Silencing of γ-gliadins by RNA interference (RNAi) in bread wheat

RNA silencing is a sequence-specific RNA degradation system that is conserved in a wide range of organisms. The elucidation of the mechanism of RNA silencing has stimulated its use as a reverse genetics tool, because RNA silencing strongly down-regulates the expression of the target gene in a sequence-specific manner. The major protein fraction of wheat grain is gluten which is largely responsible for the functional properties of dough. Gliadins contribute mainly to the extensibility and viscosity of gluten and dough, with the polymeric glutenins being responsible for elasticity. The aim of this work was therefore to silence the expression of specific γ-gliadins by RNA interference, to demonstrate the feasibility of systematically silencing specific groups of gluten proteins. The sequence of a γ-gliadin gene was used to construct the pghp8.1 plasmid. The hpRNA silencing fragment was designed on the basis of 169 base pairs (bp) in sense and antisense orientation with the sequence of the Ubi1 intron as spacer region between the repeats. Two lines of bread wheat were transformed by particle bombardment. Gliadins were extracted from 30 mg of flour, separated by acid-PAGE and determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Seven transgenic lines were obtained and all of them showed reduced levels of γ-gliadins. All seven transgenic plants were fully fertile and their grain morphology and seed weight were comparable to the control lines. MALDI-TOF MS showed that six peaks, present in the untransformed line, were missing in transgenic lines of the BW208 genotype whereas three peaks were missing in the BW2003 genotypes. The proportion of γ-gliadins was reduced, by about 55–80% in the BW208 lines and by about 33–43% in the BW2003 lines. The ELISA assay based on the R5 antibody showed reductions in total gliadins (μg/mg flour) in three of the BW208 lines and in one BW2003 line, but an increase in one BW208 line (C613).     

狗尾草U6启动子的克隆及功能鉴定

狗尾草是重要的模式植物,是研究C4光合作用的优秀模型,具有热带植物的典型特征。U6启动子主要用于构建RNAi和CRISPR表达载体,有启动干扰发夹结构的表达和基因编辑引导序列复合结构的表达的作用,该启动子具有特殊的启动位点G,能够保证转录后RNA结构的完整性。随着CRISPR 技术的发展,利用狗尾草进行基因编辑的研究日益增多和深入,目前还没有针对狗尾草U6启动子的克隆及功能鉴定。U6启动子具有种属特异性,在转基因技术中,使用转化物种本身或亲缘物种的U6启动子能取得更高的的启动效率,本研究克隆了狗尾草2个U6启动子基因,并构建不同序列长度的U6启动子序列驱动GUS基因的表达,GUS融合表达载体转化狗尾草胚性愈伤,瞬时表达验证表明,克隆出的2个狗尾草 U6启动子在狗尾草上具有很强的启动效率;并且将该载体转化狗尾草后,获得了能够稳定表达GUS基因的转基因植株;将该U6启动子用于构建狗尾草PDS基因CRISPR编辑载体,获得能够稳定表达的白化苗,说明克隆的狗尾草U6启动子能够很好的启动基因编辑载体的转录。     

HbNIN2基因在巴西橡胶树嫩皮和中脉中的RNA原位杂交分析

在橡胶树（Hevea brasiliensis）中,转化酶基因HbNIN2被证明是决定产胶细胞乳管中蔗糖代谢的关键基因,同时在叶片、树皮等多种组织中均有表达。为进一步研究HbNIN2基因的功能,利用原位杂交（in situ hybridization）技术对HbNIN2基因在橡胶树嫩皮和中脉两种组织中的表达区域与表达特点进行研究。结果显示,在橡胶树嫩皮中,HbNIN2基因主要在韧皮部中表达,而在中脉中,HbNIN2基因在除木质部外的其它部位均有表达,同时在两种组织的乳管细胞中HbNIN2基因均有明显的表达信号。比较而言,HbNIN2基因在中脉中的表达量远高于嫩皮。结合其它研究结果,推测HbNIN2基因可能参与橡胶树乳管蔗糖代谢、叶片生长发育调控等。     

RNA干扰技术在小麦中的应用研究进展

小麦是六倍体,其染色体组中存在着大量的同源基因,其育种的主要目标是获得可遗传的优良性状.RNA干扰(RNAi)是指由双链RNA介导的转录后基因沉默.该技术具有两大特点:一是可同时沉默多基因家族及多倍体中的同源基因;二是其沉默效应可在后代中稳定遗传.这使得RNAi在小麦功能基因组学和品质改良研究中的应用具有独特优势.本文对RNAi在小麦功能基因组学和品质改良研究中的应用及研究进展进行了总结,指出了目前存在的问题,并阐明了RNAi在小麦研究中的应用前景.     

Quantitative proteomic analysis of wheat grain proteins reveals differential effects of silencing of omega-5 gliadin genes in transgenic lines

Novel wheat lines with altered flour compositions can be used to decipher the roles of specific gluten proteins in flour quality. Grain proteins from transgenic wheat lines in which genes encoding the omega-5 gliadins were silenced by RNA interference (RNAi) were analyzed by quantitative 2-dimensional gel electrophoresis (2-DE). The precise effects of the genetic modifications on the proteome were assessed in four homozygous lines generated with the same RNAi construct. Two of the lines showed >80% decreases in omega-5 gliadins with only small changes in the levels of other gluten proteins. In the other two lines, omega-5 gliadins were not detectable by 2-DE. However, there were notable reductions in all omega-1,2 gliadins. Small decreases in several other gluten proteins were also detected in one of the lines. The other line showed notable decreases in three HMW-GS and an s-type LMW-GS, increases in two m-type LMW-GS and several alpha gliadins, as well as increases in both serpins and triticin. The study demonstrates that the same RNAi construct can have differential effects on the wheat grain proteome and highlights the importance of detailed proteomic analyses of transgenic grain prior to selecting lines for further assessment of flour quality and allergenic potential.     

一个八倍体小偃麦染色体组成的分子细胞学分析及品质特性鉴定

八倍体小偃麦是普通小麦遗传改良的重要种质材料,为进一步发掘和利用其优良基因,对从匈牙利科学院农业研究所引进的八倍体小偃麦BE-1的染色体组成和高分子量麦谷蛋白亚基（HMW-GS）进行了分析鉴定。细胞学观察结果表明,其体细胞染色体数目为2n=56,花粉母细胞减数分裂中期Ⅰ（PMC MI）粢色体构型2n=28Ⅱ的细胞占65．8％;分别用长穗偃麦草（Thinopyrum elongatum,D．R Dewey,2n=14,EE）、百萨薄冰草（Thinopyrum bessarabicum,A．Love,2n=14,JJ）、假鹅观草（Pseudoroegneria strigosa,A．Love,2n=14,StSt）的总基因组DNA作撂针进行原位杂交,结果发现仅在假鹅观草总基因组DNA作探针时。能够检测到BE-1 14蒂染色体明显的杂交信号,进而认为它们来源于St基因组相近的鹅观草属的物种;对八倍体小偃麦BE-1麦咎蛋白进行SDS-PAGE分析,结果表明其HMW—GS组成为1,6＋8,5＋10。红外谷物品质分析仪测试结果表明,它的蛋白质、湿面筋含量度沉淀值均超过当前优质小麦品种,可做为普通小麦品质改良的重要基因源。     

籼稻背景下抑制不同ALK等位基因表达对稻米品质的影响

【目的】稻米糊化温度是影响稻米品质的重要指标,该性状受主效基因ALK/SSII-3调控,ALK基因具有多个复等位基因,本研究旨在通过RNAi技术明确籼稻亚种中两个不同ALK等位基因的效应。【方法】以分别含有ALKc和ALKb等位基因的高糊化温度品种珍汕97B和低糊化温度品种龙特甫B为试验材料,使用RNAi技术构建ALK表达下调的转基因株系,通过对其稻米理化品质的测定来明确不同等位基因表达下调对稻米品质的影响。【结果】对不同转基因水稻目的基因的表达分析显示本研究中转基因株系的ALK基因受到了不同程度的干扰。重点分析了不同RNAi株系稻米的糊化温度,结果表明珍汕97B的RNAi转基因稻米的糊化温度极显著降低,而在低糊化温度品种龙特甫B背景中下调表达ALK基因后对糊化温度的影响较小;转基因株系与未转化亲本相比,米粉的起始糊化温度都显著降低,表现为提前糊化;在珍汕97B背景下干扰系的峰值温度与未转化亲本相比极显著降低,而在龙特甫背景下米粉的峰值温度与未转化对照相比显著降低。对不同转基因系的理化品质分析表明,ALK下调表达植株稻米的表观直链淀粉含量显著增加,下调表达ALK后会引起米粉峰值黏度和崩解值的改变。高糊化温度品种珍汕97B干扰系与未转化对照相比胶稠度呈现极显著性差异,而低糊化温度品种龙特甫干扰系的胶稠度与未转化对照相比没有差异。【结论】下调表达ALK等位基因对稻米理化品质产生显著影响,并且干扰不同等位基因的效应存在明显差异,即籼稻中的两个ALK等位基因的效应存在显著差异。     

Effects of Suppressing OsCRY1a Gene Expression on Rice Agronomic Traits

Using primers designed according to the published sequence of rice OsCRY1a gene,we obtained part of the gene fragment by PCR and constructed an RNA interference expression vector with it.To down-regulate the expression level of the gene or lead to the loss-of-function of the gene,the vector was then introduced into rice via Agrobacterium-mediated transformation.Based on the performance of the transgenic plants,the functions of the gene were analyzed and deduced.The results indicated that suppressing the expression of the gene retarded flowering for 16 d in rice with the plant height and grain length significantly increasing whereas other important agronomic traits observed remained unchanged apparently.     

Beziehungen zwischen morphologischen Merkmalen,Photosyntheserate und Ertrag bei Kartoffelgenotypen unterschiedlicher Reifegruppen. 2. Photosyntheserate und ihre Kombination mit morphologischen Merkmalen

Zusammenfassung überwiegend positive Korrelationskoeffizienten zwischen Photosyntheserate und Ertrag, aber stark schwankende Ergebnisse zwischen verschiedenen Sortimenten, weisen darauf hin, dass die Photosynthese nur als ein Faktor innerhalb eines gr?sseren Merkmalskomplexes die Produktivit?t beeinflusst. Ungünstige morphologische Merkmale, die die Strahlungsinterzeption negativ beeinflussen (geringe Staudenh?he, wenig Stengel, Lagern der Stengel), k?nnen bei (klimatisch oder durch den Bestandesaufbau bedingt) suboptimaler Beleuchtung nicht durch eine hohe potentielle Photosyntheserate kompensiert werden. Extrem ertragreiche Genotypen zeichneten sich durch eine Kombination günstiger morphologischer Merkmale mit einer hohen Photosyntheserate aus.

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Summary Previous and existing studies have found overall a positive correlation between rate of photosynthesis and yield of potato genotypes (Table 1). The large variation in the correlations of different collections is the result of the various combinations of rates of photosynthesis and morphological characters. High rates of photosynthesis cannot under sub-optimal conditions, produced by climate or canopy architecture, compensate for the negative effect of morphological characters such as low crop height and stem counts, and stem lodging on the interceptions of radiation (group 1, Table 2). Very high-yielding types have good morphological characters and high rates of photosynthesis (group 6, Table 2). An upper canopy which allows the penetration of light is better when combined with a high rate of photosynthesis than a dense one in which only the uppermost leaf layers are intensively lit (Table 2). Rates of photosynthesis under optimal conditions of isolated leaf discs, and intact field-grown plants of three genotype gave the same ranking order in each case (Table 3). There was no indication from the daily course of photosynthesis that the genotypes responded differently to environmental conditions (Fig. 1).

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Résumé Dans des travaux antérieurs ainsi que dans celui-ci, on a principalement obtenu des corrélations positives entre la photosynthèse et le rendement pour les génotypes de pommes de terre (tab. 1). Les fortes variations de caractères mesurées dans les assortiments sont expliquées par les différentes combinaisons entre la photosynthèse et les caractères morphologiques. Les paramètres morphologiques qui influencent l'interception du rayonnement de manière négative (p. ex. faible développement des fanes et tiges, verse des tiges), ne peuvent pas être compensés, lors de conditions de rayonnement suboptimales, (climat et peuplement) par un taux élevé de photosynthèse (groupe 1, tab. 2). Les types à rendement extrêmement, élevé se distinguaient par des caractères morphologiques favorables, et un taux de photosynthèse élevé (groupe 6, tab. 2). Un port des plantes permettant la pénétration de la lumière, combiné avec un taux élevé de photosynthèse semble plus favorable qu'un port compact en surface, où seules les feuilles supéreures sont exposées au rayonnement intensif (tab. 2). Des comparaisons du potentiel de photosynthèse, entre rondelles de feuilles isolées et de plantes au champ ‘in situ’ par des conditions optimales, ont révélé le même ordre de rang pour 3 génotypes (tab. 3). L'évolution journalière de la photosynthèse selon les génotypes, n'a pas montré de différence de réaction aux conditions de l'environnement selon les génotypes (fig. 1).

     

甘蓝型油菜与Brassica maurorum的异源六倍体后代及BC2细胞学分析

将甘蓝型油菜品种中油821（2n=38, AACC）与芸薹属野生资源Brassica maurorum（2n=16, MM）的三倍体杂种进行染色体加倍,得到异源六倍体（2n=54, AACCMM）。该多倍体雄性高度不育,雌性育性极低。在其花粉母细胞的减数分裂终变期,染色体平均配对构型为1.17Ⅰ+20.71Ⅱ+0.56Ⅲ+2.25Ⅳ+0.08Ⅴ+0.06Ⅵ。用甘蓝型油菜与该多倍体连续回交2次,均需借助幼胚培养才得到后代植株,BC1、BC2植株均雄性不育,雌性育性很低。BC2植株的形态特征接近甘蓝型油菜,但各植株形态有明显差异。基因组原位杂交分析表明,BC2植株含有2~5条M基因组的染色体,甘蓝型油菜染色体多配对形成二价体,附加的染色体多以单价体形式存在,或与甘蓝型油菜染色体发生联会配对。     

甘蓝型油菜Bn19070的表达与同源基因At2G19070的amiRNAi研究

基于原芯片杂交结果,油菜杂合双显性雄性核不育系差异表达基因Bn19070与拟南芥At2G19070基因高度同源,在油菜可育株雄蕊中高量表达,不育株雄蕊中表达量极低。从雄性不育基因At2G19070入手,利用amiRNAi (人工微RNA干扰)技术对其两个RNA区段分别设计靶标,转化拟南芥。在多个转基因拟南芥植株和后代中普遍观察到小孢子数量减少、成熟花粉萎缩失去活力、不结实或者少结实的不育表型。两个靶标的干扰使转基因拟南芥出现了类似的表型,基因表达抑制率都在90%以上,干扰效果好,暗示amiRNAi技术在靶标选择上具有一定的灵活性。At2G19070基因的干扰对Bn19070的功能研究有借鉴作用。     

油菜高通量根系构型定量分析与三维重建系统

根系构型是表征植物资源利用能力的重要指标之一,研究方法的改进非常重要。本文在综述现有根系构型研究手段的基础上,重点介绍了作物高通量根系构型定量分析与三维重建系统,根据油菜根系在琼脂等介质中的生长特点,对该系统进行改进,建立了油菜高通量根系构型定量分析与三维重建系统,并对该系统的可靠性进行了评价,在油菜根系构型研究上开展了应用,并探讨了需要进一步改进的方面。     

水稻端四体的分子细胞学鉴定及染色体行为分析

在水稻品种中籼3037第9染色体短臂端三体的自交后代中发现了一个变异株。该植株叶片内卷,株型偏散,结实率差。分子细胞学鉴定表明,该植株体细胞染色体数比正常植株多2条,多出的染色体均比较小。进一步用来源于水稻着丝粒特异DNA序列（RCS2）以及位于水稻第9染色体短臂上的特异性DNA序列为探针,进行荧光原位杂交分析,证明该变异株多出的染色体均为第9染色体短臂,故该变异株为第9染色体短臂端四体。对变异株的减数分裂染色体行为进行分析表明,在所观察的25个细胞中,96%的细胞增加的两个端着丝粒染色体可配对形成二价体,一般不与正常的第9染色体配对形成多价体。但额外染色体形成的二价体在中期Ⅰ容易发生提前解离的现象。     

小麦-中间偃麦草衍生系的分子细胞遗传学鉴定

对阿勃缺体与小麦-中间偃麦草部分双二倍体中2经杂交、回交选育的两个衍生系(衍生系Ⅰ和衍生系Ⅱ)研究表明,两个衍生系在形态学和细胞学上已经基本稳定,细胞学鉴定结果均为2n=44=22″。两个衍生系与阿勃二体杂交F1花粉母细胞染色体构型以及基因组原位杂交结果表明,衍生系Ⅰ为小麦-中间偃麦草异代换-附加系,衍生系Ⅱ为小麦-中间偃麦草异附加系。