脂质体介导pAcGFP-bFADD重组质粒转染Hela细胞条件的优化

用脂质体lipofectamine 2000介导携带有AcGFP报告基因的pAcGFP-bFADD质粒转染Hela细胞,并通过调整质粒DNA、脂质体的浓度及转染时间来优化转染条件,转染后12h在荧光显微镜下计数阳性细胞,MTT法检测各转染条件下Hela细胞的活性。结果表明,将2.4μg的pAcGFP-bFADD质粒与3.0μL lipofectamine 2000制备转染复合物与Hela细胞作用6h可以获得62.3%的转染效率,并且保持较高的细胞存活率（83%）。当质粒DNA与脂质体的量增加至3.2μg和6.0μL时,转染复合物对细胞的毒性作用明显增强,对细胞的抑制率可达42.98%,与对照组相比,差异极显著（P〈0.01）;条件优化后AcGFP-bFADD融合基因于转染后72h达到表达高峰,绿色荧光阳性细胞所占比率达到64%。     

氨甲喋呤(MTX)的直接碘标记方法研究

本文报道了~125I(~131I)抗肿瘤药物氨甲喋呤的制备方法,用直接碘化法——NBS、ChT、Iodogen进行标记,并对这几种方法进行了比较。结果表明,NBS法最好,标记率达90—99％。经Sephadex G-75柱凝胶过滤后得到纯产品,放化纯度为95.4％。所制得的标记产品适用于示踪研究。     

低剂量γ辐射对鸡产蛋性能的影响

1985—1990年间,用~(60)Coγ源辐照滨白鸡种蛋,结果表明,辐照促进受精蛋胚胎发育,孵化率提高6.86％,健雏率提高5.52％,雏鸡增重5—6％,提高辐照当代母鸡产蛋率8.03％,辐照二代(商品代)母鸡开产日龄提前6—7周,拉长产蛋期,72周龄产蛋量提高10.48％。     

水稻全耕层一次性基施氮肥增产的机理

本试验应用15_N示踪技术研究了氮肥不同施用方法对水稻增产的机理。结果表明:全层一次基施法与一次面施法或分次表施法相比,植株对氮素的吸收增多,碳铵利用率提高30.2—84.2％,尿素利用率提高13.8—23.7％;在土壤中的残留率,碳铵增加9.4—29.6％,尿素增加11.4—12.8％,从而有利于提高稻谷产量。     

水稻联合固氮菌质粒的鉴定及固氮(nif)基因定位

应用改进的Casse法提取质粒,鉴定了供试菌株的质粒存在情况:高NH_4~+和N_2培养的Alcaligenes faecalis A_15,Enterobacter cloacae E26,E.cloacaeEnSs和klebsiella planticola DWUL2分别携有1—2个大质粒,分子量在30—200Md之间;K.planficola和Pseudomonas saccharophila含有小质粒;K.oxytocaNG13不携质粒。供试菌株中DNA与nif探针R1 nif DH和Ec nif B-Y均具有同源性。A.faecalis A15,E.cloacae E26和K.oxytoca NG13的nif基因位于染色体上,而E.cloacae EnSs的nif基因则位于一个较大质粒上。     

大麦离体诱变效应的研究

以二稜皮大麦品种舟麦2号和秀四的幼胚及其愈伤组织为离体诱变材料,研究了γ射线不同剂量辐照后的离体培养反应、生理损伤及诱变效应。结果表明,用γ射线辐照离体大麦幼胚及其愈伤组织的适宜剂量是10—20Gy,MR_2代的突变频率可比常规诱变技术(300Gy γ射线辐照干种子)分别提高33.9％—82.1％和32.7％—229.6％;比离体培养技术分别提高84.0％—150.3％和82.4％—352.9％,而且仍可维持相对较好的培养反应。用30Gy辐照幼胚的诱变效果则可提高9—13倍,但严重抑制了离体培养物的再生能力,并导致了MR_1代严重的生理损伤。     

山茶花Camellia japonicalL辐射诱变效应

本试验用~(60)Co γ射线处理山茶花的嫩枝插条,1年生小苗和3—4年生植株,研究其辐射敏感性和诱变规律。结果表明,嫩枝插条的适宜剂量范围为1—3krad,成长植株宜控制在2krad以下。不同品种对射线敏感性不同,所引起的花色、花型突变频率亦不同,其辐度在2.5—12.0％。经3年选育,已获得观赏价值高的突变体。     

施用尿素、红萍对水稻生长及其氮素利用效率的影响

应用~(15)N示踪技术研究了尿素和红萍氮素对水稻分蘖、成穗和产量的影响以及当季水稻对氮素的利用率。结果表明:30十30kgN/ha尿素分别作基肥和分蘖期追肥(U-2),10十20十30kgN/ha尿素分别作基肥、分蘖肥和穗肥(U-3),30十30kgN/ha的红萍分别作基肥和分蘖期追肥(A-2),和不施氮肥对照(CK)处理,水稻分蘖总数分别为465.0,495.0,579.0,403.5万/ha;成穗率分别为81.9％,78.8％,69.8％,74.4％;每穗平均粒数分别为75.8,80.6,78.2,74.7粒。施红萍的稻谷产量与等氮量尿素相当。施用尿素和红萍处理,水稻生长前期平均出蘖速度明显不同,而且水稻不同时期分蘖,其成穗数和每穗粒数均随分蘖时间推移呈逐渐递减变化。试验结果表明:红萍作基肥当季的氮素利用率为41.3—48.2％,高于尿素作基肥的利用率(28.0—31.9％),但尿素作追肥(45.7—46.2％)则优于红萍作追肥(37.5—43.2％)。U-3,A-2,U-2处理当季氮素利用率分别为47.7—50.6％,39.2—45.7％,37.0—38.8％。     

辐射诱发红桔少核的细胞遗传学

研究表明,辐射诱发的少核红桔在有丝分裂前期和中期约有92.5％的细胞出现1—6对体细胞染色体联会。在对照株系中也能看到1—3对同源染色体的非交叉平行联会,仅占观察总数的31.4％,其余大多数细胞中的18条染色体则随机分散在核内;在花粉母细胞染色体行为的观察中,对照红桔终变期和中期Ⅰ约67.0％的细胞为正常的9对二价体,而辐射少核红桔却有67.3％畸变细胞类型,其中7Ⅱ+1Ⅳ构型的细胞占畸变细胞的53.4％;在后期和末期有落后染色体、染色体桥、重组核、三分体、异常四分体、五分体和九分体等畸变类型。红桔辐射少核与辐射导致染色体以易位杂合体为主的畸变有关。     

γ射线对苜蓿离体培养与植株再生的影响

以5个紫花苜蓿品种为材料,用于叶与下胚轴为外植体进行0—16kR的辐射处理,研究了不同照射量γ射线对出愈率、愈伤组织鲜重、体细胞胚诱导率和植株再生的影响。结果表明,子叶外植体的辐射敏感性高于下胚轴,2kR对愈伤组织的形成与生长、体细胞胚的产生和植株再生有不同程度的刺激作用;16kR完全抑制子叶愈、信组织的形成和生长。用英648和集宁两个品种的愈伤组织进行0—8kR的处理,结果发现,低于2kR对愈伤组织的生长和体细胞胚的形成具程度不等的刺激作用,8kR对愈伤组织具致死效应。苜蓿育种工作中离体诱变处理的最佳照射量为4—6kR。     

生物质炭与化肥配施对连作黄瓜产量及肥料利用率的影响

在如皋农科所进行黄瓜大棚试验,设置按常规施肥(计为100％,F)、减肥10％(F-10％)和减肥20％(F-20％)3个施肥水平,每个施肥水平下设置生物质炭添加量为0 t/hm2(CK)、5 t/hm2(C1)、10 t/hm2(C2)、20 t/hm2(C3)、30 t/hm2(C4)和40 t/hm2(C5)6个处...     

利用GFP基因的黄瓜T-DNA插入突变体构建与快速鉴定

为促进黄瓜功能基因组学研究,本研究开展了黄瓜T-DNA插入突变体快速构建与鉴定方法的研究。基于对黄瓜遗传转化体系农杆菌侵染活力和共培养条件的优化,本研究以绿色荧光蛋白GFP基因为插入片段,进行黄瓜T-DNA插入突变体的创制,并利用荧光显微镜进行快速鉴定,最后采用TAIL-PCR技术对突变体进行分子鉴定,确定突变位点。结果表明,选取农杆菌侵染液在活化至12~18 h时具有最佳的活力和浓度,而最佳共培养温度为23℃。采用体式荧光显微镜快速鉴定,从2 978个转化外植体中鉴定得到3株表达GFP再生苗,转化率为1.00‰。TAIL-PCR成功鉴定了一个突变体,其插入位点位于CsaV3_1G032940基因的第10个外显子中,该基因在拟南芥中的同源基因为AT4G18950 (blue light-dependent H⁺-ATPase phosphorylation 1,BHP1),参与蓝光介导的气孔开放过程,但在正常栽培环境中该突变体的T0植株与野生型无差异。本研究结果对黄瓜功能基因组学研究具有重要意义。     

不同增氧水平对夏玉米生长及氮素利用的影响

为探究有利于夏玉米生长和氮素利用的适宜灌溉水溶解氧浓度,本试验以夏玉米为供试作物,采用地下滴灌供水方式,以地下水灌溉为对照,设置10(OA10)、20(OA20)和40(OA40) mg·L^-1灌溉水溶解氧浓度3个增氧水平,研究不同增氧水平对盆栽夏玉米生长、产量和氮素利用的影响。结果表明,增氧地下滴灌显著提高了土壤溶解氧浓度,与对照相比,OA40、OA20和OA10处理土壤溶解氧浓度平均提高14.83%、9.71%和8.00%,表现为作物生长增强,作物产量和氮素利用效率均显著提高(P<0.05)。与对照相比,OA10处理的株高、叶鲜质量和叶干质量分别增加7.39%、16.30%和12.02%;OA40处理叶干质量和茎鲜质量分别增加15.82%和12.43%;OA10、OA20和OA40根系鲜质量分别增加60.00%、17.66%和52.98%,根系体积分别增加34.03%、14.56%和51.32%;OA10和OA20处理根系活力分别增加272.77%和64.44%;OA10和OA40处理产量分别增加24.46%和21.83%,水分利用效率分别提高19.10%和21.61%,百粒重分别增加17.53%和15.14%。OA10和OA40处理籽粒氮素吸收量较对照分别增加63.90%和35.27%,OA10处理籽粒氮素分配比例和氮素吸收效率分别增加21.57%和33.33%。上述差异均具有统计学意义(P<0.05)。综上,增氧地下滴灌可显著提高作物根区溶解氧浓度,促进作物生长,提高产量及氮素吸收利用,以OA10处理效果最为显著。本研究结果为增氧灌溉技术在实际生产中的合理利用提供了理论依据。     

Preparation of a tea polyphenol nanoliposome system and its physicochemical properties

Tea polyphenol is rich in green tea with diverse biological activities. However, its application in the food industry is limited due to its instability toward oxygen and light. In this study, the preparation of tea polyphenol liposome by the thin film ultrasonic dispersion method was performed in order to enhance the bioavailability of tea polyphenol. The process conditions were optimized using response surface analysis, and the optimal parameters were as follows: ratio of tea polyphenol to lecithin, 0.125:1; ratio of lecithin to cholesterol, 4:1; phosphate buffered saline (PBS) pH, 6.62; ultrasonic time, 3.5 min. The theoretical and practical entrapment efficiency were 60.36% and 60.09 ± 0.69%, respectively. Furthermore, physicochemical properties including size distribution, zeta potential, permeability, infrared spectrum and in vitro release of liposomal formulations were determined. The mean size of tea polyphenol liposome was 160.4 nm, and the ζ-potential value was -67.2. The tea polyphenol liposome was formed by physical interaction, and the in vitro release process followed a first-order equation. The results indicated that the prepared tea polyphenol liposome was stable and suitable for more widespread application.     

Production of polyclonal antibodies against ochratoxin A and its detection in chilies by ELISA

Polyclonal antibodies were produced for Ochratoxin A (OA) by injecting OA-bovine serum albumin (BSA) conjugate subcutaneously at multiple sites into a New Zealand White inbred rabbit. Antiserum could be used at a dilution exceeding 1:100 000 in an indirect competitive enzyme-linked immunosorbent assay (ELISA), and detected OA concentrations up to 0.1 ng/mL. The 50% inhibition binding (I(50)) of OA was 5 ng/mL. Antibodies did not react with ochratoxin B, coumarin, 4-hydroxycoumarin, L-phenylalanine, and aflatoxin B1. OA contamination in chilies (Capsicum annum L.) collected from commercial markets and cold storage units was determined. The mean recoveries from OA-free chilies spiked with 1 to100 microg of OA per kg of chili sample were 90-110% with a standard deviation of <10%. Of 100 chili samples tested, 26 were found to contain over 10 microg/kg of OA. In 12 samples the OA concentration varied from 10 to 30 microg/kg, in 10 samples from 30 to 50 microg/kg, in 3 samples from 50 to100 microg/kg, and in one sample it was 120 microg/kg. This is the first record in India of OA in chilies, a major component of cooked foods in this country, and it is noteworthy that OA contamination exceeded the permissible limit for human consumption of less than 20 microg/kg in over 26% of the market samples tested.     

Optimization in the preparation of coenzyme Q10 nanoliposomes

The optimal formulation of coenzyme Q10 (CoQ10) nanoliposomes and the feasibility of production in a pilot scale were investigated. The nanoliposomes were prepared by ethanol injection and sonication techniques for a desired vesicle size in the laboratory. Optimization of formulation in the preparation of CoQ10 nanoliposomes was achieved by an orthogonal array design. The best formulation was found to be phospholipid/CoQ10/cholesterol/Tween 80 (2.5:1.2:0.4:1.8, w/w) with phosphate buffer solution (pH 7.4, 0.01 M) as the hydration media. The z-average diameter (D(z)) was about 68 nm. The encapsulation efficiency was greater than 95% with a retention ratio higher than 90% and a particle size change lower than 10% after storage at 4 degrees C in the dark for 90 days. CoQ10 incorporation resulted in a dramatic increase of the microviscosity of nanoliposomes and inhibited the peroxidation of phospholipid. The D(z) of CoQ10 nanoliposomes produced in a pilot scale was about 67 nm. Results suggest that the technology developed by this investigation is practical to produce the CoQ10 nanoliposomes with the expected encapsulation quality and stability not only in the laboratory but also in a pilot scale.     

灌水量对有机基质栽培黄瓜生长及氮素利用的影响

为提高有机基质栽培水分精细化管理水平,采用醋糟基质为黄瓜栽培基质,以‘碧玉2号’黄瓜为材料,在控制灌水量(T)分别为基质最大质量含水率的40%≤T1<55%、55%≤T2<70%、70%≤T3<85%和85%≤T4<100%的条件下,研究了不同灌溉水平处理对黄瓜植株生长、干物质积累、光合作用能力、产量、水分及氮素生产率的影响。结果表明:随着灌水量的增加黄瓜产量增加但水分利用效率下降,T4灌溉处理产量最高但其水分利用效率最低,T1处理水分利用效率最高但其产量最小;T2,T3和T4处理间叶片氮质量分数差异不显著,根系氮素质量分数间差异也不显著,T1处理果实氮素质量分数显著高于其他处理,而茎、叶氮素质量分数又显著低于其他处理,不同灌溉水平处理对有机基质栽培黄瓜氮素产量生产效率影响不显著。该文研究结果可为有机基质栽培的水分精确化管理提供技术支持和依据。     

硅促进盐胁迫下黄瓜NHX1基因表达及Na+在液泡中的区隔化效应

  【目的】   硅可提高植物的耐盐性,但不同植物中硅提高耐盐性的机理并不相同。探究硅对盐胁迫下黄瓜幼苗的氧化损伤、Na+积累和激素水平的影响,以阐明硅提高黄瓜耐盐性的机制。   【方法】   以基因型为Mch-4的黄瓜幼苗为试材,进行水培试验。营养液中NaCl的胁迫浓度为65 mmol/L,施硅水平为Na₂SiO₃·9H₂O 0.3 mmol/L。在处理10天后,测定黄瓜幼苗生物量、Na+含量与分配、Na+转运相关基因表达水平及激素含量。   【结果】   施硅可改善盐胁迫下黄瓜幼苗的生长,减轻植株的氧化损伤。硅对盐胁迫下黄瓜根系和叶片Na+含量无明显影响,可显著降低根和叶中质膜Na+/H+反向转运蛋白基因SOS1的表达量,对高亲和力钾转运蛋白基因HKT1的表达均影响不大,但促进了液泡膜Na+/H+反向转运蛋白基因NHX1的表达。对盐胁迫下黄瓜叶片Na+的亚细胞定位发现,硅处理使叶绿体中Na+含量下降,而液泡中Na+含量升高。硅处理提高了盐胁迫植株根和叶片中赤霉素、生长素和细胞分裂素的水平。   【结论】   施硅可提高液泡膜Na+/H+反向转运蛋白基因NHX1的表达,将Na+区隔化于液泡中,进而降低叶绿体中的Na+含量,缓解盐胁迫下黄瓜幼苗的氧化损伤;硅还诱导产生了较多的赤霉素、生长素和细胞分裂素,其调控Na+积累和黄瓜幼苗的氧化损伤的机理还需进一步研究。     

脂质体法构建广西巴马小型猪转基因体细胞及转基因克隆胚的生产

脂质体法是一种传统的向哺乳动物细胞导入外源基因的方法,然而其转染效率受多种因素影响。本研究将从脂质体和质粒DNA量、转染暴露时间以及混合孵育时间等四方面进行探索,以寻找最佳的转染体系。试验结果表明最佳转染体系为：脂质体1.25μL、DNA 0.7μg、脂质体-DNA混合孵育0 min以及转染暴露6 h。随后我们用优化过的转染体系对新生广西巴马小型猪肾脏成纤维细胞进行转绿色荧光蛋白（green fluorescent protein,GFP）操作,获得了（26.45±2.11）%的转染率,并以此为核供体经体细胞核移植技术成功构建表达GFP的广西巴马小型猪克隆胚胎,同时证实转基因克隆胚的体外发育能力比得上非转基因克隆胚。这为我们构建用于人类疾病研究的基因修饰广西巴马小型猪奠定了基础。     

Antioxidant activity, cytotoxicity, and DNA information of Glossogyne tenuifolia

This study investigates the antioxidant activity and cytotoxicity of Glossogyne tenuifolia extract on various cancer cell lines. The 5.8s DNA of G. tenuifolia was isolated, and the species of this plant was confirmed by NCBI's DNA database. G. tenuifolia was then extracted with ethanol and separated into several fractions using the partition procedure with water, n-butanol, and ethyl acetate (EA). Among these, the EA fraction most significantly affected the activity of DPPH(*) and superoxide anion scavenging. Additionally, only the EA fraction exhibited cytotoxicity on breast cancer cells (MCF-7 and MDA-MB-231) and liver cancer cells (Hep G2 and Hep 3B). Next, the EA fraction was further separated by column chromatography, and 15 fractions were obtained. Three effective components were isolated and identified separately from the active fractions: oleanolic acid (OA) from fraction 6, luteolin from fractions 8-10, and luteolin-7-glucoside from fraction 12. The test of these three compounds on scavenging activity of DPPH(*) and superoxide anion indicates that luteolin had the highest antioxidant activity, whereas the effect of OA was negligible. Additionally, a synergistic effect between luteolin and luteolin-7-glucoside was observed. Kick-out experiments showed that the activities were vanished or decreased. Especially on MDA-MB-231 and MCF-7 cells, the cytotoxicity completely disappeared when luteolin was eliminated from fractions 8-10. These findings demonstrate that luteolin plays a crucial role in the inhibition of the growth of hepatoma cancer cell lines. Fraction 3, which did not contain luteolin, luteolin-7-glucoside, and oleanolic acid, had cytotoxicity on MDA-MB-231, MCF-7, Hep G2, Hep 3B, and A549, which implies that this fraction contained some other effective ingredients and requires further study. The investigation is currently underway in our laboratory.