大豆异黄酮及其组分含量的遗传分析与QTL检测

以栽培大豆晋豆23为母本,以山西农家品种大豆灰布支黑豆为父本杂交衍生的447个RIL作为供试群体构建遗传图谱,利用高效液相色谱法定性、定量测定样品中的异黄酮及其组分含量。采用主基因+多基因混合遗传分离分析法和Win QTLCart 2.5复合区间作图法,对大豆异黄酮及其组分含量进行混合遗传分析和QTL定位。结果表明,大豆苷、黄豆苷元、染料木素、染料木苷、大豆苷元和异黄酮总含量分别受4、4、2、3、2和2对主基因控制,并有多基因修饰。检测到44个与大豆异黄酮及其组分含量相关的QTL,与大豆苷、染料木素、黄豆苷元、大豆苷元、染料木苷和异黄酮总含量相关的QTL分别有10、9、4、7、8和6个。连续2年分别检测到与大豆苷、染料木苷、黄豆苷元和异黄酮关联,分别位于标记区间satt430~satt359、satt038~satt570、satt197~sat_128和satt249~satt285的稳定表达QTL,可尝试用于分子标记辅助育种。     

中国传统发酵酱油中大豆异黄酮和大豆皂苷质量浓度测定及抗高血压功能

采用高盐稀态发酵法酿造酱油,以高效液相色谱法(HPLC)和紫外分光光度法(UV-Vis),对酱油中的大豆异黄酮和皂苷类物质进行测定。结果表明,大豆苷质量浓度为0.035 4 mg/m L,染料木苷质量浓度为0.000 5 mg/m L,大豆苷元质量浓度为0.006 6 mg/m L,染料木素质量浓度为0.000 6 mg/m L,大豆皂苷的质量浓度为8.942 4 mg/m L。在此工艺条件下发酵的酱油时间较短,并且样品中大豆异黄酮和皂苷的质量浓度较高,研究证明,中国传统发酵酱油对瘦素、脂联素和肾素具有积极调节作用。因此,酱油是一种具有功效成分的健康食品,酱油中有效成分的测定将为下一步确定酱油对高血压的调节机制提供重要参考。     

大豆异黄酮组分HPLC快速分析技术及其在豆腐加工中的应用

大豆异黄酮育种与大豆制品加工研究需要进行大批量样品12种异黄酮组分的快速定量分析。在前人研究基础上利用Agilent 1100高效液相色谱(HPLC)系统,以大豆品种南农95C-13为材料,研究大豆苷元,大豆苷,乙酰基大豆苷,丙二酰基大豆苷,染料木苷元,染料木苷,乙酰基染料木苷,丙二酰基染料木苷,黄豆苷元,黄豆苷,乙酰基黄豆苷,丙二酰基黄豆苷等12种标准品外标法快速定量技术。从样品制备与色谱条件对分析12种异黄酮组分的准确度和分离度入手,确定分析流程,以(科丰1号×南农1138-2)的184个重组自交系(NJRIKY)为材料,研究豆腐加工中总量和各组分的变化特点。(1)样品以80%甲醇水溶液50℃超声1 h提取;色谱条件为,检测波长254 nm, 柱温36℃,流速2.0 mL min^–1, 进样量 10 μL, 流动相0.1%(V/V)乙酸水溶液(A)和100%甲醇(B),0~2 min,27% B (V/V)→2~3 min,27%~38% B→3~10 min, 38% B→10~12 min,38%~39% B→12~14 min, 39% B→14~15 min, 39~27% B梯度洗脱;在15 min内将12个组分良好分离,各组分峰面积与其相应浓度均呈良好线性关系(R²为0.9976~0.9999);加标回收率均大于99%,变异系数低于2%。(2)NJRIKY群体籽粒、豆乳、豆腐异黄酮总量和组分的大量分析验证了HPLC快速技术的效果。籽粒异黄酮总含量(3 695.00 μg g^–1)在豆乳加工中平均85.15%转入豆乳 (3 146.12 μg g^–1),14.85% (548.88 μg g^–1)进入豆渣。传统豆腐加工通过硫酸钙絮凝,只有17.32% (639.89 μg g^–1)转入豆腐,67.83% (2 506.23 μg g^–1)留在黄浆水中。豆乳中12种组分含量比籽粒稍低,均以丙酰基染料木苷含量最高;而豆腐中乙酰基染料木苷和乙酰基黄豆苷缺失,以染料木苷元与大豆苷元含量较高。大豆科丰1号与南农1138-2杂交重组后家系间的异黄酮遗传变异增大,增加了对其遗传改良的潜力。     

异黄酮含量与大豆对大豆蚜虫抗性之间的关系

利用高效液相色谱分析方法测定了10个抗感大豆品种蚜虫取食处理叶片和茎的异黄酮含量。结果表明,蚜虫取食诱导抗虫品种叶片大豆苷、染料木苷和异黄酮总含量增加,而感蚜品种异黄酮含量无明显变化。相关分析表明,大豆品种受害程度与叶片大豆苷、染料木苷和异黄酮总含量呈负相关。但茎的异黄酮含量与大豆品种的抗蚜性无明显相关。     

HPLC法同时测定安徽产野葛、粉葛中6种异黄酮成分

以安徽葛根主产区收集的6种样品(太湖野葛、岳西野葛、金寨野葛、宣城野葛、六安粉葛、亳州葛根)为试材,建立高效液相色谱法同时测定安徽野葛、粉葛中葛根素、大豆苷、染料木苷、大豆素、染料木素、刺芒柄花素6种异黄酮类成分的含量,样品前处理采用30%乙醇超声波提取,采用Phenomenex Column luna C18(2)100A(250 mm×4.6 mm,5μm)色谱柱,以甲醇(A)-水(B)为流动相,梯度洗脱,流速为1 m L/min,柱温35℃,检测波长为250 nm。标准品在范围内呈良好线性关系,相关系数R2分别为0.999 3、0.999 4、0.999 4、0.997 8、0.997 4、0.998 6,最低检出限分别为0.562 7、0.453 4、0.283 0、0.234 0、0.256 0、0.261 0μg/m L,精密度、稳定性和重现性良好,平均加标回收率分别为100.5%、99.3%、104.2%、98.6%、97.1%、104.5%。安徽产区的野葛与粉葛中葛根素等6种异黄酮含量差别显著。该方法准确、快速,操作简单,分离效果好,可用于野葛、粉葛中葛根素、大豆苷、染料木苷、大豆素、染料木素、刺芒柄花素6种异黄酮含量的同时测定。     

大孔树脂分离纯化豆酱中大豆苷元的研究

以豆酱中游离异黄酮大豆苷元为研究对象,经比较选择AB-8型大孔树脂进行大豆苷元的分离纯化。结果显示,最佳动态吸附解析条件为上样质量浓度3 mg/m L,吸附速度2.0 m L/min,洗脱速度2.0 m L/min。在此条件下,大豆苷元的纯度为71.50%。     

大豆异黄酮含量相关SSR分子标记的研究

大豆异黄酮是大豆生长过程中形成的一类次级代谢产物,是一种重要的生物活性物质。本研究以异黄酮含量差异显著的‘中豆27’(4 447 ug/g)和‘台湾75’(2 062 ug/g)为亲本,构建了包括160个家系F2:6高代重组自交系群体。采用高效液相色谱方法测定双亲及群体各单株的不同异黄酮组分的含量。异黄酮不同组别中,大豆苷类含量最高,黄豆黄素苷类含量最低。异黄酮不同存在形式中,丙二酰基葡萄糖苷型含量最多,苷元型含量最少。使用197对在双亲间表现出多态性的SSR引物对RIL群体进行扩增。而后使用SAS程序的Proc GLM命令进行异黄酮含量和标记间的方差分析,在p0.01显著水平上共检测到包括21个SSR标记的41个显著性互作。其中部分标记同时与多个不同的异黄酮成分相关;4个SSR标记与总异黄酮含量相关。本研究为高异黄酮含量育种的分子标记辅助选择奠定了基础。     

高速逆流色谱法分离澳洲薄荷中香叶木苷

为研究澳洲薄荷中香叶木苷的高效制备问题,以澳洲薄荷为原料,建立高速逆流色谱仪分离澳洲薄荷中香叶木苷的方法。澳洲薄荷干燥茎叶用65%乙醇提取,经AB-8 型大孔吸附树脂初步纯化后得到澳洲薄荷总黄酮提取物,然后采用乙酸乙酯-正丁醇-水-乙酸(3:1:3:0.005,V/V)的两相溶剂体系进行香叶木苷单体的高速逆流分离纯化：上相为固定相,下相为流动相,主机转速1600 r/min,流动相流速0.8 mL/min,柱温25℃,检测波长254 nm。分离得到的香叶木苷经液相色谱检测纯度达95.2%。     

黑龙江省大豆籽粒异黄酮含量生态差异

大豆异黄酮由于其特有的生理保健功能而受到日益广泛的关注。为了明确黑龙江省大豆籽粒异黄酮含量的生态差异规律, 2005选定5个生态条件差异明显的试验点及4个大豆品种进行试验。结果表明, 年份、地点、基因型及基因型×环境互作对大豆籽粒异黄酮总含量及3种酸解处理后所得苷元的含量具有显著效应。2006年的大豆异黄酮总含量及3种苷元组分含量的平均值显著高于其他2年;大豆籽粒异黄酮含量不同基因型间及不同地点间均差异显著,大豆籽粒异黄酮总含量及其3种酸解后所得苷元含量与纬度呈极显著正相关。据此认为,大豆籽粒异黄酮含量在黑龙江省存在优势生产区, 对大豆籽粒异黄酮含量进行品质区划是可行的。     

大豆异黄酮对奶牛脾脏与肠系淋巴结中淋巴细胞增殖的影响

为了研究大豆异黄酮对奶牛脾脏与肠系淋巴结中淋巴细胞增殖与活化的影响,并揭示大豆异黄酮提高奶牛免疫性能的作用机理。试验采用6×2（浓度×时间）实验设计,各浓度梯度的大豆异黄酮 (100.00、25.00、5.00、1.00、0.25、0.05 μg/mL)与淋巴细胞共育培养48 h和60 h后,用酶标仪在570 nm处测得各孔的OD值。结果表明：（1）添加0.05~25 μg/mL大豆异黄酮能够促进奶牛肠系淋巴结中淋巴细胞的增殖,在添加量为5 μg/mL时达到极显著的促进作用,但大豆异黄酮添加剂量达100 μg/mL时显著抑制肠系淋巴结淋巴细胞的增殖;（2）100 μg/mL和25 μg/mL大豆异黄酮添加组极显著抑制奶牛脾脏淋巴细胞的增殖(P<0.01),而1 μg/mL浓度组则显著促进奶牛脾脏淋巴细胞的增殖(P<0.05),其余各浓度组对奶牛脾脏淋巴细胞有抑制作用,但差异不显著。     

Methods for evaluation of soybean chilling tolerance at the reproductive stage under artificial climatic conditions

Several evaluation methods for soybean chilling tolerance at the reproductive stage were examined under artificial climatic conditions. Comparisons were made on plants in control and treated plots using three cultivars differing in the level of chilling tolerance. In all methods, plants were grown at 22/17°C (day/night) until first flowering, and then transferred to growth chambers at 24/17°C for control and 15/15°C for chilling treatment, respectively. A method, in which plants were grown at 20/16°C after 4 weeks of the different temperature treatments, proved comparable to the conventional one, in which chilling tolerance at the flowering stage is evaluated using natural and artificial conditions. Another method, in which the plants in chilling treatment plots were grown at 15°C until maturity, also proved usable to evaluate genotypic differences in chilling tolerance independently of maturity time.     

Genistein and Daidzein Concentrations and Contents in Seedling Roots of Three Soybean Cultivars Grown under Three Root Zone Temperatures

Daidzein and genistein are plant-to-bacterium signal compounds involved in soybean nodule formation. They can induce nod gens expression in Bradyrhizobium japonicum. The objective of this study was to determine whether the production of signal molecules was affected by low root zone temperatures (RZTs) in a manner that varied among soybean cultivars. Daidzein and genistein concentrations of soybean seedling roots were measured at three RZTs by high performance liquid chromatography (HPLC). The results indicated that daidzein content and concentration per plant were higher at 15 and 17.5°C than those at 25°C. AC Bravor had higher daidzein contents and concentrations than did Maple Glen and KG20. At 17.5°C. KG20 had higher genistein content and concentration levels than Maple Glen, and no difference existed for the two cultivars at 15 and 25 C. Daidzein contents and concentrations of Maple Glen and AC Bravor increased with harvest time. However, for cultivar KG20, the content and concentration decreased at 19 days after inoculation. Genistein contents and concentrations of the three cultivars increased under each RZT up to the last harvest. There was an interaction between soybean cultivar and RZT for root genistein and daidzein contents and concentrations. The content and concentration of daidzein in soybean seedling roots were much higher (more than five times) than those of genistein.     

In vitro germination and viability of buckwheat (Fagopyrum esculentum Moench) pollen

An in vitro method for the germination of common buckwheat pollen was developed. Pollen grains were successfully germinated in an artificial medium consisting of 0.2 g each of MnSO₄, Ca(NO₃)2.4H₂O and KNO₃, 0.04 g H₃BO₃, 15 g sucrose and 30 g polyethylene glycol (molecular weight approximately 20,000) dissolved in 100 ml of double distilled water. The viability of pollen was assessed by in vivo and in vitro germination tests at 20 °C and 25 °C over a 38 h time period. Pollen grains were collected and germinated at 4 h intervals from freshly harvested flowers grown under 16 h day length and a constant temperature. Maximum pollen viability was found 2 h and 6 h after first light when plants were maintained at 25 °C and 20 °C, respectively. Viability, as measured by germination percentage, was similar at both temperature regimes. Some pollen remained viable for approximately 34 to 38 h in intact flowers, but all pollen lost viability in less than an hour when stored at room temperature without humidity control. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effectiveness of postharvest treatment with chitosan and other resistance inducers in the control of storage decay of strawberry

This study compared the effectiveness of practical grade chitosan when used in solution with acetic, glutamic, formic and hydrochloric acids, and a water-soluble commercial chitosan formulation, in controlling postharvest diseases of strawberry. The commercial chitosan formulation and other resistance inducers based on benzothiadiazole, oligosaccharides, soybean lecithin, calcium and organic acids, and Abies sibirica and Urtica dioica extracts were also tested. The commercial chitosan formulation was as effective as the practical grade chitosan solutions in the control of gray mold and Rhizopus rot of strawberries immersed in these solutions and kept for 4 days at 20 ± 1 °C. Moreover, the treatment with commercial and experimental resistance inducers reduced gray mold, Rhizopus rot and blue mold of strawberries stored 7 days at 0 ± 1 °C and then exposed to 3 days shelf-life. The highest disease reduction was obtained with the commercial chitosan formulation, followed by benzothiadiazole, calcium and organic acids. The compounds that provided the best results in postharvest applications to control storage decay of strawberries, should be tested in further trials through preharvest treatments, applied at flowering and a few days before harvest.     

Effects of short-term storage on germinability of pistachio pollen

The effects of short‐term storage under room conditions (constant 25°C and 35% relative humidity), refrigeration (4°C) and freezing (‐20°C) on the germinability of pollen of pistachio, Pistacia vera L., were studied and the effects of desiccation and pollen age on its germinability were reassessed. It was found that: (1) weight loss as a result of desiccation was positively correlated with germinability; (2) pollen grains stored in room conditions only germinated after prehydration, which restored germinability even after 7 days of exposure; (3) refrigerated (4°C) pollen grains retained their germinability for at least a week; (4) frozen pollen grains irreversibly lost most of their germinability; (5) 2‐day‐old pollen grains, within the anthers, retained their germinability under room conditions. These findings will contribute to the improvement of pistachio pollen preservation and help to find a simple and inexpensive method for its short‐term storage, while retaining its germinability.     

Elevated Ultraviolet‐B Radiation Reduces Concentrations of Isoflavones and Phenolic Compounds in Soybean Seeds

The gradual disruption of the ozone layer in the stratosphere has resulted in increased exposure of plants to ultraviolet‐B (UV‐B, 280–315 nm) radiation. UV‐B radiation is known to affect crop growth and quality negatively. A study was conducted to determine the impact of elevated UV‐B radiation levels on the isoflavones and phenolic compound concentrations of seven soybean varieties. UV‐B radiation significantly reduced the concentration of most isoflavones and phenolic compounds in soybean seeds. Exposure to elevated UV‐B levels overall resulted in 35 % reduction in total isoflavones and 31 % in phenolic compounds concentrations. The effect on individual isoflavones and phenolic compounds depended on the compound and variety, but UV‐B overwhelmingly reduced concentrations. This study suggests that increased UV‐B radiation negatively impacted soybean quality by reducing the concentration of compounds that have health‐beneficial properties.     

Characterization of postharvest treatments with sodium methylparaben to control citrus green and blue molds

The curative antifungal activity of postharvest sodium methylparaben (SMP) treatments against citrus green (GM) and blue (BM) molds was characterized on different citrus species and cultivars artificially inoculated with Penicillium digitatum or Penicillium italicum and incubated at 20 °C and 90% RH for 7 d or stored at 5 °C and 90% RH for 8 weeks plus 7 d of shelf-life at 20 °C. Effective concentrations were selected in in vivo primary screenings with ‘Valencia’ oranges. SMP at 200 mM was tested at 20, 50 or 62 °C for 30, 60 or 150 s in small-scale trials to determine the best dip treatment conditions. Dips of 200 mM SMP at 20 °C for 60 s were selected and applied alone or in combination with 25 μL L⁻¹ of the conventional fungicide imazalil (SMP + IMZ 25). Imazalil at the very low concentrations of 25 (IMZ 25) or 50 μL L⁻¹ (IMZ 50) were also tested. Effectiveness of SMP alone at 20 °C for 60 s was significantly higher on oranges (cvs. ‘Valencia’ and ‘Lanelate’) than on mandarins (cvs. ‘Clemenules’, ‘Nadorcott’ and ‘Ortanique’), with GM and BM incidence reductions of up to 88% after 7 d at 20 °C. SMP was compatible with IMZ 25 and consistently improved its performance, irrespective of citrus cultivars and storage conditions. All treatments were less effective on ‘Clemenules’ mandarins. On ‘Valencia’ oranges stored for 8 weeks at 5 °C and 7 d at 20 °C, the combined treatment was significantly more effective than the single treatments (reductions of GM and BM incidence of about 50–60% and 90–95%, respectively). In additional tests, 200 mM SMP dips at 20 °C for 60 s did not prevent GM on ‘Valencia’ oranges wounded, treated, inoculated with P. digitatum 24 h later, and incubated at 20 °C for 7 d. In contrast, the treatments IMZ 25 and SMP + IMZ 25 showed significant preventive activity. It can be concluded from these results that SMP aqueous solutions, especially applied at room temperature, might be an interesting nonpolluting control alternative to be included in citrus postharvest disease control programs in the future.     

Genotypic differences in utilization of assimilate sources during maturation of wheat under chronic heat and heat shock stresses

Heat stress from chronic, prolonged exposure up to 32 ^°C or heat shock from brief exposure to 33 ^°C and above alters the source of assimilates for grain growth of wheat (Triticum asetivum L.). Our objectives were to identify genotypes that resist chronic heat stress and heat shock and to determine the relative contributions of photosynthesis and stem reserves to grain filling under both conditions. Twenty-eight genotypes were grown in controlled enviroments at 20/15 and 30/25 ^°C day/night in light and darkness during maturation in the first experiment, and six genotypes were grown in light at the same temperatures and at 40/35 ^°C followed by 20/15 or 30/25 ^°C in the second experimnet. Heat susceptibility indices (HSI) were calculated from grain yields of the genotypes in both experiments. The ratio of chlorophyll variable fluorescence to maximum fluorescence (Fv/Fm), a measure of the stability of photosynthesis, and carbohydrate reserves in the stems were measured in the second experiment. Photosynthesis provided 63 and 65% of assimilates in the grain at 20/15 and 30/25 ^°C, respectively, but both stable photosynthesis in some genotypes and high content of reserves in other genotypes were associated with low susceptibility to stress. The Fv/Fm ratio was decreased by heat shock and returned to normal values intolerant genotypes when the treatment was followed by 20/15 ^°C but not 30/25 ^°C. Grain yield was highly correlated among 20/15, 30/25, and 40/35 ^°C followed by 20/15 ^°C treatments, suggesting that similar plant traits were involved. We conclude that assimilates from either stable photosynthesis or high reserve levels provided for high grain yields during heat stress. Combining the two traits could improve heat tolerance of wheat but might not be feasible if other traits are impeded. This revised version was published online in July 2006 with corrections to the Cover Date.     

A Genetic Study of Frost Resistance in Wheat Callus Culture

The genetic control of frost resistance was studied in callus cultures using some of the chromosome substitution lines of the variety ‘Cheyenne’ into ‘Chinese Spring’. The survival of the calli derived from immature embryos was studied with triphenyltet-razolium chloride (TTC) and fluorescein diacetate (5DA) methods after hardening and freezing at a temperatures of -7 °C, -9 °C, -11 °C, -13 °C, and –15 °C. The donor ‘Cheyenne’ and the substitution lines 5A and 5D proved to be more frost resistant than the recipient ‘Chinese Spring1. These results are well correlated with the previously published studies when seedlings were tested under controlled conditions. Based on these results the tissue culture technique seems to be useful for testing varieties and lines for different levels of frost resistance and even for mutant selection.