紫苏FAD3基因RNA干扰载体的构建及遗传转化

本研究利用RNA干扰的方法抑制紫苏ω-3脂肪酸脱氢酶基因FAD3的表达,以期得到低ALA含量的转基因植株。根据紫苏FAD3基因(Gen Bank登录号为KC990786.1)序列设计含有不同酶切位点的特异性扩增引物,通过扩增获得长度为258 bp的RNA干扰片段,构建以Ca MV35S启动子驱动表达的紫苏FAD3基因RNA干扰表达载体p FGC5941M-FAD3I,将该载体转入根癌农杆菌GV2301后,采用农杆菌介导法进行紫苏的遗传转化,经除草剂抗性筛选和PCR检测获得1株阳性转基因植株。利用荧光定量PCR技术对野生型和转基因阳性苗的FAD3基因进行了表达分析,结果显示,阳性植株中FAD3基因的表达受到显著抑制,FAD3基因表达量降低了80%,说明此研究构建的FAD3基因RNA干扰载体对该基因表达沉默是有效的,为FAD3基因在不饱和脂肪酸代谢过程中的功能研究提供了理论依据。     

ahFAD2基因RNA干扰体的删除筛选标记载体构建及其遗传转化

将Napin启动子与含花生FAD2基因反向重复片段的RNA干扰结构连接,亚克隆至含雌激素诱导重组系统的植物表达载体pNCX相应位点中,构建完成由Napin启动子驱动的FAD2RNAi删除筛选标记表达载体pNCX-FAD2RNAi,经酶切鉴定,获得相应的启动子片段、FAD2RNAi片段及NCX-FAD2RNAi片段.利用农杆菌介导法进行遗传转化,已获得抗性丛生芽214丛.     

花生AhFAD2-1基因启动子及5'-UTR内含子功能验证及其低温胁迫应答

Δ~(12)-脂肪酸脱氢酶基因(FAD2)催化油酸生成亚油酸,是决定油酸亚油酸比值的关键基因。高油酸花生对低温更加敏感,暗示FAD2在低温响应中发挥作用。为探索花生FAD2的低温胁迫应答,本研究分析了花生FAD2-1A/B的基因结构,从普通油酸花生品种豫花9326中克隆了FAD2-1A/B启动子和内含子,并通过转化拟南芥验证了功能及其对冷胁迫的应答。结果表明, Ah FAD2-1A/B包含2个外显子和1个位于5'-UTR的内含子; Ah FAD2-1A/B基因启动子功能较弱,仅Ah FAD2-1B启动子在子叶期幼苗的子叶叶尖中观察到蓝色; Ah FAD2-1假基因5'侧翼序列具有启动子活性,可调控基因在子叶、下胚轴、种子中表达。Ah FAD2-1内含子序列具有启动子的功能,驱动基因在幼苗下胚轴及子叶中表达,同时还有提高基因表达效率和扩大表达范围的功能,是Ah FAD2-1基因表达调控必需元件;包含5'-UTR内含子的Ah FAD2-1A/B启动子的调控序列功能受低温胁迫抑制。     

甘蓝型油菜BnFAD2-C5基因启动子及内含子在表达水平的功能分析

富含油酸的菜籽油具有重要的经济价值,使得高油酸育种和形成机理的研究成为热点。油酸脱氢酶基因(FAD2基因)是控制油酸含量的关键酶基因。本文针对BnFAD2-C5基因展开研究,根据油菜和甘蓝的同源性,克隆了1257 bp启动子序列,利用GUS和GFP作为报告基因分别构建含有不同片段长度的启动子和内含子的缺失载体并转化拟南芥,经GUS染色检测发现–319 ~ –1 bp为该研究中最小启动子;采用Western技术分析启动子和内含子不同区域的功能,发现BnFAD2-C5启动子区域–1257 ~ –1020 bp和–319 ~ –1 bp能够诱导报告基因在转基因拟南芥种子发育中期高效表达,BnFAD2-C5内含子具有增强启动子转录水平的功能,该功能主要由631~1033 bp区域调控。     

CRISPR-Cas9系统敲除亚麻FAD2基因表达载体的构建

CRISPR/Cas9系统是一种新型基因编辑工具,准确编辑目标基因,广泛应用于动植物的基因编辑中。本实验构建了油用亚麻品种‘陇亚10号’FAD2基因的CRISPR/Cas9基因编辑载体,以CRISPR/Cas9基因编辑技术为切入点,根据Gene Bank中公布的FAD2基因序列(DQ222824.1)在FAD2基因外显子部分运用sg RNA在线设计软件CRISPR-P 2.0对亚麻FAD2基因做脱靶分析,在外显子区域选取2个片段作为靶序列进行敲除。以潮霉素作为筛选标记,采用Golden Gate cloning法构建针对‘陇亚10号’FAD2基因的CRISPR/Cas9基因敲除载体,命名为p YLCRISPR/Cas9-FAD2。将pYLCRISPR/Cas9-FAD2载体导入农杆菌并检测其稳定性,保存菌种以便后期利用农杆菌介导法转化‘陇亚10号’,为深入研究亚麻FAD2基因的功能提供参考。     

甘蓝型油菜PEPC基因保守序列的克隆和种子特异性ihpRNA表达载体的构建

为了通过转基因途径获得油菜种子中PEPC基因发生转录后基因沉默,通过PCR扩增分别分离得到了甘蓝型油菜种子特异性表达的Napin启动子序列（1138bp）和磷酸烯醇式丙酮酸羧化酶（PEPC）基因保守序列（181bp）,将它们分别克隆到pMD18-T载体中,利用中间载体pBS-NEI构建了PEPC基因的反向重复框。再将PEPC基因片段以正向的方式连接到一个可剪切的内含子5＇末端,以反向的方式连接到该内含子的3＇末端;然后将Napin启动子序列克隆到植物表达载体pCAMBIA1391的pUC18多克隆位点上,获得了种子特异表达的载体pCAMNapin;再将PEPase基因的反向重复序列克隆到pCAMNapin载体Napin启动子的3＇末端,构建了具有种子特异性表达的PEPC基因的ihpRNA表达载体pCAMNapin-B2-NEI-B1。通过限制性内切酶酶切对载体作了鉴定分析。     

高粱C_4型pepc基因转入大豆可改善大豆光合特性

改善作物的光合性能能够有效突破作物的产量潜力,目前国内关于转高粱C4型pepc基因大豆的研究尚未见报道。本研究以东北地区主栽大豆品种‘黑农41’为材料,通过农杆菌介导法将高粱C4型全长pepc基因转入‘黑农41’,获得了转基因植株。通过对转基因植株T0代和T1代进行了分子生物学检测、农艺性状调查和生理指标检测,结果表明目的基因已整合到大豆基因组中,T0代和T1代植株的净光合速率、气孔导度和蒸腾速率均有不同程度的提高,而胞间CO2浓度降低,其中T0代和T1代植株的净光合速率分别比对照提高了37.4%和20.7%,在T1代净光合速率增幅最大的T1-3-3,单株产量增加也最多,说明pepc基因的导入改善了转基因植株的光合特性,进而使转基因大豆产量提高。本研究可为高光效转基因大豆新品种的选育提供重要素材。     

含双边界序列植物双价表达载体的构建

转基因植物的安全性是基因工程改良作物的一个重要问题.构建含双边界序列（双T-DNA区）载体,将选择标记基因和目标外源基因分开在不同的T-DNA区,通过转基因植株有性杂交及后代分离,可在转基因后代中获得无选择标记的转基因安全植株.将2个外源基因置于同一载体上,可以提高其共转化的效率.本研究构建了1个中间载体pAHC17-PTA和1个含双边界序列的植物双价表达载体pDB13PS.pAHC17-PTA含有由Ubiquitin启动子引导的具有抗虫效果的半夏凝集素基因（PTA）.pDB13PS含2个独立的T-DNA区,在其中一个T-DNA区,含两个目的基因的完整的表达盒,一个是由Ubiquitin启动子引导的半夏凝集素基因（PTA）,另一个是由水稻胚乳特异表达启动子（Glutelin-B1 promoter）驱动的马铃薯高赖氨酸蛋白基因的cDNA（SB401）.pDB13PS的成功构建,为提高PTA和SB401的共转化效率和获得无选择标记的转基因后代植株奠定了基础.     

两种抗病基因双T-DNA区双元载体的构建

选择标记基因的删除是植物转基因工程中重要的步骤之一,双T-DNA区双元载体转化法以其共整合频率高、删除标记基因效率高的优点在植物遗传转化中应用非常广泛。本文通过构建天麻抗真菌蛋白基因、脂质转移蛋白基因双T-DNA区双元载体,为培育不含选择标记基因的抗病新材料奠定基础。通过PCR的方法克隆GAFP基因,产物纯化测序确定序列准确后与pSB130-35S载体进行BamHⅠ、SacⅠ双酶切;pCAMBIA2300-LTP、pSB130-35S经EcoRⅠ、HindⅢ双酶切,酶切产物纯化后连接,连接产物转化大肠杆菌。采用基因特异性引物进行菌液PCR鉴定阳性克隆,重组质粒pSB130-GAFP、pSB130-LTP双酶切产物大小分别为540bp、1200bp,与预期产物大小一致,pSB130-GAFP、pSB130-LTP双T-DNA区双元载体构建成功。载体pSB130-GAFP、pSB130-LTP转化根癌农杆菌后可以直接用于植物的遗传转化。     

可去除选择标记的转Bt基因抗虫玉米研究

将组成型表达的玉米泛素启动子与苏云金杆菌杀虫蛋白基因Bt连接，插入根瘤农杆菌双T-DNA质粒，构建一个T-DNA结构域含有抗潮霉素选择标记基因hyg，另一个T-DNA结构域含有抗虫基因的双T-DNA单子叶植物表达载体，用以转化农杆菌菌株，再通过共培养转化玉米胚性愈伤组织。通过潮霉素培养基抗性筛选，用特异PCR扩增和Southern杂交检测，从分化再生的T0代植株中，鉴定出4个转化Bt基因的阳性植株。目前，正结合进行田间分离纯合和DNA分子鉴定，培育去除选择标记基因的转基因抗虫玉米自交系。     

Location of a high-lysine gene and the DDT-resistance gene on barley chromosome 7

Summary The high-lysine gene in Risø mutant 1508 conditions an increased lysine content in the endosperm via a changed protein composition, a decreased seed size, and several other characters of the seed. The designation lys3a, lys3b, and lys3c, is proposed for the allelic high-lysine genes in three Risø mutants, nos 1508, 18, and 19. Linkage studies with translocations locate the lys3 locus in the centromere region of chromosome 7. A linkage study involving the loci lys3 and ddt (resistance to DDT) together with the marker loci fs (fragile stem), s (short rachilla hairs), and r (smooth awn) show that the order of the five loci on chromosome 7 from the long to the short chromosome arm is r, s, fs, lys3, ddt. The distance from locus r to locus ddt is about 100 centimorgans.     

Biological Activity and Quantification of Suspected Allelochemicals from Alfalfa Plant Parts

Autotoxicity restricts reseeding of alfalfa (Medicago sativa L.) after alfalfa until autotoxic chemical(s) breaks down or is dispersed into external environments. A series of aqueous extracts from leaves, stems, roots and seeds of alfalfa ‘Vernal’ were bioassayed against alfalfa seedlings of the same cultivar to determine their autotoxicity. The highest inhibition was found in the extracts from the leaves. Extracts at 40 g dry tissue l^?1 from alfalfa leaves were 15.4, 17.5 and 28.7 times more toxic to alfalfa root growth than were those from roots, stems and seeds, respectively. A high‐performance liquid chromatography (HPLC) analysis with nine standard compounds showed that the concentrations and compositions of allelopathic compounds depended on the plant parts. In leaf extracts that showed the most inhibitory effect on root growth, the highest amounts of allelochemicals were detected. Among nine phenolic compounds assayed for their phytotoxicity on root growth of alfalfa, coumarin, trans‐cinnamic acid and o‐coumaric acid at 10^?3 m were most inhibitory. The type and amount of causative allelochemicals found in alfalfa plant parts were highly correlated with the results of the bioassay, indicating that the autotoxic effects of alfalfa plant parts significantly differed.     

Simultaneous Determination of Four Phenolic Acids in Radix Salviae Miltiorrhizae Formula Granules by QAMS

[Objectives]This study aimed to establish a QAMS(quantitative analysis of multi-components by single-marker)method for simultaneous determination of four phenol...     

Changes in Seed Yield and Quality with Maturity in Onion (Allium cepa L., cv. 'Early Cream Gold')

Development of onion (Allium cepa L., cv. ‘Early Cream Gold’) seed under cool climate conditions in Tasmania, Australia occurred over a longer duration than previously reported, but similar patterns of change in yield components were recorded. In contrast to previous studies, umbel moisture content declined from 85 to 67 % over 57 days while seed moisture content decreased from 85 to 31 %. Seed yield continued to increase over the duration of crop development, with increasing seed weight compensating for seed loss resulting from capsule dehiscence in the later stages of maturation. Germination percentage was high and did not vary significantly from 53 to 77 days after full bloom (DAF), but mean germination time declined and uniformity of germination increased significantly over the same time period. The percentage abnormal seedlings declined with later harvest date, resulting in highest seed quality at 77 DAF. The results of this study suggest that the decision to harvest cool climate onion seed crops before capsule dehiscence will result in a loss of potential seed yield and quality.     

Water Extraction Process of Chinese Herbal Compound Man Gan Ning

[Objectives]To optimize the water extraction process of Chinese Herbal Compound Man Gan Ning and establish a method for its extraction and content determination...     

Present status and future strategy in breeding faba beans (Vicia Faba L.) for resistance to biotic and abiotic stresses

Progress is being made, mainly by ICARDA but also elsewhere, in breeding for resistance to Botrytis, AScochyta, Uromyces, and Orobanche; and some lines have resistance to more than one pathogen. The strategy is to extend multiple resistance but also to seek new and durable forms of resistance. Internationally coordinated programs are needed to maintain the momentum of this work.Tolerance of abiotic stresses leads to types suited to dry or cold environments rather than broad adaptability, but in this cross-pollinated species, the more hybrid vigor expressed by a cultivar, the more it is likely to tolerate various stresses.     

Pollen and pollination experiments. III. The viability of apple and pear pollen as affected by irradiation and storage

Summary Apple and pear pollen was irradiated with doses of 0, 50, 100, 250 and 500 krad (gamma rays) and stored at 4°C and 0–10% r.h. From the in-vitro germination percentages an average LD 50 dose of about 220 krad was estimated. For both irradiated and untreated pollen a close and corresponding lineair relationship existed between germination percentage and pollen tube growth.Irradiated pollen was much more sensitive to dry storage conditions than untreated pollen, resulting in less germination and more bursting. Apparently, irradiation caused the pollen cell membrane to lose its flexibility faster than normal. Rehydration of dry-stored, irradiated pollen in water-saturated air restored germination percentages up to their initial levels. The importance of this procedure in germination trials is stressed.     

Optimization of Extraction Technology and Determination of Content of Total Phenols of Leaves in Acanthopanax giraldii Harms

[Objectives] To determine the optimum extraction technology for total phenols of leaves in Acanthopanax giraldii Harms.[Methods]The single factor test and ortho...     

Cytoplasmic male sterility,resistance to gall mite and mildew,and season of leafing out in black currants

Summary Cytoplasmic male sterility (cms) is described in the F₁ hybrids Ribes × carrierei (R. glutinosum albidum × R. nigrum) and R. sanguineum × R. nigrum. In backcrosses to R. nigrum, progenies with R. glutinosum cytoplasm were either all male sterile, or segregated for full male fertility (F) and complete (S) and partial (I) male sterility. Ratios of F:I+S suggested that two linked genes controlled cms, F plants being dominant for one (Rf ₁) and recessive for the other (Rf ₂).Segregation for cms in relation to three linded genes, Ce (resistance to the gall mite, Cecidophyopsis ribes), Sph ₃(resistance to American gooseberry mildew, Sphaerotheca mors-uvae) and Lf ₁(one of two dominant additive genes controlling early season leafing out) indicated that Rf ₁and Rf ₂were in this linkage group. The gene order and approximate crossover values appeared to be: % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXafv3ySLgzGmvETj2BSbqef0uAJj3BZ9Mz0bYu% H52CGmvzYLMzaerbd9wDYLwzYbItLDharqqr1ngBPrgifHhDYfgasa% acOqpw0xe9v8qqaqFD0xXdHaVhbbf9v8qqaqFr0xc9pk0xbba9q8Wq% Ffea0-yr0RYxir-Jbba9q8aq0-yq-He9q8qqQ8frFve9Fve9Ff0dme% aabaqaciGacaGaamqadaabaeaafaaakeaacaWGdbGaamyzamaamaaa% baGaaiiiaiaacccacaGGWaGaaiOlaiaacgdacaGG0aGaaiiiaiaacc% caaaGaaiiiaiaacccacaGGGaGaamOuaiaadAgaliaaigdakmaamaaa% baGaaiiiaiaacccacaGGGaGaaiiiaiaaccdacaGGUaGaaiOmaiaacs% dacaGGGaGaaiiiaiaacccacaGGGaGaaiiiaaaacaWGsbGaamOzaSGa% aGOmaOWaaWaaaeaacaGGGaGaaiiiaiaacccacaGGGaGaaiiiaiaacc% cacaGGGaGaaiiiaiaacccaaaGaamitaiaadAgaliaaigdakmaamaaa% baGaaiiiaiaacccacaGGGaGaaiiiaiaacccacaGGGaGaaiiiaiaacc% cacaGGGaGaaiiiaiaacccacaGGGaaaaiaadofacaWGWbGaamiAaSGa% aG4maaaa!6E4D!\[Ce\underline { 0.14 } Rf1\underline { 0.24 } Rf2\underline { } Lf1\underline { } Sph3\]. Crossover values of 0.36 for Ce-Lf ₁, and 0.15 for Lf ₁-Sph ₃were estimated from the relative mean differences in season of leafing out between seedlings dominant and recessive for Ce and Sph ₃.It is suggested that competitive disadvantage of lf ₁-carrying gametes and/or zygotes at low temperatures may be implicated in the almost invariable deficit of plants dominant for the closely linked mildew resistance allele Sph ₃. Poor performance of lf ₁- (and possibly lf ₂-) carrying gametes and young zygotes during periods of low temperature at flowering might also account for the liability of some late season cultivars and selections to premature fruit drop (running off).     

Screening techniques and sources of resistance to parasitic angiosperms

Parasitic angiosperms cause great losses in many important crops under different climatic conditions and soil types. The most widespread and important parasitic angiosperms belong to the genera Orobanche, Striga, and Cuscuta. The most important economical hosts belong to the Poaceae, Asteraceae, Solanaceae, Cucurbitaceae, and Fabaceae. Although some resistant cultivars have been identified in several crops, great gaps exist in our knowledge of the parasites and the genetic basis of the resistance, as well as the availability of in vitro screening techniques. Screening techniques are based on reactions of the host root or foliage. In vitro or greenhouse screening methods based on the reaction of root and/or foliar tissues are usually superior to field screenings and can be used with many species. To utilize them in plant breeding, it is necessary to demonstrate a strong correlation between in vitro and field data. The correlation should be calculated for every environment in which selection is practiced. Using biochemical analysis as a screening technique has had limited success. The reason seems to be the complex host-parasite interactions which lead to germination, rhizotropism, infection, and growth of the parasite. Germination results from chemicals produced by the host. Resistance is only available in a small group of crops. Resistance has been found in cultivated, primitive and wild forms, depending on the specific host-parasite system. An additional problem is the existence of pathotypes in the parasites. Inheritance of host resistance is usually polygenic and its transfer is slow and tedious. Molecular techniques have yet to be used to locate resistance to parasitic angiosperms. While intensifying the search for genes that control resistance to specific parasitic angiosperms, the best strategy to screen for resistance is to improve the already existing in vitro or greenhouse screening techniques.