转基因不结球白菜实验地中外源基因流动及在不向作物中的自然渗入

首次报道了转基因不结球白菜实验地中 ,以除草剂抗性基因bar为标记基因的外源基因流动以及在芸薹属不同栽培作物中的基因渗入情况 .结果表明 ,外源基因能随花粉的散布流动 ,距离转基因花粉源越远 ,流动频率越低 .距离转基因白菜 0～ 3m处为花粉的主要流动区域 ,其频率占总流动率的 5 0 %左右 .2 0m隔离带后基因的流动频率仅为10m隔离带后的 1/2 0 .自然条件下 ,白菜中的外源基因在芸薹属同基因组作物中具有较高的渗入频率 ;在基因组具部分同源性的甘蓝型油菜中具一定的渗入频率 ;不能渗入甘蓝基因组中 .基因的渗入频率与作物间的亲缘性、隔离距离等多因素相关 .探讨了基因流动频率与基因渗入频率的差异 ,提出在有显性标记基因及花粉的供体、受体完全亲合的情况下 ,可以用基因的渗入频率来反应田间的基因流动情况 .图 4表 4参 12     

表达马铃薯Y病毒外壳蛋白基因的转基因烟草抗病机制

根据已报道的马铃薯Y病毒坏死株系(PVY^N)核苷酸序列，克隆了PVY^N外壳蛋白(CP)基因，通过根癌农杆菌(Agrobacterium tumefaciens)LBA4404介导的方法，转化烟草NC89，获得了38株PCR呈阳性的转基因植株．攻毒试验发现，转基因植株对PVY^N的抗性水平存在着差异，其中有4株表现为高度抗病性．Southern blot表明，PVY^N CP基因已经整合到烟草染色体中，转基因的拷贝数与植株的抗病性成正相关．Northern blot表明，PVY^N CP基因在RNA水平上得到了表达，而且PVY^N CP RNA在细胞中的积累量与转基因植株的抗病性呈明显的负相关．Western blot表明，高度抗病植株未检测到转基因CP蛋白质，而抗病和感病植株都检测到了PVY^N CP蛋白，且不同抗性的转基因植物中转基因蛋白质的积累有一定的差异．实验结果表明，表达PVY^N CP基因的转基因烟草其抗病机制类似于转录后的基因沉默，即抗病件可能是RNA介导的．     

细胞分裂素基因（T－cyt）在转基因烟草中的表达及其对生长发育的影响

将编码细胞分裂素合成关键酶──异戊烯基转移酶的Ｔ－ｃｙｔ基因分别置于ＣａＭＶ３５Ｓ启动子，ｒｂｃＳ启动子和Ｔ－ｃｙｔ基因自身启动子的调控下，构建成不同的嵌合质粒用于转化烟草，通过ＰＣＲ检测，Ｓｏｕｔｈｅｒｎ杂交和ＮＰＴⅡ的酶活检测对获得的转基因烟草进行了鉴定．Ｎｏｒｔｈｅｒｎ分析表明：ＣａＭＶ３５Ｓ启动子驱动下的Ｔ－ｃｙｔ基因在转基因烟草的根、茎、叶中均有ｍＲＮＡ的积累；ｒｂｃＳ启动子指导下Ｔ－ｃｙｔ基因在叶中转录较强，茎中次之，在根中几乎未表达．以根据抗原决定簇进行人工合成的复合抗原免疫家兔得到异戊烯基转移酶抗体，ＥＬＩＳＡ结果表明转基因烟草根中异戊烯基转移酶含量较高．Ｔ－ｃｙｔ基因的表达对转基因烟草的生长发育有明显影响：其叶绿素ａ、ｂ含量明显增加，叶衰老迟缓；顶端优势受到抑制，侧芽生长旺盛；与对照相比，其根系不发达．     

拟南芥VTE1过量表达可以增加维生素E含量和提高烟草植株耐盐行

维生素E在动物细胞内具有抗氧化等重要作用，但在植物体内的功能却鲜为人知．本实验利用CaMV 35S启动子与来源于拟南芥的编码生育酚环化酶（TC）的cDNA（VTE1）构建的嵌合表达载体，以根癌农杆菌介导的叶盘法转化烟草W38．实验结果表明，具有卡那霉素抗性的再生植株经RT-PCR检测，得到了与阳性对照一致的495bp的目标片段；转基因植株的VE含量比对照植株高2倍左右，个别株系高达11倍．实验还发现，在耐盐性实验中转基因植株对盐的抗性明显高于野生型烟草；同时，在不同盐浓度（150、250mmoL／L）胁迫下转基因植株VE含量比未转化植株增加了1．3～1．8倍，首次证明VTE1与植物耐盐性之间的关系．     

转基因棉花基因花粉散布频率及距离的研究

选用从国外引进的和我国自己培育的Ｂｔ基因抗虫棉和转ｔｆｄ Ａ基因抗除草剂棉花，研究了外源Ｂｔ基因和ｔｆｄ Ａ基因向周围环境遗传漂流的频率和距离。结果表明：无论是我国自行培育的转基因棉花，还是从国外引进的转基因棉花；无论是转Ｂｔ基因棉花，还是转ｔｆｄ Ａ基因棉花，导入到棉株体内的外源基因均可向周围环境漂流，其最高漂流频率为１０．４８％，最远漂流距离可达５０ｍ。防止外源基因向周围环境扩散的最有效方法是     

绿萍LmPDS基因RNAi载体的构建及遗传转化

PDS基因编码控制类胡萝卜素生物合成过程中的关键酶,其被沉默或抑制性表达会使叶片组织出现斑驳、黄化、白化的现象.构建绿萍(Lemna minor)LmPDS基因RNAi载体及完成遗传转化研究,对于实现RNAi技术在绿萍中的应用具有重要意义.选取595 bp LmPDS基因片段为干涉片段,利用中间载体pUCCRNAi将克隆的LmPDS干涉片段正反向插入植物表达载体pCAMBIA2300-35S-OCST中.将成功构建的RNAi载体通过农杆菌介导法转入绿萍愈伤,经愈伤抗性筛选、植株再生、植株扩培、标记基因NPTII检测等过程,获得植株70株,其中4株为转基因阳性植株.再经荧光定量PCR分析、番茄红素含量检测、表型观察转基因植株.结果显示,转基因阳性植株LmPDS相对表达量明显下降,为对照的28.8%-40.5%,其番茄红素含量也明显下降,且叶片出现了明显的白化表型.本研究成功构建绿萍LmPDS基因RNAi载体,实现了RNAi在绿萍中的应用,结果可为进一步的绿萍基因功能研究提供材料和方法.(图11表1参30)     

铁皮石斛外源lea3基因的转化及耐盐性分析

利用基因枪法将来源于大麦(Hordeum vulgare L.)的抗旱耐盐基因lea3导入铁皮石斛(Dendrobium candidum Wall.ex Lindl)的类原球茎(PLBs)中,经膦丝菌素类除草剂PPT筛选2次获得抗性PLBs,于无选择压下分化获得不定芽,再经PPT筛选和生根壮苗培养获得转化植株.对转化植株进行除草剂PPT叶片涂抹检测和lea3基因的PCR检测,表明lea3基因已整合到6个株系7株铁皮石斛转化植株基因组中,转化频率为1.05%.与对照相比,获得的转基因植株的耐盐胁迫能力明显增强,表明遗传转化lea3基因可用于石斛抗旱耐盐新品种的选育.     

优良籼型恢复系转基因植株的获得及其遗传稳定性

报道了用基因枪转化法将雪花莲凝集素基因(gna)转移到优良籼型杂交稻恢复系蜀恢527中．PCR、PCR Southern blotting和Southern blotting等分子检测证明，外源基因以多拷贝单位点方式整合到受体基因组中并稳定遗传到转基因第三代(T2)．转基因第一代植株(TO)在株高和结实率上与相应的组培、种子实生苗植株相比，发生明显的不可遗传的变异，随着繁殖代数的增加，转基因植株恢复到与对照植株一致．Western blotting表明，目的基因在转基因植株中正确表达．蛋白活性分析显示，外源基因在转基因植株中合成的蛋白具有凝血生物活性，含量占可溶性总蛋白的0．1％左右．本文还对转基因技术在杂交稻育种中的应用进行了讨沦．图4表2参18     

基因污染与生态环境安全

论述了基因污染可能对生态环境安全造成的影响。转基因作物有可能污染天然物种的基因库；有可能破坏传统农作物品种的遗传多样性，加速作物种植品种的单一化及基因资源的流失；使抗除草剂作物的基因向野生或半野生植物漂移的可能性加大，有可能创造出“超级杂草”；带有病毒型转基因作物的基因重组，有可能创造出新的植物病毒，导致新的病害；BT转基因抗虫作物分泌的毒素，在食物链中传递，很难加以控制，对非目标生物造成危害，并最终破坏自然生态系统的平衡。文章还从不同角度分析了基因污染的严重性。     

转Osepsps抗草甘膦水稻不同生境适合度及对稻田杂草的影响北大核心CSCD

携带适合度优势基因的转基因作物如果长期存活于栽培系统之外,抗性基因可能转移到野生近缘种并带来生态问题.以转Osepsps基因水稻EP为材料,以非转基因受体"明恢86"为对照,通过田间定位试验,研究半野生生境下转Osepsps基因水稻的营养生长、繁殖能力以及稻田杂草的种类、频度和密度等指标变化,分析转Osepsps基因在半野生生境中的生态适合度以及对稻田杂草群落的影响.连续2年试验的结果显示:在常规栽培和半野生生境下,转基因稻与非转基因稻的株高、分蘖数、单株穗数、单株总粒数、单株实粒重和千粒重等指标没有显著差异;但结实率显著受环境影响,半野生生境下转基因水稻2年结实率为50.33%和17.72%,相比对照的68.82%和30.27%均显著下降(P<0.05);2012年转基因稻单株实粒数为335粒,显著少于非转基因对照的548粒(P<0.05).转基因稻与非转基因稻稻田的杂草种类均为9科9属9种;栽培稻田杂草密度和频度显著低于半野生稻田;相同生境下转基因稻与非转基因稻稻田杂草频度、密度总体上没有差异.本研究表明,与非转基因对照相比,转epsps基因稻生态适合度没有显著提高,在半野生生境下有所下降,其生态风险不高于栽培稻.     

Ecological and evolutionary mechanisms for low seed: ovule ratios: need for a pluralistic approach?

Central to the ecology and evolution of a broad range of plants is understanding why they routinely have submaximal reproduction manifested as low seed : ovule and fruit : flower ratios. We know much less about the processes responsible for low seed : ovule ratios than we do for fruit : flower ratios. Current hypotheses for low seed : ovule ratios are largely drawn from those for fruit : flower ratios, including proximate (ecological) causes of pollen limitation, resource limitation, and pollen quality, as well as the ultimate (evolutionary) hypothesis of "bet hedging" on stochastic pollination. Yet, such mechanisms operating on fruit : flower ratios at the whole-plant level may not best explain low seed : ovule ratios at the individual-flower level. We tested each of these proximate and ultimate causes for low seed : ovule ratios using the specialized pollination mutualism between senita cacti (Pachycereus schottii) and senita moths (Upiga virescens). Seed : ovule ratios were consistently low (approximately 0.61). Such excess ovule production by senita likely has a strong genetic component given the significant differences among plants in ovule number and the consistency in ovule production by plants within and among flowering seasons. Excess ovule production and low seed : ovule ratios could not be explained by pollen limitation, resource limitation, pollen quality, or bet hedging. Nevertheless, phenotypic selection analyses did show significant selection gradients for increased ovule number, suggesting that other evolutionary processes may be responsible for excess ovule production and low seed : ovule ratios. In contrast, low fruit : flower ratios at the whole-plant level were explained by an apparent equilibrium between pollen and resource limitation. Thus, mechanisms responsible for low fruit : flower ratios at the whole-plant level are not necessarily in accord with those of low seed : ovule ratios at the individual-flower level. This suggests that we may need to adopt a more pluralistic approach to seed : ovule ratios and consider alternative hypotheses, including a greater array of proximate and ultimate causes. Initial results of this study suggest that floral allometry, selection on correlated floral traits, stigma clogging with pollen grains, and style clogging with pollen tubes may provide promising avenues for understanding low seed : ovule ratios.     

不同虫压下转Bt基因水稻与非转基因水稻生态适合度差异

为了解转基因水稻的基因扩散效率和潜在生态风险,以转Bt抗虫水稻Bt63、R1、R2和非转基因常规水稻Ⅱ优838为试材,采用高、低两个不同虫害胁迫水平和转基因与非转基因水稻相间种植方式,通过观察水稻植株营养生长、结实以及对螟虫危害的抗性表现等差异,研究比较了Bt外源基因插入后对水稻植株适合度的影响.结果表明:在低虫害胁迫条件下,转Bt基因水稻在植株分蘖数、生物量鲜重等营养生长指标方面与非转基因对照品系间无明显差异,但株高、穗长、穗重等指标不及对照,且R2和Bt63与对照间差异显著;在高虫害胁迫条件下,3个转Bt基因水稻品系的分蘖数、穗长、穗重等指标明显高于对照.而不同转基因品系株高适合度效应不同,这可能与受体本身的特性相关.3种转基因水稻的单株结实粒数、千粒重与对照在两种虫害胁迫条件下均无显著性差异,Bt基因对受体植株的结实影响不明显.在高虫害胁迫条件下,3种转Bt基因水稻的抗虫能力均显著优于非转基因水稻,表明Bt基因对受体植株的抗虫性影响显著.同时,本研究结果还表明转Bt基因水稻的适合度代价较小,预示了抗虫转基因水稻外源Bt基因在一定环境条件下具有逃逸的可能,但这种风险比较小.     

农杆菌介导的抗除草剂基因转入两系杂交稻恢复系的研究

利用农杆菌水稻高效转化系统，成功地将外源ＢＡＲ基因转入实用性两系要交稻恢复系Ｒ１８７和秀水０４，经过除草剂Ｂａｓｔａ田间涂布试验，其阳性率达到９０％以上。对转基因植株同步进行的ＰＣＲ特异扩增和总ＤＮＡ Ｓｏｕｔｈｅｒｎ检测结果也证实抗Ｂａｓｔａ的植株中含有ＢＡＲ基因。讨论了应用转ＢＡＲ基因的方法提高两系杂交水稻制种纯度的条件。图２表１参３     

异质环境下入侵植物薇甘菊的适应性与繁殖特性

薇甘菊（Mikaniamicrantha H.B.Kunth）为世界上最具有入侵性和危害性的外来入侵物种之一,对其控制与管理已成为长期以来世界性难题。了解入侵植物薇甘菊在异质环境下的适应性与繁殖特性对安全有效预警、监测和防治该入侵物种具有重要意义。本文于薇甘菊盛花期,根据薇甘菊入侵生境的光照条件、群落结构和生长方式差异,在薇甘菊常见的4种入侵生境（林地、荒地、农田和和河边）选取10个样地,调查研究了不同生境条件下薇甘菊的开花结实和繁殖分配,并运用植物叶片功能性状的研究方法研究了不同生境条件下薇甘菊的比叶面积（SLA）、叶干物质比例（LMF）和茎干物质比例（SMF）的3种叶片功能性状。研究结果表明,生境条件对薇甘菊的开花结实和繁殖分配具有显著影响。其中抛荒农田（样地Ⅷ）和河流边缘（样地Ⅸ和Ⅹ）薇甘菊的总花数、种子量、生殖枝茎生物量分配和花生物量分配均显著大于其他生境,显示在有利于其生长的条件下,薇甘菊种群倾向于有性繁殖;而在不利于薇甘菊生长的低光照林地（样地Ⅰ）和种间竞争强的农田红薯地（样地Ⅵ）生境条件下,薇甘菊的总花数和种子量明显小于其他生境,但营养枝叶生物量分配显著大于其他样地,而茎生物量分配值则处于中间值,表明在不利于其生长的生境条件下,薇甘菊通过提高营养枝茎的生物量分配和叶的生物量分配来适应,其种群则更倾向于克隆繁殖。通过分析不同生境条件下薇甘菊的叶片功能性状,结果表明,在不利于薇甘菊生长的低光照林地（样地Ⅰ）和种间竞争强的农田红薯地（样地Ⅵ）生境条件下,薇甘菊的叶面积和茎干物质比例（SMF）显著小于其他样地,且彼此差异不显著;但比叶面积（SLA）和叶干物质比例（LMF）则显著大于其他样地,显示薇甘?     

Specialized bees fail to develop on non-host pollen: do plants chemically protect their pollen?

Bees require large amounts of pollen for their own reproduction. While several morphological flower traits are known to have evolved to protect plants against excessive pollen harvesting by bees, little is known on how selection to minimize pollen loss acts on the chemical composition of pollen. In this study, we traced the larval development of four solitary bee species, each specialized on a different pollen source, when reared on non-host pollen by transferring unhatched eggs of one species onto the pollen provisions of another species. Pollen diets of Asteraceae and Ranunculus (Ranunculaceae) proved to be inadequate for all bee species tested except those specialized on these plants. Further, pollen of Sinapis (Brassicaceae) and Echium (Boraginaceae) failed to support larval development in one bee species specialized on Campanula (Campanulaceae). Our results strongly suggest that pollen of these four taxonomic groups possess protective properties that hamper digestion and thus challenge the general view of pollen as an easy-to-use protein source for flower visitors.     

Nectar secondary compounds affect self-pollen transfer: implications for female and male reproduction

Pollen movement within and among plants affects inbreeding, plant fitness, and the spatial scale of genetic differentiation. Although a number of studies have assessed how plant and floral traits influence pollen movement via changes in pollinator behavior, few have explored how nectar chemical composition affects pollen transfer. As many as 55% of plants produce secondary compounds in their nectar, which is surprising given that nectar is typically thought to attract pollinators. We tested the hypothesis that nectar with secondary compounds may benefit plants by encouraging pollinators to leave plants after visiting only a few flowers, thus reducing self-pollen transfer. We used Gelsemium sempervirens, a plant whose nectar contains the alkaloid gelsemine, which has been shown to be a deterrent to foraging bee pollinators. We found that high nectar alkaloids reduced the total and proportion of self-pollen received by one-half and one-third, respectively. However, nectar alkaloids did not affect female reproduction when we removed the potential for self-pollination (by emasculating all flowers on plants). We then tested the assumption that self-pollen in combination with outcrossed pollen depresses seed set. We found that plants were weakly self-compatible, but self-pollen with outcrossed pollen did not reduce seed set relative to solely outcrossed flowers. Finally, an exponential model of pollen carryover suggests that high nectar alkaloids could benefit plants via increased pollen export (an estimate of male function), but only when pollinators were efficient and abundant and plants had large floral displays. Results suggest that high nectar alkaloids may benefit plants via increased pollen export under a restricted set of ecological conditions, but in general, the costs of high nectar alkaloids in reducing pollination balanced or outweighed the benefits of reducing self-pollen transfer for estimates of female and male reproduction.     

Foliar damage to parental plants interacts to influence mating success of Ipomoea purpurea

Studies of how herbivory affects plant fitness often determine whether damage to one parent alters reproductive output (i.e., seed set or paternity) but ignore the possibility that the outcome may be different if both parents were damaged (i.e., the presence of maternal x paternal damage interactions). Using inbred lines of the common morning glory, Ipomoea purpurea, I conducted a series of greenhouse experiments to test whether foliar damage from a generalist insect herbivore, Trichoplusia ni, alters male and female fitness components when neither parent, one parent, or both I. purpurea parents had been damaged. In a single-donor experiment, flowers on both damaged and undamaged maternal plants received pollen from either damaged or undamaged paternal plants. I. purpurea flowers were more likely to be aborted when they received pollen from damaged paternal plants, or when maternal plants were both damaged and grown under low-resource conditions. Foliar damaged plants also produced less seed and pollen than undamaged plants, although seed mass and pollen viability were not affected by damage. In a multiple-donor experiment, flowers on damaged and undamaged maternal plants simultaneously received pollen from damaged and undamaged paternal plants, and F1 seeds were analyzed for paternity. Damaged paternal plants had reduced siring success compared to undamaged paternal plants, and this discrepancy was most pronounced when competition occurred on damaged maternal plants. Thus, damaged maternal plants were more "selective" than undamaged maternal plants. Although previous studies have demonstrated that herbivory can alter fruit and seed production and paternity patterns, this is the first study to show that the magnitude of herbivore damage experienced by both parents can interact to influence maternal and paternal mating success.     

Heterologous overexpression of Populus euphratica BAK1;1 gene enhanced resistance to Pst DC3000 in tobacco北大核心CSCD

BRI1-ASSOCIATED RECEPTORKINASE1(BAK1), a leucine-rich repeat (LRR) receptor protein kinase, plays a significant role in brassinosteroid (BR) signaling. Furthermore, it combines with other LRR-RLKs protein to initiate immune response in plants. The objective of this study was to (1) investigate the function of the Populus euphratica BAK1;1 gene in the resistance of transgenic tobacco to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) and (2) discuss the regulation pathway of PeBAK1;1 involved in the resistance to plant pathogen. We cloned the cDNA sequence of the P. euphratica PeBAK1;1 gene, constructed the pBI121-35S::PeBAK1;1 over-expression vector, and then transformed it into wild-type tobacco by Agrobacterium-mediated transformation to obtain PeBA K 1;1-overexpressing transgenic tobacco plants. The bioinformatic analysis showed that the PeBAK1;1 protein contained all the structural features of the plant SERK family. The phylogenetic tree showed that PeBAK1;1 has the highest sequence homology with PtBAK1. The gene expression profile results indicated that the expression of PeBAK1;1 in the root was higher than that in the leaf and stem. The wild-type tobacco plants showed an obvious susceptibility to Pst DC3000, whereas the transgenic plants exhibited enhanced resistance to Pst DC3000. Compared with that of the wild-type (WT), the real-time PCR and quantitative real-time PCR analysis revealed that the expression of pathogenesis-related genes (including PR1, PR3, PR4, and PR5), BAK1-interacting receptor kinase 1 gene, and BONZAI1 gene was upregulated in 35S::PeBAK1;1 transgenic tobacco plants. In conclusion, the PeBAK1;1 gene plays a positive regulatory role in 35S::PeBAK1;1 transgenic tobacco against Pst DC3000, which can enhance the resistance of plants to pathogen. © 2018 Science Press. All rights reserved.     

Nonadditive effects of floral display and spur length on reproductive success in a deceptive orchid

Pollinators may mediate selection on traits affecting pollinator attraction and effectiveness, and while nonadditive effects of traits influencing the two components of pollination success are expected when seed production is pollen limited, they have been little studied. In a factorial design, we manipulated one putative attraction trait (number of flowers) and one putative efficiency trait (spur length) previously shown to be subject to pollinator-mediated selection in the deceptive orchid Dactylorhiza lapponica. Removal of half of the flowers reduced pollen removal, proportion of flowers receiving pollen, fruit set, and fruit mass compared to unmanipulated plants, while spur-tip removal increased fruit set and fruit mass but did not affect pollen removal or proportion of flowers receiving pollen. The effect of spur-tip removal on fruit mass was stronger among plants with intact number of flowers compared to plants with experimentally reduced number of flowers. The results demonstrate that number of flowers and spur length are direct targets of selection and may affect female fitness nonadditively. More generally, they show that the adaptive value of a given trait can depend on floral context and illustrate how experimental approaches can advance our understanding of the evolution of trait combinations.     

同源四倍体水稻亲本材料的结实研究

研究了2份二倍体水稻和29份同源四倍体水稻(2n=4x=48)亲本(其中4份低代材料、25份高代材料)在2004年和2005年的结实情况.与二倍体对照相比,同源四倍体材料的花粉育性、结实率均不同程度下降,尤其低代材料的花粉育性(59.6%～65.3%)和结实率(26.8%～33.5%)大幅度降低;但经过选择改良,大多数材料的花粉育性和结实率均不同程度提高.F检验表明,同源四倍体不同品种间各性状均差异显著,说明同源四倍体材料具有很大的改良潜力.从1996年到2005年对部分同源四倍体水稻进行了连续选择改良,并对其结实率进行T检验,结果表明经过9a的选择改良,同源四倍体水稻结实率显著提高.本文还对同源四倍体水稻各性状间的相关性进行了分析,结果表明,结实率与花粉育性、穗着粒数、穗实粒数极显著相关,理论产量与花粉育性、有效穗数、穗着粒数、穗实粒数、结实率及千粒重极显著相关.表3图1参18