玉米醇溶蛋白γ-zein基因载体构建及亚细胞定位

用PCR方法从pROK.TG1LK扩增得到γ-zein基因的全长,克隆到中间载体pMD18-T,获得了重组质粒pMD18-γ-zein,分别用Nco Ⅰ和Bgl Ⅱ 2种限制性内切酶酶切重组质粒和经过改良的pCAMBIAI 1302载体,经回收、连接、转化、鉴定后,构建了由35S驱动的绿色荧光蛋白(GFP)融合的植物表...     

pCB-zeolin-GFP表达载体的构建及瞬时表达

为利用荧光蛋白基因GFP检测外源基因在转基因植株中的表达和定位,构建含有GFP基因的植物表达载体pCB-zeolin-GFP。在目的基因的开放阅读框（ORF）两端设计引物,并引入酶切位点和保护碱基,用PCR方法从pDHA扩增得到zeolin基因的全长,克隆到中间载体pMD18-T,分别用NcoⅠ和BglⅡ2种限制性内切酶酶切重组质粒和经过改良的pCAMBIAI1302植物表达载体,经回收、连接、转化、鉴定后,利用基因枪转化法将重组载体转入洋葱表皮细胞,通过共聚焦显微镜检测绿色荧光蛋白在洋葱表皮细胞中的瞬时表达。构建了zeolin基因与绿色荧光蛋白（GFP）融合的植物表达载体pCB-zeolin-GFP,并在洋葱中得到了表达。构建的融合植物表达载体pCB-zeolin-GFP正确,该载体的成功构建为今后进行基因转移、基因功能研究及培育新品种奠定了基础。     

红掌漆酶基因Lac的表达载体构建与剑麻转化

用RT-PCR从红掌品种亚利桑那的cDNA中扩增出漆酶基因Lac并得到的基因的全长.将Lac片段克隆到pMD18-T载体,然后将此基因插入pBI121的CaMV35S启动子后取代原载体中的GUS基因构成表达载体pBI121-Lac.通过冻融法将表达载体导入根癌农杆菌(Agrobacterium tumefaciens)LBA4404,并转化剑麻获得转基因植株.对转化植株进行的基因组PCR分析结果表明,Lac基因已成功的整合到剑麻的基因组中.转漆酶基因植株的获得为剑麻抗性的提高提供了可能性,并且为以后研究漆酶的具体功能提供了方便.     

果实特异启动子调控薄皮甜瓜ACC合成酶反义基因植物表达载体的构建

从成熟的薄皮甜瓜(齐甜1号)果肉组织中提取总DNA,经PCR扩增得到约0.5kb的ACC合成酶基因的片段,将其克隆到质粒载体pMD18-T中,测序表明,该基因为583bp,编码194个氨基酸;从番茄(东农706)叶片组织中提取总DNA,经PCR扩增得到约2.2kb的E8基因片段,将其克隆到质粒载体pGEM-TEasy中,测序表明,该基因为2192bp;以pCAM2301为起始植物表达载体,pCAM-GT为中间载体,成功构建了果实特异启动子(E8)调控薄皮甜瓜ACC合成酶反义基因植物表达载体,采用冻融法将其转入根癌农杆菌LBA4404,得到了完整的Ti质粒表达载体系统。     

禽脑脊髓炎病毒VP1基因植物表达载体的构建

以提取的禽脑脊髓炎病毒总RNA为模板,以P1和P2为引物,反转录为cDNA,再以cDNA为模板进行PCR扩增.将扩增产物克隆pMD18-T载体中,获得重组质粒pMD18-T-VP1,PstⅠ/BamHⅠ双酶切及PCR确认正确后,利用pMD18-T-VP1重组质粒为模板,以P3和P4为引物,经PCR扩增获得了大小约810 bp的产物.VP1基因和 pBI121植物表达载体用SmaⅠ和SacⅠ双酶切,经纯化后连接,再转化到EHA105农杆菌中,酶切和测序鉴定结果表明,成功地构建了重组质粒pBI121-VP1,为进一步利用转基因植物生产VP1蛋白奠定了基础.     

猪IgG Ⅱ类Fc受体基因真核表达重组质粒的构建及表达

利用本研究小组克隆的猪IgG Ⅱ类Fc受体cDNA(swFcγRⅡ)基因序列(DQ026064)设计引物,应用RT-PCR技术,从猪外周血白细胞cDNA中扩增了完整的960 bpswFcγRIIORF序列,利用基因重组技术将swFcγRIIORF基因亚克隆到真核表达载体pcDNA3,经PCR及双酶切鉴定:扩增出901 bp的PCR产物;KpnI/EcoR I双酶切,切出5 376和901bp DNA片段,表明重组质粒pcDNA/swFcγRⅡ构建成功。然后脂质体法转染COS-7细胞,用玫瑰花环试验鉴定了swFcγRII配体亲和特性。     

香蕉ACC氧化酶反义基因植物表达载体的构建及其转化烟草的研究

用限制性内切酶Xba Ⅰ和Sac Ⅰ从克隆载体pUCm-ACO上切下约1.2 kb的香蕉ACO基因,将其定向连接在经相同酶切的质粒载体pBI-121上,构建成植物表达载体pBI-aACO.在此基础上用EcoR Ⅰ和HindⅢ从在pBI-aACO上切下大小约2.3 kb的目的基因35Sp-aACO-NOSt,将其连接在质粒载体pCAMBI-A2301载体上,构建成香蕉ACO反义基因植物表达载体pCB-aACO.采用直接转化法将pCB-aACO导入根癌农杆菌菌株EHA105,采用该菌株转化普通烟草.在Kanamycin选择压力下获得的烟草转化不定芽和完整植株,经过GUS组织化学法检测、以及PCR和RT-PCR方法鉴定,验证了该植物表达载体的报告基因,选择基因和目的基因都得到了表达,证明该植物表达载体构建成功.此项研究为下一阶段用该反义基因转化香蕉品种以改良香蕉果实耐贮运性打下基础.     

岩栖蝮蛇毒纤溶酶基因克隆、序列分析及真核表达载体的构建与鉴定

克隆岩栖蝮蛇（Gloydius saxatilis）纤维溶解酶（Fibrinolytic enzyme,FLE）基因,分析基因序列并对该基因的功能进行分析;构建真核表达载体并鉴定。从岩栖蝮蛇毒腺细胞中提取总RNA,通过RT-PCR法扩增纤溶酶基因,与pMD18-T载体连接进行TA克隆,再进行序列的测定并且对该序列进行分析。测序鉴定后将纤溶酶基因克隆入真核表达载体pEGFP-C1,构建该基因的真核表达载体pEGFP-FLE,进行双酶切、PCR和测序鉴定。扩增得到包括完整开放读码框在内的长为774 bp的纤溶酶基因序列,成功克隆了编码岩栖蝮蛇纤溶酶的基因序列,开放读码框编码257个氨基酸,其分子质量约为30 KDa,成功构建了真核表达载体pEGFP-FLE。成功克隆岩栖蝮蛇毒纤溶酶基因并构建该酶的真核表达载体为该基因的真核表达及后续研究提供依据。     

球孢白僵菌HFW-05几丁质酶基因的克隆与序列分析

通过对粉虱、小菜蛾高效的球孢白僵菌HFW-05几丁质酶基因的克隆及序列分析,旨在从分子水平上了解HFW-05菌株的几丁质酶特性。根据已发表的几丁质酶基因序列(EU828354)设计几丁质酶基因全长引物,扩增HF-WBbchit1全长基因,与大肠杆菌(Escherichia coli)克隆载体pMD19-T连接获得含有HFWBbchit1全长基因的重组质粒pMDchit1并测序。该基因的编码区包括1 047 bp,编码了348个氨基酸,分子量约为36.795 kDa,进行同源性比较分析结果表明:其基因序列与球孢白僵菌菌株NCIM1216几丁质酶基因(EU828354)和球孢白僵菌菌株Bb0062几丁质酶基因(AY145440)的核苷酸同源性最高,同源性达到98%。氨基酸序列与球孢白僵菌菌株Bb0062几丁质酶(AAN41259)同源性达到99%。     

葡萄糖氧化酶基因植物表达载体的构建

为进一步开展转基因研究创造条件,用限制性内切酶将葡萄糖氧化酶(GOD)基因从pMD/GO载体上切下,定向连接到植物表达载体pROKⅡ的CaMV35S启动子下游和NOS终止子上游,成功地构建了GOD基因植物表达载体pROK/GO。利用冻融法将此表达载体导入根癌农杆菌LBA4404中,提取转化质粒,经PCR和酶切鉴定表明,GOD基因植物表达双元载体构建成功。     

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.     

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.     

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.     

A note on the origin of Kalanchoë x vadensis Boom & Zeilinga (Crassulaceae)

Summary K x vadensis is a hybrid of K. blossfeldiana and K. marmorata obtained after doubling the number of chromosomes.     

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...     

Avoidance of leaf rust fungi in wild relatives of cultivated cereals

Summary Avoidance of rust fungi that was based on poor appressorium induction was previously found in Hordeum chilense. In the present study 95 accessions of Triticeae were screened for avoidance of Puccinia hordei. The percentage of appressorium formation per germinated spore ranged from 6 to 90%. On none of the 41 accessions of Aegilops, Agropyron, Elymus, Secale, Thinopyrum or Triticum studied was the rate of appressorium formation lower than 25%. Lower rates of appressorium formation were, however, found on accessions of wild barley species Hordeum brachyantherum, H. marinum, H. parodii and H. secalinum. Its implications in cereal breeding are discussed.     

Variation in forage grasses as hosts of the root-knot nematode Meloidogyne naasi (Franklin) and selection for resistance in ryegrass

Summary Reactions of 13 grasses to Meloidogyne naasi varied with species; ryegrasses, fescues and their hybrids were generally susceptible and cocksfoot and timothy resistant. Marked variation in host resistance levels occurred between genotypes within cultivars.Selection of single plants, followed by tests on replicate tillers, identified resistant and susceptible genotypes in both Italian and perennial ryegrass cultivars. Resistant plants had few nematode-induced galls and fewer females and eggs than susceptibles. There was more or less continuous variation, with many genotypes intermediate between extremes of resistance and susceptibility. Selected resistant and susceptible genotypes are of use in assessing variation in nematode populations and as controls for breeding and selection programmes.     

Current and future strategies in breeding lentil for resistance to biotic and abiotic stresses

Lentil production is limited by lack of moisture and unfavorable temperatures throughout its distribution. Waterlogging and salinity are only locally important. Progress has been made in breeding for tolerance to drought through selection for an appropriate phenology and increased water use efficiency and in breeding for winter hardiness through selection for cold tolerance.The diseases rust, vascular wilt, and Ascochyta blight, caused by Uromyces viciae-fabae, Fusarium oxysporum f. sp. lentis, and Ascochyta fabae f. sp. lentis, respectively, are the key fungal pathogens of lentil. Cultivars with resistance to rust and Ascochyta blight have been released in several countries and resistant sources to vascular wilt are being exploited. Sources of resistance to several other fungal and viral diseases of regional importance are known. In contrast, although the pea leaf weevil (Sitona spp.) and the parasitic weed broomrape (Orobanche spp.), and to a lesser extent the cyst nematode (Heterodera ciceri), are significant yield reducers of lentil, no sources of resistance to these biotic stresses have been found. Directions for future research in lentil on both biotic and abiotic stresses are discussed.     

Breeding forage grasses for organic conditions

Herbage yield trials are necessary to test if candidate varieties are well suited for organic conditions. In order to elucidate a particular suitability for organic conditions, we run such trials in parallel under both organic and conventional conditions. Here, we report on second and third year results of the sowings 2004 and 2005. These trials are part of a targeted breeding programme for organic conditions. Irregular colonisation of pure grass plots with adventitious plants, in particular white clover, was recognized as a major constraint to the quality of the results obtained under organic conditions. The coefficients of variance in dry matter yield almost doubled when compared to conventional trials, and significant differences in annual yield among varieties were rare. However, when herbage yield was corrected for the estimated fraction of adventitious plant colonization at each cut, the number of instances with statistically significant differences for annual yield among varieties increased more than fourfold. Dry matter yields under organic and conventional conditions were significantly and positively correlated. However, analysis of variance showed a significant culture type by variety interaction for annual dry matter yield in 12 out of 24 trials. When diploid and tetraploid varieties of the same Lolium species were tested in one common trial, only tetraploid varieties were identified as being particularly well suited for organic conditions, while in these trials, the great majority of apparently poorly suited varieties were diploid. This suggests that breeding tetraploid Lolium varieties is promising when aiming at a favourable response to organic agriculture.     

The performance of double or mixed pollinations with compatible and self-incompatible or incongruous pollen of pear and apple

Summary Six pear and five apple trials were carried out to ascertain the outcome of combinations of compatible pollen (C) with self (S) or incongruous pollen (I) as to the pollination index (PI=seeds/pollinated flower). The PI of the mixture C+I (1:5) was consistently depressed as compared to that of the control C. The results of the double pollinations S/C and I/C were affected by the temperature at pollination; their PI's at <15°C were twice as high as those at >15°C, being well above and below the PI of C in the former and latter case respectively. The opposite was true for the C/S combination, the PI of which increased with the pollination temperature; the PI of C/I did not differ much from the PI of C, irrespective of temperature. The conclusion was reached that the interaction previously and presently found between compatible and self-incompatible pollen also exists to a fair extent between compatible and incongruous pollen. However, in pear neither the mentor nor the pioneer pollen technique proved to aid its hybridization with apple, the formation of self seed was not observed either. In apple the production of apple × pear hybrids was likewise doubtful, but the double pollinations S/C and C/S formed 4–10% self seed.