转基因甘蔗BtG-2的T-DNA侧翼序列分析及其转化事件特异性检测

转基因甘蔗BtG-2是利用农杆菌介导法把Cry1Ac-2A-gna融合抗虫基因导入‘新台糖22号’的转基因甘蔗株系,具有良好的抗虫特性和农艺性状。为了明确转基因甘蔗BtG-2的分子特征及其检测方法,推进其生物安全性评价工作,以BtG-2的T2代为研究材料,利用Southern杂交检测外源基因在转基因甘蔗基因组内的拷贝数;利用染色体步移技术分离外源基因在甘蔗基因组中插入位点的侧翼序列,并建立了该转化体高效灵敏的特异性PCR检测方法。结果表明：Southern杂交检测证明外源T-DNA以单拷贝方式插入BtG-2株系;经过3次的热不对称巢式PCR扩增,获得外源基因T-DNA左边侧翼序列984 bp和右边侧翼序列705 bp;以这2个序列和相应的T-DNA的左右端序列分别设计3对检测引物对,建立了BtG-2株系的转化事件特异性PCR检测方法,扩增效率最高的引物对LS011/LA451和RS160/RA588分别扩增到440 bp和428 bp的特异片段。其中T-DNA左侧设计的LS011/LA451检测引物对扩增的灵敏度高、特异性强,能够在甘蔗BtG-2基因组DNA相对含量为0.1%的模板中检测出转基因目的成分,相当于9个单倍体基因组拷贝数。本研究完成了转基因株系BtG-2的分子特征及其转化事件特异性检测,为该转基因甘蔗及其衍生产品的检测和身份识别提供技术依据。     

Comparison of flagellin and an oil-emulsion adjuvant in inactivated Newcastle disease vaccine in stimulation of immunogenic parameters

The present study was designed to investigate the potential application of native (N) and recombinant (truncated modified [tmFliC] and full-length [flFliC]) flagellin proteins along with inactivated Newcastle disease virus (NDV). Fifty six SPF chickens were immunized twice with PBS (control), inactivated NDV (Ag), inactivated NDV/flFliC (AgF), inactivated NDV/tmFliC (AgT), inactivated NDV/N (AgN), commercial vaccine containing Montanide (Vac) and Vac/N (VacN), with a two-week interval. Blood was collected weekly and spleens were harvested after chickens were sacrificed. Interleukin-6 (IL-6) and tumor necrotic factor-α (TNF-α) gene expression in peripheral blood mononuclear cells were analyzed by Real-Time PCR. Antibody response was assessed by haemagglutination inhibition (HI). Cellular activity was quantified by MTT assay. Results showed that the most IL-6 and TNF-α gene expression was observed in AgF group (P < 0.01). The lowest gene expression among vaccinated groups was observed in Ag group for IL-6 and Ag and Vac group for TNF-α. The highest HI titer was observed in Vac, VacN, AgF and AgT groups. The AgF group showed the highest cellular activity (P < 0.01). In conclusion, flagellin-adjuvanted groups showed a pro-inflammatory effect and acted similarly to or better than the Vac group. Hence, flagellin can be proposed as a potential adjuvant for ND vaccine.     

Genome wide association studies (GWAS) of element contents in grain with a special focus on zinc and iron in a world collection of barley (Hordeum vulgare L.)

Genome wide association studies (GWAS) were carried out to map Quantitative Trait Loci (QTL) associated with element contents in the grain using 336 spring barley. Of the elements analyzed, Fe content ranged from 21.9 to 91.0 mg kg⁻¹, Zn from 10.4 to 54.5 mg kg⁻¹, Ba from 0.2 to 8.9, Ca from 186.4 to 977.5, Cu from 1.5 to 9.8, K from 353.2 to 7721.5, Mg from 1049.8 to 2024.2, Mn from 8.1 to 22.9, Na from 55.9 to 627.9, P from 2272.9 to 5428.8, S from 880.7 to 1898.0, Si from 19.1 to 663.2, and Sr from 0.35 to 2.62 mg kg⁻¹. GWAS were carried out using 6519 SNP markers and multiple elements in MLM:PCA + K model in TASSEL software. Population analyses showed two sub-populations, primarily based on row types. GWAS for row types showed association with INTERMEDIUM-C, a modifier gene for lateral spikelet fertility in the 4H chromosome, validating current GWAS approach. GWAS also showed that 2 QTL for Ba, 2 for Ca, 4 for Cu, 11 for Fe, 2 for K, 3 for Mg, 6 for Mn, 4 for Na, 3 for S, 5 for Si, and 3 for Zn were mapped in barley chromosomes. The QTL identified in the current study are valuable for breeding nutrient dense barley cultivars in the future, especially Zn and Fe.     

An investigation on the effect of heat-moisture treatment on baking quality of wheat by using response surface methodology

Effect of heat-moisture treatment on quality properties of two bread wheats (cvs. Tosunbey and Bayraktar) were investigated by using response surface methodology (RSM). Temperature and moisture conditions in the experimental design were in the range of 55–95 °C and 13–19%. Heat-moisture treated grains were milled into flour and quality properties were determined. The optimum moisture-temperature combination for the highest dry gluten, Zeleny sedimentation, Alveograph W and bread volume values were estimated as 14%-63 °C for Tosunbey and 19%-55 °C for Bayraktar samples. Alveograph W seems to be a good indicator of baking quality for wheats treated at higher temperatures. In order to describe the relationship between the dependent and independent variables (moisture, temperature), the response values were fitted by second order polynomial models. Significance analysis showed that the effect of both moisture and temperature on dry gluten content, sedimentation and falling number values for Tosunbey; falling number and damaged starch values for Bayraktar were significant (p < 0.05). The effect of temperature on Farinograph water absorption, W and P/G, bread volume and firmness values were significant for both cultivars (p < 0.05). It can be concluded that improvement in baking quality can be achieved and flours with different properties can be produced by heat-moisture treatments on wheat.     

Variation in the development of secondary dormancy in oilseed rape genotypes under conditions of stress

Summary A substantial amount of seed is left in the fields before and during harvest of oilseed rape. Although this crop exhibits little or no primary dormancy, the absence of certain environmental cues that promote germination of imbibed seeds induces secondary dormancy. The work reported investigated the extent to which environmental stress conditions, including osmotic stress, low oxygen stress and anaerobiosis, induce secondary dormancy in oilseed rape, and examined the variation in development of secondary dormancy between and within genotypes. Osmotic stress was most effective in inducing dormancy. Anaerobic treatment produced very few dormant seeds, as did an atmosphere low in oxygen and high in nitrogen. The development of secondary dormancy under osmotic stress varied considerably between and within genotypes. Dormancy ranged from almost zero to about 60% for winter genotypes and about 85% for spring types. Within genotypes, variations occurred between seed lots and years of harvest. Temperature variations affected the percentage of dormant seeds. More dormant seeds were likely to be produced with incubation under water stress at 20 °C than at 12 °C. In winter genotypes, fewer dormant seeds were produced when incubation temperature and germination test temperatures differed. Thus, incubating at 20 °C and 12 °C, followed by germination tests at 20 °C and 12 °C, respectively, produced most dormant seeds. Also, in the winter genotypes, the potential development of secondary dormancy was positively correlated with the pattern and speed of germination of untreated seeds.     

云南省马铃薯晚疫病菌的交配型、抗药性及生理小种分布的研究

 从云南省陆良县、建水县和南涧县采集2000年秋播的马铃薯晚疫病标本,分离获得124个致病疫霉菌株,连同1999~2000年分离自昆明、曲靖等地的春播马铃薯晚疫病的10个菌株共134个,采用常规技术对这些菌株进行了交配型、抗药性及生理小种的研究。交配型测定结果为:陆良县的18个菌株中有3个为A₂交配型,其余15个菌株为A₁交配型,建水县和南涧县的106个菌株及昆明、曲靖1999~2000年的10个菌株都是A₁交配型。测定了83个菌株对瑞毒霉(metalaxyl)和烯酰吗啉(dimethomorph)的抗药性,结果有12.0%的菌株对瑞毒霉表现抗性,16.9%表现中抗,71.1%表现敏感,其中陆良、昆明和曲靖的抗瑞毒霉菌株的比例大于建水和南涧的抗药性菌株的比例;所测定的83个菌株都对烯酰吗啉表现敏感。生理小种鉴定结果表明,云南晚疫病菌为3.4、0、3和4生理小种,分别占鉴定菌株总数的48.0%、32.5%、15.6%和3.9%。但南涧仅发现3.4和0小种,而昆明有0、3、3.4和4小种,陆良有0、3和3.4小种,可见昆明和陆良地区的晚疫病菌生理小种比南涧地区的生理小种的组成更趋多样。     

哈茨木霉几丁质酶诱导及其对水稻纹枯病菌的拮抗作用

 在实验室条件下分别以几丁质和水稻纹枯病菌(Rhizoctonia solani)细胞壁作唯一碳源诱导哈茨木霉(Trichoderma harzianum)菌株NF9、TC3和P1产生几丁质酶,用硫酸铵沉淀法制备几丁质酶粗提液。上述木霉菌株内切几丁质酶活性(对胶体几丁质浑浊度的减少率)分别为79.8%、74.4%和76.0%,均显著高于非诱导的阳性对照。培养第5 d几丁质诱导的木霉菌株NF9和TC3内切几丁质酶活性显著高于由水稻纹枯病菌细胞壁诱导的酶活性。体外测定表明,通过诱导的木霉菌株TC3、NF9和P1几丁质酶粗提液对水稻纹枯病病菌的拮抗圈直径可达38、21和23 mm,与非诱导的阳性对照比较有显著性差异。对木霉几丁质酶拮抗作用的特点及生防应用进行了讨论。     

Differential levels of antioxidants in paraquat-resistant and -susceptible Erigeron canadensis biotypes in Korea

To characterize the biochemical differences in paraquat-resistant and -susceptible biotypes of Erigeron canadensis L. collected from Korea, we investigated the constitutive levels of various antioxidants such as antioxidant enzymes and low molecular weight antioxidants in leaves, as well as after paraquat treatment. The activities of superoxide dismutase, peroxidase, ascorbate peroxidase, and catalase were higher in the paraquat-resistant biotype than in the paraquat-susceptible biotype. Reduced ascorbic acid content was higher in the resistant biotype, but the content of reduced glutathione was higher in the susceptible biotype. These results indicate that one of the paraquat-resistant mechanisms in E. canadensis in the present study might be related to protecting the activities of antioxidant enzymes, such as superoxide dismutase, peroxidase ascorbate peroxidase, and catalase, as well as the contents of low molecular weight antioxidants such as ascorbate and glutathione.     

开食料中不同NDF水平对犊牛生长性能、瘤胃内环境及血清生化指标的影响

为探索开食料中不同NDF水平对犊牛生长、瘤胃发酵参数以及血清生化指标的影响,选用初生重相近(42±2.5) kg、饲喂足量初乳的中国荷斯坦犊牛60头,其中公犊牛36头,母犊牛24头。随机分为A、B、C和D 4个处理组,每个处理15头,其中9头公犊牛+6头母犊牛,试验犊牛于犊牛岛(1.5 m×3.4 m)单独饲养。15日龄开始分别饲喂NDF水平不同的4种开食料,分别为:A处理10%,B处理15%,C处理20%和D处理25%,70日龄断奶,试验期112 d。结果表明:1)0~42日龄和70~112日龄,B组日增重均高于A、C和D组(P<0.05),分别高出45.69、39.79、117.13 g·d^-1和35.33、153.29、 145.93 g·d^-1。70~112日龄,B、C和D组开食料采食量显著高于A组(P<0.05),分别高出255.20、252.48和392.27 g·d^-1。A(0.61)和B(0.60)组犊牛的饲料转化率显著高于C(0.55)和D(0.54)组(P<0.05);2)犊牛瘤胃pH在35和70日龄时,C组显著高于其他3组(P<0.05),112日龄时,A和B组显著高于C和D组(P<0.05);3)瘤胃NH₃-N含量在90日龄前差异不显著(P>0.05),但在112日龄时, B、C和D组较A组显著降低16.72%、44.19%和52.10%(P<0.05);4)C和D组乙酸比例显著高于A和B组(P<0.05);B组丙酸比例较A、C和D组高出4.24%、3.18%和6.43% (P<0.05),乙酸/丙酸显著低于A、C和D组(P<0.05);5)各处理组间血清总蛋白、葡萄糖和尿素氮含量无显著差异(P>0.05)。1~3月龄犊牛开食料中NDF适宜水平为15%有利于提高断奶后日增重,促进饲料利用效率,改善瘤胃内环境,且对犊牛血清生化指标无不利影响。     

Effect of process conditions and amylose/amylopectin ratio on the pasting behavior of maize starch: A modeling approach

The effects of different process conditions on the pasting behavior of the 14%, w/w suspensions of high amylose, waxy and normal maize starches at mixing speeds of 50, 160 and 250 rpm with the heating rates of 2.5, 5 and 10 °C/min were investigated. In addition, the impact of the starch mixture with an amylose-amylopectin ratio of 0–70% at 160 rpm and a heating rate of 5 °C/min on the pasting parameters was studied. According to the results, when stirring speed decreased from 250 rpm to 50 rpm, the peak viscosity dramatically increased. Furthermore, both heating and stirring rates significantly affected the pasting properties (p < 0.05). The amylose content of maize starch had a negative correlation with peak viscosity, trough viscosity, breakdown viscosity, final viscosity, and setback viscosity. Besides, syneresis values decreased as amylose content decreased from 70% to 0%. According to the kinetic modelling of pasting curves, starch coefficients were found to be higher than 1 for all starches, indicating that the penetration of water into starch granules increased granule swelling rate. The findings of the present study confirmed that both process conditions and amylose/amylopectin ratio can be optimized without necessity of starch modification to obtain the products with the desired quality.