Cloning of a flower-specific expression promoter from Arabidopsis thaliana and its plant expression vector construction

Given the sequence of Chs gene promoter from Arabidopsis thaliana reported in GenBank （AF248988）, a pair of specific PCR primers was designed with the Primer Premier 5.0 software. PCR products of about 0.5 kb were successfully amplified with the genome DNA of A. thaliana as a DNA template and Taq polymerase as DNA polymerase. The purified PCR products were ligated to the pMD18-T vector. The sequencing result showed that the Chs promoter from A. thaliana was 531 bp long. Sequence alignment analysis based on the DNAMAN software revealed that the sequence similarity between the cloned promoter and target promoter （AF248988） was up to 100%. Online PLACE analysis indicated that the Chs promoter contained cis-elements such as TATA-box, CAAT-box, pollen-box, G-box, ACGT-containing element, R response element, Myb recognition element and TACPyAT-box. At the same time, a plant expression vectorpAtChs：：GUS which fused the Chs promoter and the marker gene GUS was successfully constructed.

Construction of Plant Expression Vector for Recombinant Human Epidermal Growth Factor (hEGF)

ObjectiveThis study aimed to construct plant expression vector for recombinant human epidermal growth factor （hEGF） and further to provide a basis for the expression of hEGF in peanut hairy root system. MethodAccording to the hEGF sequence in GenBank, hEGF was synthesized artificially; subsequently, hEGF gene was ligated with green fluorescent protein （GFP） gene, and their ligation product was then amplified with primers flanked with corresponding endonuclease cleavage sites, followed by double digestion by Sal I and EcoR I of the amplified products; next, pRI 101 AN DNA was extracted and digested by both Sal I and EcoR I; susequently, the digestion products of hEGF and GFP ligation fragment by Sal I and EcoR I and the digestion products of pRI 101 AN plasmid DNA by Sal I and EcoR I were ligated, and their ligation product was transformed into Escherichia coli XL10-Gold, followed by extraction of DNA from the recombinants exhibiting green fluorescence, which was then identified by enzymatic digestion and PCR, and the verified recombinant plasmid DNA was named pBZG101. ResultHuman epidermal growth factor gene （hEGF） and green fluorescent protein gene （GFP） were successfully ligated, and their ligation fragment was successfully ligated to pRI 101 AN DNA, finally with the acquirement of the plant expression vector for recombinant human epidermal growth factor-（pBZG101）. ConclusionThe plant expression vector for recombinant human epidermal growth factor-（pBZG101）- was successfully constructed in this study.

Cloning of H6H Gene from Atropa belladonna and Construction of the Efficient Plant Expression Vector

[Objective] The aim was to clone H6H gene from Atropa belladonna and construct an efficient plant expression vector.[Method] The coding sequence of H6H（Hyoscyamine 6β-hydroxylase）was cloned from Atropa belladonna with RT-PCR.Then,the sequence was subcloned into the reconstructed plant binary expression vector p2301 to construct the recombinant vector p2301-H6H,which was then introduced into Agrobacterium tumefaciens strain LBA4404 and Agrobacterium rhizogenes strain C58C1,respectively.[Result] The engineering bacteria p2301-H6H-LBA4404 and p2301-H6H-C58C1 which could be directly used in genetic improvement were obtained.[Conclusion] The present research provided basis for the increasing of alkaloid content of Atropa belladonna by plant genetic engineering technology.

Construction of Plant Virus Expression Vector pClYVV/CP/W and Expression of GFP

[ Objective] The study was to report the construction of plant virus expression vector pCIYVV/CP/W and the expression of green fluorescent protein（GFP） with pCIYVV/CP/W, and to develop effective plat virus vector for plant bioreactor to produce useful protein. [ Method] A section of multiple cloning sites among NIb/CP genes in pCIYVV genome and deoxyribonucleotide polylinker of cleavage recognition sequence containing viral protease Nla were cloned with infectivity full-length cDNA of clover yellow vein virus （CIYVV）, and pCIYVV/CP/W vector was constructed, GFP gene was inserted into pCIyVV/CP/W to construct the pCIYVV/CP/W/GFP vector. The transcription situation of recombinant virus clone was detected by RT-PCR, and targeted gene products expressed by recombinant virus clone were detected with western blot （WB）. [Result] The broad bean seedling inoculated with pCIYVV/CP/W/GFP expressed the same symptom as wild type CIYVV, morbidity was of 100%, the result showed that recombinant virus clone pCIYVV/CP/W/GFP didn＇t suppress, insertion of foreign gene didn＇t destroy the open reading frame of pCIYVV/CP/W. Foreign gene can keep living in F, progeny virus genorne steadily, recombinant virus clone pCIYVV/CP/W/GFP could steadily express GFP in progeny virus at least.[ Conclusion] The useful plant virus vector was provided for useful protein expressing.

Cloning of CBF3 Gene from Arabidopsis thaliana and Construction of Plant Expression Vector

[Objective] The aim was to clone CBF3 gene from Arabidopsis thaliana and construct plant expression vector pCAMBIA1301-Rd29A-CBF3.[Method] CBF3 gene and stress-inducible promoter Rd29A were amplified from the genomic DNA of A.thaliana for the construction of plant expression vector.[Result] Sequencing results showed that the cloned CBF3 gene had 750 bp,and showed 100% identity with the sequence published on GenBank.The promoter Rd29A had 1 425 bp,and showed 100% identity with the sequence published on GenBank.[Conclusion] Based on the binary vector pCAMBIA1301,the plant expression vector pCAMBIA1301-Rd29A-CBF3 was constructed successfully,which could materially improve the salt resistance,drought-tolerance,cold resistance of plants.

Numerical Analysis of Stochastic Vector Borne Plant Disease Model

We are associating the solutions of stochastic and deterministic vector borne plant disease model in this manuscript.The dynamics of plant model depends upon threshold number P^(∗).If P^(∗)<1 then condition helpful to eradicate the disease in plants while P^(∗)>1 explains the persistence of disease.Inappropriately,standard numerical systems do not behave well in certain scenarios.We have been proposed a structure preserving stochastic non-standard finite difference system to analyze the behavior of model.This system is dynamical consistent,positive and bounded as defined by Mickens.

Cloning of XET gene from Anthocephalus chinensis and its plant expression vector construction

A full-length cDNA sequence of xyloglucan endotransglycosylase gene （XET）, abundantly expressed in the cambium of Anthocephalus chinensis was cloned by conserved PCR, rapid-amplification of cDNA ends and by chromosome walking. Analytical results of the DNA sequence show that a 912 bp complete open reading frame （ORF） encoded a 303-amino acid protein was in the 1205 bp full cDNA sequence. The deduced amino acid sequence of AcXET, which contained the conserved specific EIDFE catalytic site sequence to XETs was homologous to the other known XET proteins. In order to study the gene function of AcXET and obtain transgenic plants, a plant expression vector pBIAcXET was constructed by recombinating the AcXET fragment from the cloning vector pMD19AcXET and the binary vector pBI121 between the XbaI and SmaI sites. The fragment ofAcXET gene was inserted between the CaMV 35S promotor and the coding region of the GUS gene in pBI121. The identification results show that the plant expression binary vector pBIAcXET was constructed successfully. These results lay the foundation for studying the molecular mechanism ofAcXET gene during wood formation.

TURNIP MOSAIC VIRUS COAT PROTEIN GENE: CLONING AND CONSTRUCTION OF THE PLANT VECTOR

Complementary DNAs to turnip mosaic virus (a radish Raphanus stativus isolate) genomicRNA were synthesized using oligo (dT) as primer and cloned into the vector λ-ZAP Ⅱ. Afterhybridization with a single-stranded cDNA probe and the sequencing of inserted DNA,positive clones with poly--A tails were obtained. One clone containing 1429-base pair insertwas sequenced. The coat protein gene was identified based on the molecular weight of theTuMV coat protein and the consensus sequences of the polyprotein processing sites ofpotyviruses. The 5’ end of the coat protein gene was modified by PCR to introduce aninitiation codon, ATG, and two restriction enzyme sites. The gene was then manipulatedinto a binary vector pBIN437 which was derived from pBI121, and the plant expressionvector is being used to transform Brassica napus.

Cloning the Promoter of BcNA1 from Brassica napus and Fad2 Gene from Arabidopsis thaliana and Construction of the Plant Expression Vector

The upstream regulatory region of a seed specific gene was isolated from the genomic DNA of Brassica napus by PCR amplification. The cloned fragment contained 1755 nucleotides, and shared a sequence homology of 99.6% with the reported data. The coding region of oleic acid desaturase gene was then cloned from Arabidopsis thaliana. The sequencing analysis indicated that the sequence of the PCR product was just the same as reported before. In addition, the plant expression vector harboring the seed specific promoter and trans Fad2 gene was constructed.

Construction of Plant Expression Vector of Heat Shock Factor Gene AtHsfA1a from Arabidopsis

[ Objective ] Heat shock factors （HSFs） are the major transcription factors of eukaryotic heat shock responses. This study aims to investigate the adversity stress tolerance functions of Arabidopsis heat shock factor AtHsfAla, which has important significance for in-depth understanding of adversity stress tolerance mechanisms of plants and further utilization of heat shock factor genes. [Method] Genomic DNA of Arabidopsis was extracted with CTAB method and purified to obtain Arabidopsis DNA samples for in vitro site-specific recombination cloning （ Gateway cloning） to construct plant expression vector of heat shock factor AtHs- fAla. Firstly, donor vector pDONR 201/AtHsfAla was constructed based on attB and attP site-specific recombination method （BP reaction）, to identify E. coli transformants harboring correct sequence of AtHsfAla by sequencing; secondly, plant expression vector pBTWG2/AttlsfAla overexpressing Arabidopsis heat shock factor AtHsfAla was constructed based on attL and attR site-specific recombination method （LR reaction）, to screen E. coli transformants harboring target plasmid. [ Result] Plant expression vector of Arabidopsis heat shock factor gene AtHsfAla was constructed successfully. [ Conclusion] This study not only provided experimental materials for acquiring transgenic plants overexpressing heat shock transcription factor AtHsfAla, but also laid the foundation for further investigation of the diversity of adversity stress tolerance functions reanlated by HSFs.

Cloning and Bioinformatics Analysis of ZmERECTA-LIKE1 and Construction of Plant Expression Vector

[Objective] This study was conducted to clone and analyze ERECTA-LIKE1 gene in Zea mays by PCR and bioinformatics methods and to construct plant expression vector p Cambia3301-zm ERECTA-LIKE1. [Method] zm ERECTA-LIKE1(zm ERL1)gene was obtained using RT-PCR, and physical-chemical properties were analyzed by bioinformatics methods, including domains,transmembrane regions, N-Glycosylation potential sites phosphorylation sites, and etc. [Result] Bioinformatics results showed that zm ERL1 gene was 2 169 bp, which encoded a protein consisting of 722 amino acids, 11 N-glycosylation potential sites and 42 kinase specific phosphorylation sites. According to CDD2.23 and TMHMM Server v. 2.0 software, there were leucine-rich repeats,a PKC domain and a transmembrane region in this protein. The theoretical p I and molecular weight of zm ERL1 encoded protein was 6.20 and 79 184.8 using Compute PI/Mw tool. Furthermore, we constructed the plant expression vector p Cambia3301-zm ERECTA-LIKE1 by subcloning zm ERL1 gene into p Cambia3301 instead of GUS. [Conclusion] The results provide a theoretical basis for the application of zm ERL1 gene in future study.

Global Dynamic Analysis of a Vector-Borne Plant Disease Model with Discontinuous Treatment

This paper proposes a vector-borne plant disease model with discontinuous treatment strategies. Constructing Lyapunov function and applying non-smooth theory to analyze discontinuous differential equations, the basic reproductive number R0 is proved, which determines whether the plant disease will be extinct or not. If R0 R0 > 1 , there exists a unique endemic equilibrium which is globally stable. The numerical simulations are provided to verify our theoretical results, which indicate that after infective individuals reach some level, strengthening treatment measures is proved to be beneficial in controlling disease transmission.

大白菜BrCYP83B1基因的克隆及表达分析

【目的】细胞色素P450家族是十字花科植物硫苷合成重要的酶系,其中CYP83亚家族主要参与核心结构的合成,旨在探究大白菜(Brassica rapa ssp.pekinensis)CYP83B1基因的功能。【方法】利用RT-PCR技术克隆BrCYP83B1基因,通过生物信息学软件分析其编码蛋白理化性质、同源性及启动子顺式作用元件,利用RT-qPCR技术分析BrCYP83B1的表达模式,并构建其植物超表达载体。【结果】BrCYP83B1 cDNA序列全长为1500 bp,编码499个氨基酸,编码蛋白属于细胞色素P450超家族,主要定位于细胞质,二级结构主要由α-螺旋和无规则卷曲构成,与甘蓝型油菜、青花菜的CYP83B1蛋白具有较高的同源性。启动子分析表明,该基因启动子区域包含水杨酸、脱落酸及茉莉酸甲酯等激素响应的顺式作用元件,说明BrCYP83B1基因表达可能受激素调控。RT-qPCR分析结果表明,BrCYP83B1基因在大白菜的根、茎、叶、花和果中均有表达,且以叶中的表达量最高;茉莉酸甲酯够显著促进该基因的表达,而水杨酸处理对其表达具有一定的抑制作用,脱落酸处理下基因先上调后又下调。【结论】BrCYP83B1可能参与大白菜对激素的响应调控。

番茄两种虫媒传播的重要病毒病研究进展

近年来,番茄黄化曲叶病毒和番茄褪绿病毒对番茄生产造成了巨大的危害,这两种病毒都主要通过烟粉虱传播。本文对番茄黄化曲叶病毒病和番茄褪绿病毒病的扩散趋势、引起这两种病毒病的病原物特性及其发病症状差异、传播这两种病毒的主要媒介昆虫、烟粉虱媒介的危害及其研究概况、烟粉虱生物型的演化及其鉴定方法、烟粉虱和番茄两种病毒的互作以及烟粉虱媒介和番茄两种病毒病的防控等方面进行综述,以期为番茄病虫害的防控提供科学指导。

环保型水培种植载体性能的试验研究

为筛选出适合立体水培生产的可降解环保型种植载体,本研究对不同种植载体的原料特性、机械特性及栽培效果进行了分析。结果表明,与花泥型、淀粉型种植载体相比,生物质炭型种植载体的机械性能较好,其中生物质炭1的跌落损失率<3.5%、粘附性<0.2 N·mm、回弹率>75%、内聚性>0.5 N·mm、硬度>80 N。其水培特性符合种植标准,pH值为6.68,吸水率为330.52%,保水率为93.64%,经过6周降解后的降解率为9.23%。其在育苗过程中的发芽率为66.67%;植株的壮苗指数最高,为3.91,种植后的EC值为667.33 ppm。通过主成分分析可知,竹炭+竹纤维为原料的生物质炭型载体的综合性能最好,为环境友好型种植载体的研究和应用提供了科学依据和技术支持,具有重要的理论和实践价值。

基于图像处理的水培生菜冠层图像叶面积估测研究

为实现精准、高效、无损地获取植物工厂环境下水培生菜相关长势参数叶面积(Leaf area,LA),基于数字图像处理和机器学习回归方法建立单株水培生菜冠层图像LA估测模型。首先,通过智能手机获取2个生菜品种不同生长期的冠层可见光图像,利用Photoshop图像处理软件将原始图像统一剪裁为900像素×900像素大小,采用中值滤波(MedianBlur)法对剪裁后的图像进行去噪运算,将RGB图像转化为HSV颜色空间,再采用mask掩膜法分割彩色图像;然后,利用图像法获取单株生菜LA实测值,构建以LA实测值为因变量,以生菜冠层投影面积(Projected leaf area,PLA)为自变量的线性回归(Linear regression,LR)模型和以全局图像特征(颜色、形状、纹理等)为自变量的支持向量回归(Support vector regression,SVR)、多元线性回归(Multiple linear regression,MLR)和随机森林(Random forest,RF)等LA估测模型进行对比分析;最后,采用决定系数(Coefficient of determination,R^(2))和均方根误差(Root mean square error,RMSE)评估模型的准确性。结果表明:RF模型估测效果最好,对于生菜品种‘绿萝’单株LA估测结果的R^(2)为0.9714、RMSE为8.89 cm2,对于品种‘碧霄’估测结果的R^(2)为0.9201、RMSE为23.34 cm2。本研究验证了RF回归模型能够较准确地估测生菜单株叶面积,可为植物工厂水培生菜LA无损估测提供新的解决方案和研究基础。

CTM2-GFP转基因拟南芥构建及细胞定位研究

通过筛选突变体库发现的镉耐受突变体植株可能参与了植物Cd胁迫的调控。文章通过同源重组方法构建pART27-CTM2-GFP载体,并将pART27-CTM2-GFP重组质粒转入农杆菌GV3101,采用浸花转染法转入野生型拟南芥植株;通过抗性筛选和聚合酶链式反应(polymerase chain reaction,PCR)鉴定获得CTM2-GFP转基因阳性植株,进行抗性分离鉴定获得纯合转基因植株;最后通过qPCR检测CTM2基因表达,筛选最佳的CTM2-GFP转基因株系;通过观察CTM2-GFP转基因植株中绿色荧光蛋白(green fluorescent protein,GFP)的荧光发现CTM2为细胞膜定位蛋白。研究结果为深入研究CTM2基因在调控植物镉耐受分子机制中的作用提供了遗传材料支撑。

基于无人机遥感通信农业种植数测量系统设计

为了快速且准确地获取农业种植数量信息,基于无人机遥感通信技术对农业种植数测量系统进行了设计。系统主要由无人机飞行平台、飞行控制系统、遥感设备、地面监控站、无线电通讯系统和数据处理系统等几部分组成。对遥感图像信息的颜色和纹理特征的提取方式进行设计,并采用支持向量机(SVM)对种植数进行计算,使系统可以准确测量农业种植数。为了验证测量系统的性能,对其进行种植数测量试验,结果表明:系统对样本点统计的精度较高,可以用于大面积农作物种植数的测量。

拟南芥35S∶NAS2载体构建和转基因株系的筛选

为了研究基因NAS2和BZIP44在拟南芥缺铁胁迫响应中的遗传关系,文章以野生型(wild type,WT)拟南芥为材料构建35S∶NAS2/WT转基因植株,以拟南芥突变体bzip44为材料构建35S∶NAS2/bzip44转基因植株;提取野生型拟南芥的RNA,以其反转成的cDNA作为模板克隆NAS2的CDS区域,将双酶切后的目的片段与同样双酶切的pART27质粒进行连接,得到的连接产物转入大肠杆菌DH5α感受态中,通过菌落聚合酶链式反应(polymerase chain reaction,PCR)鉴定和测序得到阳性单克隆,然后将重组质粒转入农杆菌感受态GV3101中,获得阳性单克隆菌落;用浸花法分别侵染野生型拟南芥和纯合的拟南芥bzip44突变体植株,通过筛选和鉴定得到纯合的35S∶NAS2/WT和35S∶NAS2/bzip44转基因植株。研究结果为基因NAS2和BZIP44在拟南芥缺铁胁迫响应中的遗传关系提供参考。

基于误差修正和VMD-ICPA-LSSVM的短期风速预测建模

精准的风速预测是将风能大规模应用到电力系统中的关键,而风速序列的随机性和波动性等特点使得风速预测难度增加.为增强风速序列的可预测性,采用Logistic混沌映射策略、自适应参数调整策略以及引入变异策略对食肉植物算法(CPA)进行改进,并提出了基于误差修正和VMD-ICPA-LSSVM的短期风速预测模型.首先将气象因子作为最小二乘支持向量机(LSSVM)的输入对风速进行预测,获得误差序列.再利用K-L散度自适应地确定变分模态分解(VMD)的参数,并对误差序列进行分解.结合改进食肉植物算法(ICPA)优化LSSVM可调参数的方法来预测分解的子序列.叠加各子序列预测结果后对原始预测序列进行误差修正,进而得到最终风速预测值.实验结果表明,与其他模型相比,所提模型有着更好的预测精度和泛化性能.