转基因小麦Puroindoline蛋白SDS-PAGE 方法的研究与应用

小麦嘌呤吲哚蛋白(Puroindoline)是优质遗传特性硬度的生化标记,对小麦磨粉品质起决定性作用.对目前分析此类蛋白的两类Triton-X-114和Tricine-SDS-PAGE系统进行了优化和比较,建立了详细的适合半子粒分离Pin蛋白的电泳方法.采用此法检测转pinA基因的硬粒小麦P34的后代以及转基因硬粒小麦之间杂交子代的外源基因pinA的表达情况,取得了良好的效果.该方法为Puroindoline蛋白的进一步研究奠定了基础,为小麦品质生化和分子标记辅助育种提供了方法.     

籽粒硬度Pinb基因反义表达载体构建及遗传转化

籽粒硬度是小麦品质改良的重要目标性状.利用含有Ubi启动子、Bar表达盒的基础质粒pAHC25,构建了含有Pinb基因的植物反义表达载体pAHantipB,其中pAHantipB载有小麦籽粒硬度Pinb基因的反向序列,可用于小麦遗传转化,利用花粉管通道法将其转入软质小麦品种京411,获得1株转基因植株,T1代转基因植株抗性筛选及PCR12检测结果表明,转化率为0.17%.基因组DNA斑点杂交证实了外源基因在受体小麦基因组中的整合.     

转基因下游MAR在稳定转化的CHO细胞中对基因表达的调控作用

[目的]探讨转基因下游MAR在稳定转化的CHO细胞中对基因表达的调控作用。[方法]将PCR扩增得到的人β-珠蛋白MAR插入到真核表达载体pCATG的下游，构建转基因表达盒3’端含MAR的表达载体。酶切鉴定正确后，转染CHO细胞，G418筛选出稳定转化的细胞株，ELISA分析转基因的表达水平，半定量PCR分析转基因相对拷贝数。[结果]结果表明，表达盒3’端含MAR序列能使转基因表达水平降低，其基因拷贝数则有一定程度的增加。转基因下游β-珠蛋白MAR在一定程度上能抑制外源基因的表达水平；外源基因表达量与基因拷贝数不成正比，未呈现出“拷贝数依赖性”。[结论]该研究结果为进一步研究MAR的调控机制奠定基础。     

不同质地土壤对小麦籽粒容重及部分面粉品质的影响

[目的]研究不同质地土壤对小麦籽粒容重及面粉品质的影响。[方法]采用池栽试验,将3个小麦品种(强筋:藁麦8901、中筋:豫麦49和弱筋:洛麦1号)分别栽培于粘、壤和砂3种质地的土壤中,研究不同质地土壤对小麦籽粒容重及面粉品质的影响。[结果]粘土种植条件下洛麦1号和豫麦49的籽粒容重最高(分别为789.07、84.0 g/L),而藁麦8901的籽粒容重最低(791.0 g/L)。3种筋型小麦蛋白含量和沉降值在粘土种植条件下均达到最高值,且蛋白含量在粘土和壤土间差异不显著,而沉降值在3种土壤中差异显著,壤土种植时最低;面粉出粉率为粘土种植条件下最高,壤土次之,砂土最低。[结论]不同质地土壤对不同筋型小麦的影响不尽相同。     

利用实时荧光P CR技术快速检测玉米籽粒中转基因成分

[目的]利用Taq Man特异性荧光标记法快速检测玉米籽粒中转基因成分。[方法]外源基因选取具有一定代表性的Ca MV35S启动子、NOS终止子、BAR、Cry IA(b)基因,数据结果由实时荧光定量PCR反应的Ct值判定。[结果]用该方法检测玉米籽粒中未含有转基因成分,可作为结果判定。[结论]该方法在样品量大时极具优势,高效,准确,对环境污染较小。     

番茄rbcS3A启动子调控的外源基因在转基因番茄中的表达模式(英文)

[目的]研究番茄rbcS3A启动子调控的外源基因在转基因番茄中的表达模式。[方法]以PCR方法克隆番茄Rubisco小亚基启动子rbcS3A,并构建Rbcs3A-Gus嵌合表达载体p1300RG,将重组载体用农杆菌介导的方法导入到番茄中,最后通过对阳性苗进行Gus活性检测,分析rbcS3A启动子驱动的外源基因在番茄中的表达模式。[结果]试验获得15株PCR阳性苗,阳性苗的获得率为33.3%;Gus活性染色结果显示,番茄rbcS3A控制的外源基因能够在转基因番茄成熟的叶片中高效表达,在果实等生殖器官中表达较弱,在种子中不表达。[结论]调整后的试验方案更有利于获得阳性苗;证实了启动子rbcS3A能够驱动外源基因在番茄营养器官叶片中高效表达而在种子等生殖器官中不表达或表达极低。     

转TaDREB3a大豆品系KD1遗传稳定性分析及抗旱性鉴定

利用基因工程技术将抗旱基因转入大豆,培育高抗旱大豆品种,可提高大豆总产量.抗旱转基因大豆KD1是通过农杆菌介导法将来源于小麦TaDREB3a基因导入大豆受体品种垦农18中获得的转基因大豆材料.利用PCR方法,对转基因大豆KD1连续3代(T2～T4代)转基因大豆进行特异性PCR检测验证.结果表明,TaDREB3a和bar基因在KD1后代中稳定遗传.进一步对外源基因作表达分析,连续3代(T2～T4)qRT-PCR检测结果表明,外源基因TaDREB3a和标记基因bar在转基因大豆叶、茎、根和籽粒中均表达且稳定遗传,且TaDREB3a基因在叶片和根中表达水平较高.Western blot结果表明,TaDREB3a和bar基因翻译的外源蛋白在转基因材料后代中稳定表达.对连续2代转基因材料抗旱性分析表明,KD1在芽期具有较强抗旱性,且其抗旱性状显著高于对照且稳定遗传.对转基因大豆KD1作靶标及非靶标除草剂抗性及耐性分析得知,外源基因bar在转基因大豆中稳定表达且在受体垦农18中表达TaDREB3a基因未改变非靶标除草剂抗性.     

Pina和Pinb融合基因表达载体的构建及其在硬粒小麦中的转化

 【目的】构建Pina、Pinb融合基因表达载体，验证Pina和Pinb基因的功能，为利用转基因技术改良小麦籽粒质地提供理论依据和基础材料。【方法】以软质小麦京411作为Pina和Pinb基因的供体，将Pina、Pinb基因融合后，与基础质粒pG4AB上的Ω和poly(A)等增强基因表达的调控序列及pAHC25上的Ubiqutin启动子和bar基因表达盒相连接，构建Pina、Pinb融合基因植物高效表达载体。利用基因枪转化硬粒小麦幼胚，并对硬粒小麦幼胚再生体系进行探索。【结果】在构建完成Pina、Pinb融合基因植物高效表达载体pFUBPaPb的基础上，转化硬粒小麦幼胚16 000多枚，经biolaphos筛选、PCR鉴定和斑点杂交鉴定，得到再生植株2 390株，35株为阳性植株。共得到T1代籽粒356粒，对种子量较多的16个单株籽粒进行蛋白表达分析，有2株检测到基因表达产物，其籽粒SKCS硬度值比受体品种分别降低了10和12。培养基SD2适于硬粒小麦幼胚愈伤组织的诱导，MS+8 mg•L-1玉米素可获得较为理想的再生频率。【结论】Pina和Pinb的共同表达降低了硬粒小麦籽粒硬度，具有软化籽粒质地的功能。     

不同方法转基因动物的外源基因甲基化分析(英文)

[目的]检测不同克隆方法所获得的转基因绵羊的外源基因及其启动子的甲基化水平,以及蛋白表达的差异。[方法]通过体细胞克隆、干细胞克隆和卵周隙内注射法将外源基因增强型绿色荧光蛋白(enhanced green fluorescent protein,eGFP)和成纤维细胞内生长因子5(fibroblast growth factor5,FGF5)分别导入绵羊体内,获得转基因绵羊,以CMV为启动子,利用完全重亚硫酸盐沉淀测序法检测绵羊尾部组织外源基因启动子及编码区甲基化状态,Western blot检测外源基因的蛋白表达水平。[结果]启动子区的甲基化水平最高的是干细胞克隆,其次是体细胞克隆,卵周隙注射最低;eGFP编码区的甲基化水平以卵周隙注射最高,其次是干细胞克隆;FGF5编码区甲基化水平体细胞克隆高于卵周隙注射法。外源基因蛋白的表达量同其启动子区域的甲基化水平呈负相关关系。[结论]该研究结果提示不同的克隆方法可能会影响外源基因的甲基化水平,从而影响到外源基因的表达。     

转反义trxs基因小麦籽粒发育过程中基因表达差异

为探讨反义trxs基因在转基因抗穗发芽小麦中的作用机制,以转反义trxs基因和非转基因小麦开花后5~30 d的籽粒为材料,采用mRNA差异显示技术分析了转基因与非转基因小麦在籽粒形成过程中的基因表达差异。结果表明,在籽粒发育过程中,转基因与非转基因小麦存在明显的基因表达差异,差异可归纳为转基因增强型、转基因减弱型、转基因特异型和沉默型4种类型,而且特异型表达明显高于增强型和减弱型;差异条带数随种子成熟度的增加而不断增多,到花后第25天达到最高(77条),然后略有下降,且在胚乳中的差异明显高于胚中的。     

Web2.0,以个人为中心的互联网时代的到来

本文介绍了Web2．0的知识和背景，对Web2．0出现的基础以及典型技术和应用作了阐述，同时对其作出了前景展望。     

Virus Location and Lymphocyte Apoptosis in Lymph Nodes of Piglets Infected with PCV-2

The present study has been performed to understand the location of the virus, type of apoptotic cells, and their relation to lymph nodes of piglets infected with porcine circovirus type Ⅱ （PCV-2）. Nine 32-day-old conventional piglets free of infection with PCV-2 were used, and distributed into three groups： control group （n = 3）, piglets inoculated with PCV-2 alone （PCV-2, n = 3）, and PCV-2 inoculated and KLH immunostimulated group （PCV-2 ＋ KLH, n = 3）. Superficial inguinal lymph nodes from all piglets were collected for histological examination after 32 days postinoculation, and immunohistochemistry for PCV-2 detection. Location of apoptotic cells was detected with TdT-mediated dUTP nick end labeling （TUNEL） and cell cycle, and the apoptotic rates were measured by flow cytometry. The characteristic histopathological lesions of the piglets in PCV-2 and PCV-2 ＋ KLH were lymphocyte depletions in the cortex and paracortex of the lymph nodes, epithelioid-like macrophage infiltration, and intracytoplasmic inclusion bodies presented in epithelioid-like macrophages. PCV-2 was mainly found in epithelioid-like macrophages by immunohistochemistry. In the lymph nodes, lymphocytes presented higher apoptotic rates in the cortex by TUNEL, special B-cell areas, and similar apoptotic cells were found in this compartment in the control. The apoptotic rates of the lymph nodes were 0.41, 3.34, and 4.88% in the control, PCV-2, and PCV-2 ＋ KLH groups by flow cytometry, respectively. The apoptotic rates of lymph nodes for PCV-2 and PCV-2 ＋ KLH piglets were significantly higher than those for the control group （P〈0.05 and P〈0.01）. The proliferation index （PI） was 0.17_＋0.01, 0.12_＋0.01 and 0.12_＋0.04 in the control, PCV-2, and PCV-2 ＋ KLH group, the PI of the control group was higher than that of the other groups, but without the statistical difference. PCV-2 can induce lymphocyte depletion in lymph nodes of piglets by blocking cell proliferation and promoting apoptosis. This is one o     

市级综合气象信息分析与管理系统的设计与实现

以抚顺地区3个国家基本站50余年气象资料为依托，采用Microsoft Excel工作表实现由气候和农业气候2部分构成的气象与农业综合信息处理系统的基本思路、方法、内容、用途和使用情况。为了实现气候分析及应用的科学化、客观化、系统化、规范化、程序化及连续化的业务目标，按业务流程设计与实现的，包括资料处理、气候趋势模拟、气候预测、气候鉴定、季节性信息化处理、农作物产量预报、气候生产潜力分析等一系列内容在内的系统建设。关键是解决了大量的计算问题，建立了区域性通用模式。     

Multi-variable grey model (MGM (1,n,q)) based on genetic algorithm and its application in urban water consumption

Urban water consumption has some characteristics of grey because it is influenced by economy, population, standard of living and so on. The multi-variable grey model （MGM（1,n））, as the expansion and complement of GM（1,1） model, reveals the relationship between restriction and stimulation among variables, and the genetic algorithm has the whole optimal and parallel characteristics. In this paper, the parameter q of MGM（1,n） model was optimized, and a multi-variable grey model （MGM（1 ,n,q）） was built by using the genetic algorithm. The model was validated by examining the urban water consumption from 1990 to 2003 in Dalian City. The result indicated that the multi-variable grey model （MGM（1,n,q）） based on genetic algorithm was better than MGM（1,n） model, and the MGM（1,n） model was better than MGM（1,1） model.     

Molecular detection of virulence genes in Campylobacter species isolated from livestock production systems in South Africa

Campylobacter species are a major cause of foodborne bacterial infections in both developed and developing countries worldwide. Campylobacter jejuni is responsible for the majority of infections. This study was conducted to identify virulenceassociated genes in Campylobacter species isolated from livestock production systems in South Africa. A total of 250 fecal samples consisting of cattle(n=50), chickens(n=50), goats(n=50), sheep(n=50) and pigs(n=50) were randomly collected from livestock in Eastern Cape and KwaZulu-Natal provinces of South Africa between April and October 2018. The samples were analyzed for the presence of virulence genes in Campylobacter species using molecular PCR-based methods. It was found that 77 and 23% of Campylobacter jejuni and Campylobacter coli respectively were isolated from all the livestock samples. There were positive significant(P0.05) correlations amongst all the virulence genes that were investigated. Chisquare and Fisher's exact tests were implemented to test for the effect of livestock species on the presence or absence of virulence genes. The study demonstrated that most of livestock species can potentially cause zoonotic infections and food poisoning due to the high prevalence of Campylobacter. The high prevalence of virulence genes highlights the significance of Campylobacter in livestock production systems in South Africa. This requires the implementation of one-health approaches to reduce the impact of foodborne and zoonotic diseases for the welfare of human and animal health.     

不同施肥水平对木薯氮磷钾养分积累、分配及其产量的影响

 【目的】通过比较不同施肥水平下木薯氮磷钾养分积累、分配和产量的差异，探讨粤北坡岗地优质高产木薯氮磷钾养分的最佳用量。【方法】以木薯品种南美119为材料，采用“3414”方案，在粤北翁源进行大田试验。试验共设14个处理。【结果】施肥处理的氮素主要分配到地上部，不施肥处理的氮素则主要分配到根部；不同施肥处理的磷素均主要分配到地上部；不施肥处理和不施钾处理的钾素主要分配到根部，不施氮处理的钾素在根、冠间分配较均衡。在木薯不同生长阶段，植株氮磷钾含量均呈下降趋势，但施氮、磷、钾化学肥料处理植株氮磷钾含量的下降速度小于不施氮、磷、钾化学肥料的处理。处理N2P2K2的氮、磷、钾含量和积累量在各生育期均为最大，不施肥处理（N0P0K0）的氮、磷、钾含量和积累量在各生育期均为最小（P＜0.05）。产量最高的是N2P3K2处理，为22 694.06 kg·hm-2，其次是N2P2K2处理，为21 417.87 kg·hm-2。【结论】木薯产量、氮磷钾养分积累及其在根冠间的分配，对施肥水平高度敏感, 适当比例的氮磷钾肥配合施用既可以显著增加木薯植株的氮磷钾含量和积累量，又可以提高产量。在粤北坡岗地木薯生产中，氮素最重要，在氮素得到满足的条件下，磷素较钾素更重要。在本试验条件下，氮磷钾养分最佳用量为358.80 kgN·hm-2、89.10 kg P2O5·hm-2和187.50 kg K2O·hm-2。     

红苕饲粮中添加铜和/或喹乙醇对猪生产性能及某些血液生化指标的影响

选体重约38公斤的长白(♂)×成华(♀)杂交猪20头,随机分为四组,饲予四种不同的饲粮,研究饲粮中添加铜和/或喹乙醇对猪生产性能和某些血液生化指标的影响。结果表明:基础组、加铜组、加喹乙醇组和两者同时加组全期日增重分别为674、761、796、678克,日采食量分别为2.50、2.65、2.74、2.40公斤,饲料利用率分别为3.73、3.49,3.44、3.54,添加铜和/或喹乙醇对试验第65天血清铜兰蛋白、尿素氨及屠宰率、背膘厚和眼肌面积无显著影响(p>0.05);添加铜使65天血清碱性碱酸酶活性极显著增高(p<0.01),添加喹乙醇有使之升高的趋势,两者同时添加时,碱性酸磷酶活性降低。     

Current progress on genetic interactions of rice with rice blast and sheath blight fungi

Analysis of genetic interactions between rice and its pathogenic fungi Magnaporthe oryzae and Rhizoctonia solani should lead to a better understanding of molecular mechanisms of host resistance, and the improvement of strategies to manage rice blast and sheath blight diseases. Currently, dozens of rice resistance (R) genes against specific races of the blast fungus have been described. Among them, ten were molecularly characterized and some were widely used for breeding for genetic resistance. The Pi-ta gene was one of the best characterized rice R genes. Following the elucidation of its molecular structure, interaction, distribution, and evolution, user friendly DNA markers were developed from portions of the cloned genes to facilitate the incorporations of the Pi-ta mediated resistance into improved rice varieties using marker assisted selection (MAS). However, rice blast is still a major threat for stable rice production because of race change mutations occurring in rice fields, which often overcome added resistance based on single R genes, and these virulent races of M. oryzae pose a continued challenge for blast control. For sheath blight, progress has been made on the exploration of novel sources of resistance from wild rice relatives and indica rice cultivars. A major quantitative trait locus (QTL), named qSB9-2, was recently verified in several mapping populations with different phenotyping methods, including greenhouse methods. The ability to identify qSB9-2 using greenhouse methods should accelerate the efforts on the qSB9-2 fine mapping and positional cloning.     

仙人掌超氧化物歧化酶提取与纯化研究

以仙人掌为试验材料,采用硫酸铵分级沉淀法得到超氧化物歧化酶（Superoxide Dismutase,SOD）粗酶液。然后用Sephadex（葡聚糖凝胶）G-75层析柱层析对粗酶液进行进一步纯化,最后用邻苯三酚法测定酶活力,并对酶活力主要影响因素（包括pH值、温度、抑制剂）进行了研究。结果显示仙人掌SOD酶比活力为751.56U.mg-1。pH值为7.5~8.5时酶活力最大,稳定性也好。温度维持在25~30℃时酶活力最高。由于过氧化氢和氰化钾均能使仙人掌SOD的酶活力显著下降,因而可以初步判断从仙人掌中提取到的SOD是Cu.Zn-SOD。     

锌对镉·汞复合污染下豌豆幼苗生长的影响

以豌豆为试验材料,采用水培方法研究了锌对镉、汞复合污染下的豌豆种子萌发与生长的影响。结果显示,锌浓度≤100.0 mg/L时促进豌豆种子的萌发,锌浓度为200.0和300.0 mg/L时抑制豌豆种子的萌发。当镉、汞的浓度为3.0 mg/L时,100.0 mg/L锌的解毒效应最明显,表现为能够促进镉、汞胁迫下豌豆种子萌发和幼苗生长,降低豌豆幼苗根尖细胞畸变率,提高豌豆幼苗的叶绿素含量及过氧化物酶活性;但当锌浓度≥200.0 mg/L时,与镉、汞协同迫害豌豆幼苗的生长。