植株的相对电导率始终保持在较低水平。短暂冰冻处理（-20'C，4O分钟）后置室温下4天，pBl121-6转化烟草总叶绿素含量损失最多，对照次之，而pB1121-13转化烟草中多数植株总叶绿素损伤量都低于其他两种烟草。从这两个抗寒性测定实验，可判定pBl l 2 l—I 3烟株最抗寒，对照次之，而pB1121-6烟株最不抗寒。 由于pB1121-13是增强转基因烟草中SAD活性，而pB1121-6是削弱SAD基因的表达，因此．本研究首次证明通过SAD的基因工程可以改变植物的抗寒性。

细胞分裂素的测定及其转基因植物的基因表达和调控的研究

细胞分裂素是一类重要的植物激素，它参与调节许多植物的生命活动过程。本文从几个方面研究了细胞分裂素的作用。 在细胞分裂素的活性测定中，通过改进尾穗蔸苋红素合成法建立了一种简便、准确的生物试法，同时还建立了根据物理化学和免疫学原理而测定细胞分裂素的HPLC和ELISA方法，使得细胞分裂素的定量更加准确。经过对上述三种方法的相互验证实验表明，同时采用二种方法可以保证细胞分裂素分析的准确和可靠。 细胞分裂素可以促进黄瓜子叶的扩张。利用离体黄瓜子叶，分析BA诱发其扩张与子叶内源细胞分裂素之间的关系，实验证明，BA能促进玉米素及其核苷的迅速积累，进而诱发子叶的扩张。上述结果还表明，黄瓜子叶可能具有合成细胞分裂素的能力。 荸荠球茎是一种贮藏器官，但实验测定发现其中含有细胞分裂素的生理活性形式——异戊烯基腺嘌呤核苷(iPA)，而且合成它的前体腺嘌呤的含量也十分丰富，考虑到球茎与种子的类似之处，推测它可能做为合成细胞分裂素的一个源，而且其合成途径可能有别于植物其它组织。 农杆菌中的异戊烯基转移酶(ipt)基因是负责细胞分裂素生物合成的关键基因。将ipt基因克隆后对其启动子进行了改造，分别构建了如下三种基因：(1) ipt启动子+ipt编码区和3，区(ipt)，(2)磷酸核酮糖羧化酶小亚基启动子SSU 301+ipt编码区和3，区(SSU -ipt)，(3)豌豆种子特异性启动子viciln+ipt编码区和3，区(vic-ipt)。上述三种基因经农杆菌介导转化烟草，获得了16株再生植株，经Southern杂交证明其中15株的基因组上含有正常整合的ipt基因。Northern杂交表明有13株转基因烟草中的ipt基因能转录出大小正常的ipt mRNA并促进了细胞分裂素的生物合成。 实验表明，转基因烟草中ipt基因的表达受到多种因素的调控。首先启动子决定了ipt基因的表达模式，SSU -ipt基因的表达受光的诱导，黑暗中这种基因的转录完全停止，而vic-ipt基因的表达是种子特异性的，它不在烟草营养生长器官如根、茎、叶和愈伤组织中表达。第二，生长素能降低ipt基因的表达活性。第三，在整体植物的根中，存在某些反式因子，能够控制ipt基因的过量表达，这其中可能涉及到细胞内的蛋白因子、基因的甲基化作用及细胞分裂素的反馈调节等。 vic-ipt基因在烟草种子中的特异性表达导致种子内形成了一个细胞分裂素合成的源(source)。对种子中营养物质积累的研究表明，ipt基因的表达促进了种子干物质的积累，其中作用最明显的是增加种子内蛋白质的合成。转入vic-ipt基因后的烟草种子其萌发率没有显著变化，但幼苗的生长速率明显加快，这表明细胞分裂素能调节植株的生长。 通过Northern杂交检测转基因烟草中基因表达的调控，实验证明，ipt基因的表达明显抑制一组植物病理相关蛋白(PR)基因的转录活性，这组基因编码：几丁质酶，β-1，3一葡萄糖苷酶，伸展蛋白和渗调蛋白。对这些调控作用的生理学意义还有待进一步探索。 上述结果表明，在高等植物中，除了传统上认为根是合成细胞分裂素的部位之外，其它组织和器官也具有合成细胞分裂素的能力，其中合成能力最强的是一些离体组织和贮藏器官。农杆菌中的细胞分裂素生物合成基因(ipt)能够在高等植物的基因组中正常的整合和表达，并受到植物体内生理、发育等多种因素的调控，而与整体植物的正常生理过程协调一致。ipt基因的表达还能够调节植物体的生长和发育，包括种子发育时营养物质的积累、幼苗的生长和某些相关基因的表达。对上述问题的深入研究,必将促进细胞分裂素及其相关生理学和发育学研究的进展。