花生PEPC基因反义表达载体构建及对花生的遗传转化

磷酸烯醇式丙酮酸羧化酶(PEPC)是控制花生蛋白质/油脂含量比例的一个关键酶.本研究利用PCR方法扩增出了PEPC基因片段,并将其克隆到pMD18-T Simple载体,序列分析表明克隆片段的长度为728bp.将该PEPC基因片段反向插入pCAMBIA1301-35S双元表达载体,构建了带PEPC反义基因的双元载体并进行花生的转化,目前已获得转基因植株,收获T1代种子.     

花生AhFUSCA3基因的原核表达及在非生物胁迫下的表达分析

根据本实验室已获得的花生AhFUSCA3基因(NCBI登录号JX420284)序列,设计特异引物,构建原核表达载体,进行了重组蛋白表达分析,获得了分子量为42KD左右的目的蛋白条带;利用荧光定量PCR(Quantitative Real-time RT-PCR)方法,检测了AhFUSCA3基因在低温、高盐和干旱胁迫条件下的表达情况。结果显示,AhFUSCA3基因在低温和高盐处理的花生叶片中表达量明显上调,但在干旱处理的叶片中表达量则有明显下降。以上结果表明AhFUSCA3基因可能参与了花生对低温、高盐和干旱的抗性调控。     

大豆GmAYI基因的克隆及初步功能预测

根据拟南芥At1G74730基因的保守序列设计引物,利用RT-PCR技术在大豆中克隆得到GmAYI基因。对GmAYI基因的结构域和启动子元件进行分析,结果表明:大豆GmAYI基因位于4号染色体上,含跨膜结构域,属于膜蛋白。该基因启动子区域含有激素相关应答元件和胚乳表达所必须的顺式作用元件。推测该基因可能参与调控大豆生长发育及外界环境胁迫应答等过程。进而对14个大豆品种中GmAYI基因的转录水平进行了定量分析,对GmAYI基因在14个大豆品种中的转录水平与单株粒数、单株粒重、百粒重、株高等产量相关性状进行相关性分析,结果表明:GmAYI基因的表达量与百粒重呈显著负相关,推测GmAYI可能是参与大豆产量相关性状调控的基因。     

一种适用于油菜、大豆、花生、芝麻四种油料作物种子的RNA提取方法

本研究在商业化RNA提取试剂盒的基础上,通过以下3方面的改进使之成功应用于油菜、花生、芝麻和大豆的各个时期的种子RNA提取。1）增加抗氧化剂巯基乙醇和PVP;2）根据不同作物的油脂含量调整氯仿的比例;3）改进RNA沉淀方法。与常规的CTAB、TRIZOL及文献报道的RNA提取方法比较结果显示,本方法提取的RNA在完整性、得率及质量上都优于上述方法,并成功应用于后期荧光定量表达分析。FAD2基因在四种作物种子不同发育时期的差异表达结果进一步证明了此方法的有效性。     

大豆GmNCED1基因的克隆及表达模式分析

本研究以抗逆性强的大豆旱碱一号为材料,首次从中克隆了编码9-顺式-环氧类胡萝卜素双加氧酶（9-cis-epoxycarotenoid dioxygenase,NCED）的GmNCED1基因全长cDNA片段,该基因编码区含有1 836个核苷酸,编码611个氨基酸。通过同源性比对分析发现GmNCED1与花生、菜豆等双子叶豆科植物NCED的同源性高达84%以上。同时,利用荧光定量PCR分析发现高盐、低温、干旱、外源ABA以及NAA处理均可以诱导该基因在大豆叶片及根中表达。本研究初步揭示GmNCED1基因在植物逆境胁迫中的作用,为基因工程育种提供优质的候选基因。     

油菜种子发育过程中总RNA量及线粒体nad6基因转录水平的变化

以油菜为材料,在摸索出合适的种子总ＲＮＡ提取技术基础上,研究其发育过程中总ＲＮＡ量的变化,然后结合定量斑点杂交技术,研究种子发育过程中线粒体ｎａｄ６基因转录水平的变化规律。结果表明,采用的总ＲＮＡ提取方法具有操作简单、纯度高、降解少、电泳图谱理想等优点。角果宽度在２．８０ｍｍ以下的种子总ＲＮＡ量随发育而平稳地增加,角果宽度在２．８０ｍｍ以上的种子总ＲＮＡ量随发育而迅速降低。除３．８５ｍｍ角果外,发育过程中的种子总ＲＮＡ量均高于叶片的。线粒体ｎａｄ６基因的ＲＮＡ定量斑点杂交结果显示叶片ｎａｄ６基因的转录量很低,而发育中各个阶段的种子ｎａｄ６基因转录量很高,并有随发育加强的趋势。     

Real-time PCR检测大豆CONSTANS基因在不同光照条件下的表达

采用实时荧光定量PCR方法检测了不同感光大豆品种的CO(CONSTANS) 基因在不同光照条件下的mRNA水平变化.结果表明:早熟品种东农49和晚熟品种东农42各自在短日照(8h/16h 光/暗)下会比长日照(16 h/8 h 光/暗)下表达有所增强,同时东农49中CO基因的表达要高于东农42,它们的表达峰值均出现在光暗交界处.东农42由长日照或短日照移入完全光或完全暗条件时,暗下CO基因的表达量剧增,并且大致维持之前长日照和短日照的表达规律.CO基因的表达有很强的昼夜节律性,暗下利于其表达,并且在不同感光品种中表达有所不同.     

山羊GDF-8基因的表达差异与发育性变化分析

为探索GDF-8基因在山羊不同生长阶段的肝脏、心脏、肾脏、脂肪和骨骼肌中mRNA表达发育性变换规律,采用SYBR Green实时荧光定量PCR技术对2月龄、3月龄和4月龄的海南本地山羊进行检测。结果显示,GDF-8基因在3月龄的心脏组织中mRNA表达量最高,其次是肾脏和骨骼肌;在2月龄和4月龄时5种组织中GDF-8基因表达量较低,尤其是在心脏组织中几乎不表达;在肝脏和脂肪中的表达依次为4月龄＞2月龄＞3月龄,且各月龄之间表达差异极显著(p＜0.01);心脏、肾脏和骨骼肌的表达依次为3月龄＞4月龄＞2月龄,其中,肾脏在不同月龄段表达差异极显著(p＜0.01),而心脏和骨骼肌在2月龄和4月龄之间表达差异不显著(p＞0.05)。     

青稞Hva1基因的表达模式及表达载体的构建

为了解青稞种子胚胎成熟晚期丰富蛋白(LEA)基因Hva1与青稞抗旱性的关系,以抗旱性强的旱地紫青稞和抗旱性弱的大麻青稞为材料,研究了不同浓度PEG胁迫下Hva1基因在两个青稞品种中的表达模式。结果表明,Hva1基因在抗旱性不同的两个青稞品种中的表达均呈单峰模式,且抗旱性强的旱地紫青稞表达峰值和对应的PEG胁迫浓度均高于抗旱性弱的大麻青稞;PEG胁迫浓度大于13.18%时,旱地紫青稞的Hva1基因表达量高于大麻青稞。可见,Hva1基因的表达在青稞的抗旱中起到重要的作用,为此我们成功构建了青稞Hva1基因的过量表达载体,期望为抗逆性基因在青稞中的应用和抗旱新品种的选育奠定基础。     

甘蓝型油菜三种种子储藏蛋白和丙酮酸羧化酶基因片段的克隆及hpRNA表达载体的构建

利用PCR从甘蓝型油菜(B rassica napusL. )华双4号基因组DNA中扩增出种子储藏蛋白cruciferin、nap2 in、oleosin和丙酮酸羧化酶( PEPC) 基因片段,再以扩增出的片段为模板设计引物从一端扩增出4个相应的小片 段,然后将同一基因的大小两个片段反向连接,插入到种子特异表达载体2300 - nap多克隆位点的nap in启动子和 nos终止子之间,构建成可以在油菜种子中转录表达发夹RNA (Hairp in RNA, hpRNA)结构的植物表达载体。     

Genome-wide identification of TPS genes in sesame and analysis of their expression in response to abiotic stresses

Trehalose and its precursor, trehalose-6-phosphate, play critical roles in plant metabolism and response to abiotic stresses. Trehalose-6-phosphate synthase(TPS) is a key enzyme in the trehalose synthesis pathway. Hence this study identified TPS genes in sesame(Si TPSs) and examined their expression patterns under various abiotic stresses. Totally, ten Si TPSs were identified and comprehensively characterized. Si TPSs were found to be unevenly distributed on five out of 13 sesame chromosomes and...     

芝麻主要过敏原油质蛋白的基因克隆和原核表达

为克隆黑、白芝麻主要过敏原油质蛋白Oleosins的基因并在原核细胞中对其进行表达,本研究分别提取黑、白芝麻的总RNA,逆转录合成cDNA,根据序列设计简并引物,扩增芝麻主要过敏原油质蛋白Oleosins基因的完整开放阅读框,并与pET-28a载体连接,测序鉴定后转化大肠杆菌E.coliBL21(DE3)及用IPTG诱导表达。结果表明克隆获得黑、白芝麻主要过敏原油质蛋白Oleosins的全长基因约为438bp的开放阅读框(包括终止密码子),编码145个氨基酸。所编码的蛋白相对分子质量约为15.5kDa,为小分子量蛋白,等电点为5.18,与理论值相符。序列同源性分析发现其与数据库中已知的黑、白芝麻油质蛋白Oleosins基因同源性很高(GenBank黑、白芝麻油质蛋白基因登录号分别为EU999158和EU999159),并在DE3中高效表达。     

芝麻赤霉素合成相关基因鉴定与表达分析

赤霉素的生物合成是多种酶促反应的过程,与植物植株发育、逆境响应密切相关。为分析芝麻基因组中赤霉素合成相关基因的分布、结构和活性特征,本研究以芝麻基因组序列和不同组织转录组为对象,通过生物信息学方法,从中共鉴定出32个赤霉素合成相关基因,分属7个不同的基因家族,分布在除5号染色体外芝麻所有的染色体上。不同基因家族蛋白质肽链长度存在较大差异,其中CPS家族的蛋白质序列最长,平均包含776个氨基酸残基;KAO家族蛋白序列长度的变异最大,在401-834个氨基酸之间。所有芝麻赤霉素合成相关基因编码的蛋白质预测为亲水性蛋白。与拟南芥、水稻、大豆、葡萄、高粱、番茄等物种相比,芝麻中赤霉素合成相关基因数目处于中等水平,但CPS家族中芝麻的基因数目明显多于其他物种,进化分析表明,在分析的7个物种中,芝麻与番茄具有较近的进化关系。在不同组织中,7个相关基因家族表达模式不同。KS家族基因在种子、茎尖和叶片中表达相对较高;CPS家族基因在心皮和种子中活性较强,但在茎尖和叶片中几乎没有表达;KAO和KO家族基因在不同组织中整体上具有相对较高的活性,而KS、GA3ox家族的基因在不同组织中整体表达量较低。就不同组织而言,种子中的GA合成相关基因活性整体较高;而在根尖、茎尖、叶片等与芝麻植株形态建成密切相关的3个组织中,这些基因表达以根尖较突出、其次为茎尖,在叶片中的活性整体较低。本研究为解析芝麻赤霉素合成相关基因的结构特征及其在不同组织中的作用特点提供了基因信息和理论参考。      

油料作物内标准基因研究与应用

内标准基因（Endogenous Reference Gene）是指具有植物物种专一性且拷贝数恒定、不显示等位基因变化的保守DNA序列。可用于对基因组中某一目的基因进行定量分析和验证PCR反应体系中是否存在抑制物质。基于其物种特异性,内标准基因还能用于食品搀假使杂的判定。油料作物涉及我们日常生活的方方面面,油料制品的鉴别和转基因油料作物的检测急需使用内标准基因。目前,油料作物内标准基因的研究与应用取得了长足进展,但是还没有对它们进行综合比较和分析的报道。本文根据国内外有关油料作物内标准基因的研究,阐述了各物种已开发的内标准基因。并对这些基因从引物位置、扩增片段的大小、拷贝数等特征进行比较和分析。本文综述了油料作物内源特异参照基因的研究概貌和存在问题,有助于对油料作物及其他作物内源特异参照基因的进一步开发和深入研究。     

Molybdenum-enriched soybean seeds enhance N accumulation, seed yield, and seed protein content in Brazil

High soybean yields require large amounts of nitrogen (N), which can be obtained mainly from symbiotic N₂ fixation. However, the efficiency of this biological process can be limited by micronutrient deficiencies, especially of molybdenum (Mo). In Brazil, soybean generally responds positively to fertilization with Mo in soils of low fertility and in fertile soils depleted of Mo due to long-term cropping. The micronutrient can be supplied by seed treatment, however toxicity of Mo sources to Bradyrhizobium strains applied to seed as inoculant has been observed, resulting in bacterial death and reductions in nodulation, N₂ fixation and grain yield. Therefore, use of seeds enriched in Mo could be a viable alternative to exterior seed treatment, allowing elite inoculant strains of Bradyrhizobium to sustain high rates of biological N₂ fixation. We demonstrated the feasibility of producing Mo-rich seeds of several soybean cultivars, by means of two foliar sprays of 400 g Mo ha⁻¹ each, between the R3 and R5 stages, with a minimum interval of 10 days between sprays. As a result of this method, considerable increases in seed-Mo content were obtained, of as much as 3000%, in comparison to seeds obtained from plants which received no Mo. In field experiments performed in soils with low N content and without any N-fertilizer supply, inoculation of Mo-rich seeds produced plants with increased N and Mo contents in the grain and higher yields of total N and of grain. In most cases, Mo-rich soybean seeds did not require any further application of Mo-fertilizer.     

Genomic analysis of sugar transporter genes in peanut (Arachis hypogaea): Characteristic,evolution and expression profiles during development and stress

Peanut(Arachis hypogaea L.) is an economically significant crop with aerial cleistogamous flowers and subterranean geocarpic fruit(pods). The formation of peanut pod requires movement of the embryo from air to ground and then development in the soil, which is a complex biological process involving transport and accumulation of sugars. Sugar transport proteins(STP) mediate the transport of monosaccharides in various physiological processes, including fertilization, ovary formation, and seed devel...     

Changes in fatty acids composition during seed growth and physicochemical characteristics of oil extracted from four safflower cultivars

Fatty acid contents at different stages ofmaturity and physicochemical characteristics of oilextracted from mature seeds of four safflowercultivars (S208, S400, S541, and S303) were studied. Results indicated that for all cultivars bothsaturated and unsaturated fatty acid contentsfluctuated with seed growth and development. Palmiticacid content decreased up to day 20 after which itstarted to increase for the S400, S541, and S303cultivars, while for S208 it progressively decreased.Stearic acid fluctuated with seed growth anddevelopment. Oleic acid showed slight changes withseed growth and development, while linoleic aciddecreased with seed growth and development for theS400, S541, and S303 cultivars while for S208 afterday 30, it started to increase significantly. For allcultivars, glyceride contents varied among thecultivars with maximum values of 97.70, 2.80 and0.20 for tri-, di- and monoglycerides, respectively. Physicochemical investigation of mature seed oilsshowed that the color, density, refractive index, free fatty acids, peroxide value, saponificationvalue and unsaponifiable matter were similar for allcultivars while viscosity, iodine value, and acetoneinsoluble matter varied among the cultivars.     

Morphological and oil content variation in seeds of Azadirachta indica A. Juss. (Neem) from northern and western provenances of India

Seed morphology (seed length and 20 seed weight) and oil content was studied in Azadirachta indica A. Juss. (Neem) of five provenances of northern and western India. Trees with wide ranges of girths were considered for study. Maximum average oil content was observed in trees from Hisar provenance. Seed oil content in most of the provenances was not consistently and significantly correlated with morphological parameters of seeds. Age of the tree had no significant effect on the oil yield.     

油料作物种子形成及老化过程中的生理生化变化

油料作物种子形成过程首先是积累糖、淀粉,接着大量形成脂肪、蛋白质等,从糖类的积累转入脂肪积累伴随着EMP和HMP两条途径;主要物质的合成和积累速率与各自相关调控酶活性变化基本一致。种子老化能导致种子活力下降,影响发芽出苗、植株生长发育和产量、品质。抗氧化相关酶活力下降,质膜过氧化作用及其产物增加,导致膜结构和功能受损,内溶物质外渗,有害物质积累,代谢作用紊乱,是种子老化、活力丧失的生理因素。本文提出了油料作物种子形成及老化过程生理生化研究中存在的不足及今后的重点。