Construction of Fusion Expression Vector Carrying GFP and ZmCIPK

[Objective] The aim was to isolate the CBL-interacting protein kinases(CIPK)from maize(Zea mays L.)and construct the fusion gene expression vector which consisted the ZmCIPK8 and GFP.[Method] The ZmCIPK8 cDNA was successfully cloned by using RT-PCR method.And then,it was connected to the pBlueScript SK(pSK)plasmid,which contained the GFP gene.So that the fusion gene vector pSK-CIPK-GFP was obtained.Then,the fusion gene was connected into the efficient plant expression vector PBI121 to construct the fusion gene expression vector PBI-CIPK-GFP.At last,the recombined expression vector was transformed to Agrobacterium tumefaciems LBA4404 to produce the engineering strain LBA4404-PBI-CIPK-GFP.[Result] The fusion gene expression vector which consisted of GFP and ZmCIPK8 gene and engineering strain LBA4404-PBI-CIPK-GFP were successfully constructed.[Conclusion] The results lays a foundation for further study of subcellular localization of ZmCIPK8,which can help to clarify the molecular mechanism of regulation serious stresses,and also provides an important basis for the research on resistance stress engineering of maize.     

Study on Red Coat Color Gene and Prediction of the Secondary Structure in Chinese Holstein

The nucleotide sequence of the melanocortin-l-receptor （MC1R） gene was studied with the help of the polymerase chain reaction （PCR）, in which the protein structure in Chinese Holstein was predicted, and the molecular mechanism of the red coat color was investigated. Polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） was performed to genotype the individuals. The bioinformatics and biotechnology softwares were used to predict the secondary structure of MC1R. The results showed that the EE genotype was the dominant genotype in Chinese Holstein Black and White herd, whereas, it was ee in Chinese Holstein Red and White herd. The secondary structure of the mutational MC1R protein was changed and the deletion mutation caused an earlier termination in translation, which led to the formation of the red coat color. The allele E was mainly associated with the black coat color, whereas, e was associated with red.     

Design and Experiment of Slave Computer Control System for Applying Variable-rate Liquid Fertilizer

In order to increase the applying rate of liquid fertilizer and reduce environmental pollution, a slave computer control system for applying variable-rate liquid fertilizer was designed. The system used SMC as core processor and electrically controlled pressure regulator as execution component. The characteristic equation of the system was obtained by using classical control theory. Results indicated that the characteristic equation met the requirements of routh-criterion, which indicated the working process of the system was stable. Performance of the slave computer was verified via bench tests. Results demonstrated that there was no significant influence on the response from interclass error. The fertilization error was less than 0.9, and the fertilization accuracy was larger than 97%. The liquid fertilizer emitted by the fertilizing devices had no significant difference in uniformity, which met the demands of the slave computer control system for applying variable-rate liquid fertilizer.     

Anatomical Study an the Development of Strawberry Fruit

The development of strawberry fruit,two cultivars-Gelila and Xiaoshi,was systematically studied by anatomical and embryological methods.The growth dynamics of fruit showed that the two cultivars performed similar changes with their fresh weight volume and structure,it included the first slow growth stage,the rapid growth stage and the second solw growth stage.The fruit was a typical polyachene with a ovule which belonged to a Poligonum type,the growth process of pollen tube from stigma to embryo sac was clearly observed by flourescence microscopy.Embryo development was similar to that of most dicotyle,and the mature embryo was orthotropous;Endosperm development was a nuclear type,and gradually disappeared with development:Seed coat consisted of 3-4 layer cells,and pericarp was composed of 6-8 layer cells which differentiated from ovary wall cell;The structure of receptacle was a typical stem which consisted of epidermis,cortex and vascular cylinder.With development,the cortex and pith of fruit contained plentiful parenchyma tissue,and they were mainly edible sections.     

Effect of Beak Trimming Stress on the Apoptosis and Its Related Protein Expression of Chicken Spleen

Beak trimming was a part of routine husbandry for poultry industry,which was the most efficient and economic way to reduce the incidence of cannibalism.However,the controversy about young chicken trimm...     

Effect of Different Growth State of Broccoli （Brassica oleracea var. italica） on Low Temperature Induction

The productive organ of brocooli is curd which is consisted of main scape which was breviate and fleshy and some numbers of branch curds, whereas every branch curd consist of first grade lateral scape which was breviate and fleshy and other grades lateral scape and flower buds[1].The size and weight of broccoli was main determ ined by the num bers and bulk of branch curds which were associated with the developm ental extent differentiation by them selves closely. So floral bud differentiation …     

Modeling and Kinematics Simulation of Hydro-mechanical Split Path Steering Device of Tracked Vehicle Based on CATIA

Based on the working principle of hydro-mechanical split path of tracked vehicle, a operating gear was developed which was controlled by steering wheel and match with transmission case. Then CATIA software was used to build the three-dimensional model and carry out dynamic simulation of the mechanism. The result indicates that the design of the mechanism fulfills the request.     

Factors Hindering Pakistani Farmers' Choices Towards Adoption of Crop Insurance

This study was conducted to analyze the factors that negatively influence Pakistani farmers’ willingness to participate in crop insurance. Probit model was applied to identify the significant factors which influenced our dependent variable "not willing to participate". The results of the analyses showed that crop insurance premium was the most influencing factor which had positive and significant impact on dependent variable. Similarly dissatisfaction with crop loan insurance scheme, lacking of knowledge about crop insurance, believing of being against Islamic rules and time taking process was also found to be positive and significantly influenced the dependent variable. While limited decision power and limited perils were not found to be significant in the results.     

Application of Interval Algorithm in Rural Power Network Planning

Rural power network planning is a complicated nonlinear optimized combination problem which based on load forecasting results, and its actual load is affected by many uncertain factors, which influenced optimization results of rural power network planning. To solve the problems, the interval algorithm was used to modify the initial search method of uncertainty load mathematics model in rural network planning. Meanwhile, the genetic/tabu search combination algorithm was adopted to optimize the initialized network. The sample analysis results showed that compared with the certainty planning, the improved method was suitable for urban medium-voltage distribution network planning with consideration of uncertainty load and the planning results conformed to the reality.     

Research on Anti-noise Processing Method of Production Signal Based on Ensemble Empirical Mode Decomposition(EEMD)

The grain production prediction is one of the most important links in precision agriculture. In the process of grain production prediction, mechanical noise caused by the factors of difference in field topography and mechanical vibration will be mixed in the original signal, which undoubtedly will affect the prediction accuracy. Therefore, in order to reduce the influence of vibration noise on the prediction accuracy, an adaptive Ensemble Empirical Mode Decomposition (EEMD) threshold filtering algorithm was applied to the original signal in this paper: the output signal was decomposed into a finite number of Intrinsic Mode Functions (IMF) from high frequency to low frequency by using the Empirical Mode Decomposition (EMD) algorithm which could effectively restrain the mode mixing phenomenon; then the demarcation point of high and low frequency IMF components were determined by Continuous Mean Square Error criterion (CMSE), the high frequency IMF components were denoised by wavelet threshold algorithm, and finally the signal was reconstructed. The algorithm was an improved algorithm based on the commonly used wavelet threshold. The two algorithms were used to denoise the original production signal respectively, the adaptive EEMD threshold filtering algorithm had significant advantages in three denoising performance indexes of signal denoising ratio, root mean square error and smoothness. The five field verification tests showed that the average error of field experiment was 1.994% and the maximum relative error was less than 3%. According to the test results, the relative error of the predicted yield per hectare was 2.97%, which was relative to the actual yield. The test results showed that the algorithm could effectively resist noise and improve the accuracy of prediction.     

超表达OsSsr1基因增强烟草耐盐性

本研究从水稻品种R109中克隆了OsSsr1基因（GenBank Accession No.NM₀01065574）,并用土壤根癌杆菌介导的方法,把OsSsr1基因转入本氏烟（Nicotiana benthamiana）中,提高了转基因植株的耐盐性。生理指标测定结果表明:在盐胁迫下,转基因和野生型植株中的丙二醛（MDA）、脯氨酸的含量以及过氧化物酶（POD）和超氧化物歧化酶（SOD）的活性都呈上升趋势,但转基因烟草中MDA含量升高速率明显低于野生型,而脯氨酸的含量以及POD、SOD活性的升高速率明显高于野生型烟草。因此,OsSsr1转基因烟草耐盐性的增强,可能是脯氨酸含量和活性氧清除能力的提高引起的。     

盐胁迫对转ZmPP2C2基因烟草和野生型烟草叶片膜脂过氧化及体内保护系统的影响

以转ZmPP2C2基因烟草和野生型烟草为材料,研究了盐胁迫对两种类型烟草叶片膜脂过氧化及体内保护系统的影响。结果表明,在相同的盐浓度(NaCl 150 mmol/L)下,随着处理时间的延长,两种烟草叶片的相对电导率、MDA的含量、可溶性糖、脯氨酸的积累以及POD的活性都呈上升趋势。但转基因烟草相对电导率和MDA含量较野生型烟草低,可溶性糖、脯氨酸的积累以及POD活性的增加明显高于野生型烟草。SOD的活性均是先上升后下降,但转基因烟草下降幅度小,且保持比较高的活性。     

过表达细叶百合LpNAC6基因增强烟草的耐盐性

目的NAC转录因子是一类具有多种生物功能的新型转录因子，在植物抗逆响应中发挥着重要作用。本文旨在克隆并研究细叶百合LpNAC6基因在逆境胁迫下的表达模式，探究其在烟草中响应盐胁迫的功能。方法本研究采用同源克隆技术克隆得到细叶百合LpNAC6基因，利用生物信息学软件对LpNAC6基因进行分析；通过基因枪法对LpNAC6蛋白进行亚细胞定位；利用实时荧光定量PCR的方法分析LpNAC6基因在不同非生物胁迫和不同组织中的表达模式；构建植物表达载体pBI121-LpNAC6-GFP转化烟草，通过对转基因烟草进行盐胁迫处理验证LpNAC6基因的功能。结果LpNAC6基因长909 bp，编码302个氨基酸，存在一个高度保守的NAM结构域，属于NAC基因家族。LpNAC6为不稳定亲水性蛋白，无信号肽和跨膜结构域，有5个糖基化位点和20个磷酸化位点，亚细胞定位在细胞核。LpNAC6基因与黄褐棉的NAC转录因子进化关系最近。细叶百合中LpNAC6基因对ABA、干旱、低温及盐胁迫均有响应。盐胁迫下，过表达LpNAC6基因的转基因烟草其SOD、POD、CAT的活性和叶绿素、脯氨酸、可溶性蛋白的含量均显著高于野生型。结论细叶百合LpNAC6基因能够响应ABA、干旱、低温、盐胁迫等非生物胁迫，其过表达能够提高转基因烟草在盐胁迫下的代谢活力和抗氧化酶活性，从而增强烟草耐盐性。      

过表达毛白杨线粒体APX基因烟草提高抗逆能力的研究

  目的  为了研究毛白杨线粒体APX（PtomtAPX）在抗逆过程中的作用,本研究对过表达PtomtAPX的转基因烟草进行抗逆研究。  方法  对过表达PtomtAPX烟草和野生型烟草进行干旱、盐、氧化胁迫处理后,测量相对含水量、叶绿素含量、丙二醛含量、APX活性、AsA消耗量、NADP/NADPH比值和SOD活性。  结果  通过对比转基因烟草植株与野生型植株的生长差异发现,在氧化胁迫、盐胁迫和干旱胁迫下,转PtomtAPX基因烟草的APX活性、相对含水量、叶绿素含量、AsA消耗量、NADP/NADPH比值升高量均明显高于野生型,其中转PtomtAPX基因烟草的APX活性为野生型的1.77倍,平均相对含水量为野生型的1.15倍,叶绿素含量为野生型的1.6倍,AsA消耗量为野生型的1.11倍,NADP/NADPH值为野生型的1.18倍,表明过表达PtomtAPX转基因植株清除活性氧的能力更强。  结论  在非生物胁迫下,PtomtAPX能消除H₂O₂,防止细胞损伤,在植物抗逆中发挥重要作用。      

水分胁迫对转ZmPP2C2基因烟草膜脂过氧化及抗氧化酶活性的影响

以转ZmPP2 C2基因烟草和非转基因烟草为材料(NC89),采用聚乙二醇(PEG-6000)处理,研究水分胁迫对转ZmPP2 C2基因烟草叶片膜脂过氧化及抗氧化酶活性的影响。结果表明:在15%PEG-6000模拟水分胁迫条件下,随着处理时间的延长,2种烟草叶片的相对电导度、MDA含量及POD活性都呈上升趋势。但转基因烟草相对电导度和MDA含量上升较非转基因烟草低,POD活性的增加明显高于非转基因烟草。胁迫10 d时与胁迫0 d相比,转基因烟草和非转基因烟草POD分别上升了483.54和212.38%,2种烟草SOD活性均表现为先上升后下降,但转基因烟草下降幅度小,且保持比较高的活性。试验结果说明,干旱胁迫下,转ZmPP2 C2基因烟草叶片具有较高的抗氧化酶活性,膜脂过氧化程度低于非转基因烟草。     

盐胁迫对棉花幼苗保护酶系统活性的影响

在盐胁迫条件下，对耐盐性不同的２个陆地棉品种幼苗子叶和胚根的保护酶活性变化进行了比较。结果表明：盐胁迫条件下，２个品种ＰＯＤ，ＳＯＤ活性在胚根部位均有所增强，子叶部位则有不同程度的降低；耐盐性较强的枝棉３号在盐胁迫初期胚根部位ＰＯＤ、ＳＯＤ活性上升幅度大于耐盐性较弱的鲁棉１０２４，但随盐胁迫时间延长，枝棉３号ＰＯＤ、ＳＯＤ活性又开始下降，而鲁棉１０２４则持续上升；枝棉３号子叶部位ＰＯＤ、ＳＯＤ活性经调节之后均接近对照，而鲁棉１０２４的ＰＯＤ、ＳＯＤ活性则表现为持续下降。结果提示出棉花幼苗对盐渍的适应调节主要表现在根部，但棉花品种耐盐性差异则表现在子叶部位。     

过表达AtNEK6转基因烟草的耐旱耐盐性研究

NEK6基因编码一种丝/苏氨酸蛋白激酶,具有调控植物细胞周期蛋白基因表达和乙烯信号转导的功能,与植物抵抗逆境胁迫相关。将拟南芥AtNEK6基因导入烟草,利用甘露醇和NaCl模拟干旱和盐胁迫环境对T2代转基因烟草进行研究。结果表明,在无胁迫处理条件下,转基因烟草根长、侧根数和鲜重显著高于野生型;在干旱和盐胁迫条件下,转基因烟草根长、侧根数和鲜重均极显著高于野生型,表现出较强的耐旱耐盐性;胁迫处理的转基因烟草叶片叶绿素荧光参数值(Fv/Fm)、SOD和CAT活性以及Pro含量极显著高于野生型,MDA含量极显著低于野生型,进一步证明了AtNEK6基因能够促进烟草的生长和提高转基因烟草对于干旱与盐碱的抵抗能力。     

胡杨PeREM1.3过表达提高烟草耐盐性的机制

目的盐害作为一类非生物胁迫严重危害了农作物的生存以及产量。Remorin作为一类植物特有的蛋白质在植物适应环境过程中具有重要功能。本研究克隆了胡杨remorin蛋白PeREM1.3的编码基因PeREM1.3,并研究PeREM1.3基因在植物耐盐性中的作用。方法笔者将基因构件35S::PeREM1.3转入模式植物烟草中, 在盐胁迫条件下,通过生理生化的方法对表达PeREM1.3的转基因烟草进行基因功能的分析。结果研究显示PeREM1.3蛋白定位于细胞质膜上,其编码基因PeREM1.3的开放阅读框(ORF)长600 bp,编码199个氨基酸。胡杨中PeREM1.3能够响应盐胁迫和渗透胁迫表达上调。结果表明,在烟草中过表达PeREM1.3明显地提高了耐盐性。过表达PeREM1.3的烟草转基因株系中抗氧化物酶如SOD、POD和CAT活性显著提高,降低了活性氧水平,调控活性氧平衡。另外植物抗逆相关基因SOS1、HAK、NHA1、VAG1和PMA4的转录水平显著增高,调控K+/Na+平衡。结论这些结果说明PeREM1.3蛋白通过维持植物的活性氧平衡和K+/Na+平衡来提高植物的耐盐性。      

过量表达棉花GhSAP1提高转基因烟草的耐盐性

【目的】SAP（stress-associated protein）是一类涉及胁迫应答和调控的锌指蛋白。克隆并分析其对逆境胁迫应答的反应,为棉花抗逆育种提供候选基因。【方法】通过电子克隆方法并经RT-PCR验证获得棉花锌指蛋白基因GhSAP1,将带有目的基因的载体质粒通过PEG介导转化棉花叶肉原生质体细胞,瞬时表达分析其编码蛋白的特性及亚细胞定位信息。通过qRT-PCR技术分析目标基因的组织表达特征及非生物胁迫条件下的表达特性。通过农杆菌介导叶盘转化法,经组织培养获得转基因烟草进行异位表达,检测其与抗逆相关的生理指标,证明其提高植物抗逆能力。【结果】GhSAP1 ORF长度为543 bp,编码一条含180个氨基酸残基的多肽。其理论等电点8.97,分子量19.3 kD,具有典型的A20/AN1锌指结构域。亚细胞定位显示该基因编码的蛋白定位于细胞核上。不同组织器官表达分析显示,GhSAP1在根、茎、叶中的表达量高,而在开花后5 d的胚珠中表达量很低。GhSAP1受盐胁迫诱导,高盐处理2 h后表达量最高。利用含100 μg•mL-1 Kan的培养基进行抗性筛选转基因后代,结合目标基因的PCR与RT-PCR验证,获得转GhSAP1的转基因烟草阳性株系10个。目标基因GhSAP1均能在烟草基因组中整合并异位过量表达。随机选择3个转基因烟草株系,盐胁迫处理显示,发芽一周的种子在含200 mmol•L-1 NaCl培养基上胁迫12 d,转基因植株幼苗平均成活率为81.7%,比非转基因对照植株增加近3倍,其平均根长为3.05 cm,极显著高于非转基因对照。利用GhSAP1表达较高的L2纯系进行成株期烟草盐胁迫处理30 d,转基因烟草后代的SOD活性和可溶性总糖含量增幅分别为23.89 U•g-1 FW和8.37 %,均显著高于非转基因对照;与未胁迫处理相比,转基因植株的叶绿素含量降低幅度仅为0.17 mg•g-1 FW,而非转基因对照降低了0.39 mg•g-1 FW;盐胁迫处理后,转基因植株的MDA含量增幅为7.42 nmol•g-1FW,而非转基因对照增幅达20.85 nmol•g-1FW;在盐胁迫下,转基因植株具有更强的K+根部运输到植株绿色组织能力,其地上部的K+/Na+为1.23,显著高于非转基因对照。【结论】GhSAP1在参与植物的逆境应答反应机制中具有重要作用,过量表达GhSAP1可显著提高转基因烟草的耐盐能力。     

盐胁迫对转SacB、BADH基因的美丽胡枝子部分逆境生理指标的影响

为了探讨盐胁迫下外源基因对植物渗透调节的影响,以同时培育的转入果聚糖蔗糖转移酶（SacB）基因、转甜菜碱醛脱氢酶（BADH）基因及未转基因的美丽胡枝子盆栽苗为材料,研究不同浓度(0、0.5%、1.0%、2.0%)NaCl处理对3种试验材料的耐盐性及盐胁迫下的脯氨酸、甜菜碱、可溶性糖、丙二醛含量、过氧化物歧化酶活性的影响。结果表明:在未进行盐胁迫时,3种试材的这几项指标含量没有明显差异,但随着盐胁迫强度的增加,两种转基因的美丽胡枝子在积累脯氨酸、可溶性糖能力方面明显强于非转基因植株,转入BADH基因的美丽胡枝子在积累甜菜碱上要强于非转基因植株及转入SacB基因的植株;尽管在盐胁迫强度增大的情况下,3种植株的过氧化物歧化酶活性增强了,但两种转基因植株的过氧化物歧化酶活性并没有明显大于非转基因植株;两种转基因植株可明显抑制丙二醛在植物体内的快速积累。