草鱼TGF-β1、Smad4基因的克隆及真核过表达和RNA干扰表达载体的构建

为研究TGF-β1/Smad4信号通路在草鱼(Ctenopharyngodon idellus)肌肉发育过程中的作用机制,首先克隆了草鱼TGF-β1、Smad4基因开放阅读框(ORF,Open reading frame)序列各1 134和1 644bp。其次,在TGF-β1、Smad4序列两端分别加上相应的酶切位点,同时对pcDNA3.1(+)真核表达载体进行双酶切,将带酶切位点的片段正向插入到真核表达载体pcDNA3.1(+)中,构建TGF-β1、Smad4基因的真核过表达载体pcDNA3.1(+)-TGF-β1、pcDNA3.1(+)-Smad4。另一方面,根据TGF-β1、Smad4基因序列全长分别设计3对长度为48bp的shRNA,然后将构建好的shRNA插入到载体pRNA-U6.1/Neo中,即可成功构建TGF-β1、Smad4基因的RNA干扰表达载体pRNA-U6.1/Neo-TGF-β1和pRNA-U6.1/Neo-Smad4。     

植物蛋白融合HA标签通用载体构建与应用

以植物表达载体pCamE(GenBank No.:JX841315)为基本骨架,HA蛋白YPYDVPDYA为标签序列,构建可分别融合目标蛋白N端与C端表达载体pCHAN与pCHAC,并增加有利外源基因插入的酶切位点,使之成为含有7个常见限制性内切酶(PstⅠ、SpeⅠ、XbaⅠ、Bam HⅠ、KpnⅠ、SmaⅠ、ApaⅠ或SalⅠ)单一识别位点的通用型标签载体;为验证该载体实用性,将项目组自主克隆的大豆紫色酸性磷酸酶GmPAP14(GenBank No.:JN967626)连接至上述载体,并转入拟南芥;经PCR检测与测序分析获得T2转基因株系,HA标签抗体Western blotting结果发现转pCHAN-GmPAP14、pCHAC-GmPAP14拟南芥可获得目的蛋白融合HA标签杂交条带,而对照无该条带出现,证明2个HA标签载体能够稳定融合外源基因编码蛋白N端与C端,且可在转基因植物中正常翻译表达,为植物蛋白分离纯化及相关领域研究提供了稳定可靠的通用型融合标签载体资源。     

盐生植物小花碱茅CYP86A基因的RNAi载体构建

拒盐型盐生植物小花碱茅根系具有高度木栓化的内皮层可能是其抵御盐胁迫的重要策略之一。本研究以盐胁迫下小花碱茅幼苗的根为材料,采用RT-PCR方法扩增到木栓质生物合成关键基因CYP86A的RNAi靶片段,以中间载体pHANNIBAL和植物表达载体pART27为基础,采用酶切连接结合In-Fusion的克隆方法成功构建了以35S启动子驱动,含PtCYP86A基因片段反向重复序列的RNAi植物表达载体pARC,为利用RNAi技术深入探究根系木栓化在小花碱茅拒盐中的功能奠定基础。     

Control of potato virus Y (PVY) in seed potatoes by oil spraying,straw mulching and intercropping

Potato virus Y (PVY) is the potato virus with the highest economic impact on seed potato production. Insecticides are efficient in controlling aphids, which are the vectors of this virus, but rarely limit virus spread in the field. Straw mulching and mineral oil spraying are known as alternatives to insecticides to reduce PVY incidence, but important year‐to‐year variation in efficacy has been observed with both of these techniques. Preliminary studies revealed the efficacy of intercropping in controlling PVY spread, but more data are needed to validate this observation. A four‐year field trial was conducted in Switzerland to assess the potential synergistic effect of combining mineral oil spraying with straw mulching to increase the protection of seed potato crops against PVY spread. Furthermore, the efficacy of intercropping with oat and hairy vetch was examined as a novel way to control in‐field PVY spread. The present work demonstrates that the modes of action of mineral oil and straw mulching are complementary and reduce the year‐to‐year variation observed with oil and straw when used alone as PVY control agents. The results also demonstrate the efficacy of intercropping for the control of PVY, and the mode of action of this novel control method is discussed. Overall, this work shows that it is possible to increase the protection of potato fields against PVY spread by combining control strategies with different modes of action that complement each other, such as mulching, oil spraying and intercropping.     

Big Data for weed control and crop protection

Farmers have access to many data‐intensive technologies to help them monitor and control weeds and pests. Data collection, data modelling and analysis, and data sharing have become core challenges in weed control and crop protection. We review the challenges and opportunities of Big Data in agriculture: the nature of data collected, Big Data analytics and tools to present the analyses that allow improved crop management decisions for weed control and crop protection. Big Data storage and querying incurs significant challenges, due to the need to distribute data across several machines, as well as due to constantly growing and evolving data from different sources. Semantic technologies are helpful when data from several sources are combined, which involves the challenge of detecting interactions of potential agronomic importance and establishing relationships between data items in terms of meanings and units. Data ownership is analysed using the ethical matrix method to identify the concerns of farmers, agribusiness owners, consumers and the environment. Big Data analytics models are outlined, together with numerical algorithms for training them. Advances and tools to present processed Big Data in the form of actionable information to farmers are reviewed, and a success story from the Netherlands is highlighted. Finally, it is argued that the potential utility of Big Data for weed control is large, especially for invasive, parasitic and herbicide‐resistant weeds. This potential can only be realised when agricultural scientists collaborate with data scientists and when organisational, ethical and legal arrangements of data sharing are established.     

利用酵母同源重组系统快速构建柑橘叶斑驳病毒的侵染性克隆

【目的】构建柑橘叶斑驳病毒（Citrus leaf blotch virus,CLBV）中国分离株的侵染性克隆,为从分子水平解析其致病机理打下基础。【方法】以GeneArt® pYES1L Vector为模板,PYES2117F、PYES2117R为引物扩增含有酵母相关复制起始位点的片段pYES1L-2117,利用限制性内切酶Sac II单酶切双元载体DK1317-2并回收其大片段,利用In-Fusion HD Cloning Kit重组连接双元载体DK1317-2骨架和片段pYES1L-2117,得到可以在酵母-农杆菌-大肠杆菌中复制的三元穿梭载体pCY。以马铃薯X病毒(Potato virus X,PVX)全长cDNA侵染性克隆为模板,pCY-PVX-F、pCY-PVX-R为引物扩增PVX全长,采用限制性内切酶Stu I、Sma I酶切质粒pCY,采用醋酸锂转化法将获得的PVX基因组全长cDNA与线性化的pCY载体共转化酵母菌YPH501,通过同源重组获得PVX基因组全长cDNA克隆。通过农杆菌介导接种本生烟验证所构建克隆的侵染性,从而建立基于酵母同源重组的病毒侵染性克隆快速构建体系。在此基础上,以CLBV中国分离物（CLBV-HBYD）全长cDNA为模板,分段扩增其基因组,得到片段CLBV-1、CLBV-2;利用所建体系对CLBV-1、CLBV-2及pCY载体片段进行同源重组。得到重组质粒pCY-CLBV后,经农杆菌介导接种本生烟和锦橙幼苗,并利用RT-PCR和Northern blot检测其侵染性。【结果】建立了酵母-大肠杆菌-农杆菌的三元穿梭载体PCY,该载体全长10 347 bp,含有酵母、农杆菌、大肠杆菌的复制位点,能在酵母-农杆菌-大肠杆菌稳定复制,可用于在酵母中通过同源重组快速构建病毒基因组全长cDNA克隆,也可用于通过农杆菌介导直接接种植物寄主。利用该体系获得了CLBV中国分离株的基因组全长cDNA克隆16个。随机选取1个克隆进行了序列测定（GenBank登录号：MG572236）,其基因组全长8 747 nt,包含3个开放阅读框(open reading frame,ORF),ORF1为5 889 nt、ORF2为1 089 nt、ORF3为1 092 nt。全基因组序列比对显示,CLBV-HBYD与已登录的9个CLBV分离物的核酸序列一致性为79％-98％,其中与柑橘来源的EU857540一致性最高,为98%,与猕猴桃来源的JN983454、JN983455、JN983456和JN900477的一致性均为79%。在利用MEGA6 软件构建的系统发育树上,CLBV-HBYD与柑橘来源的CLBV分离株聚为一簇,而猕猴桃来源的CLBV分离株聚为另一簇。将所获16个CLBV全长cDNA克隆转化农杆菌,以pCY空载体为阴性对照注射接种本生烟。接种20 d后,抽提RNA进行RT-PCR检测,结果表明11个克隆的接种植株检测出CLBV特异性条带;随机选取5个阳性克隆进一步进行了Northern blot杂交,结果显示pCY-CLBV 1、2、3、14、15接种的本生烟可以检测到CLBV特异性条带,而对照样本未检测到任何条带,表明这些克隆均为侵染性克隆。【结论】构建了基于三元穿梭载体pCY的酵母重组克隆体系,利用该体系获得了中国CLBV分离株的适于农杆菌介导接种的基因组全长cDNA侵染性克隆。     

葡萄园植保机器人路径规划算法

为提高植保机器人葡萄园作业在垄行识别和路径规划中的准确度和可靠性,该文提出了一种基于支持向量机(support vector machine,SVM)的多支持向量配比权重进行葡萄园垄行识别与农业机器人作业路径规划的算法。该算法利用Kalman滤波器对由激光雷达扫描获取的粗大实况果园数据信息进行预处理,校正数据中的噪声离群点,然后结合SVM,获得垄行环境中的分割超平面和分类边际线,最后根据样本点与分类边际线存在的几何间隔关系判别各点所占相对权重,获取垄线安全预估测位置并进行农业机器人作业导航线的规划拟合。通过对多个实际样本的试验与测试,拟合导航线与实际垄行中心线平均角度偏差为0.72°,相对植保机器人的平均距离偏差为4.22 mm。试验结果表明,该算法能够有效的识别与定位植保机器人所需导航线的位置,拟合的导航线满足葡萄园植保机器人准确作业的要求。     

山黧豆CASase基因DNA全长序列的扩增及CRISPR/Cas9敲除载体的构建

【目的】克隆山黧豆β-腈基丙氨酸合成酶基因(CASase)DNA全长序列,构建CRISPR/Cas9敲除载体,为筛选"低毒、高硫"山黧豆品系奠定基础。【方法】以萌发4d山黧豆幼苗根部为材料,提取其基因组DNA,利用PCR克隆CASase基因全长;经序列同源性、内含子、外显子、sgRNA靶位点和脱靶分析后,在CASasecDNA上选取5个sgRNA靶位点进行寡核苷酸链设计和体外活性检测;从5个sgRNA靶位点中取活性较高的4个sgRNA,利用酶切连接法构建基因敲除载体pPLHACas9-sgRNA20r、pPLHACas9-sgRNA68r、pPLHACas9-sgRNA225f和pPLHACas9-sgRNA256f。【结果】获得了CASase基因3 143bp的DNA全长序列,该序列编码β-腈基丙氨酸合成酶;在CASase cDNA中选取5个sgRNA靶位点在体外均能有效剪切靶序列;菌落PCR检测结果显示,4个CRISPR/Cas9基因敲除载体构建成功。【结论】克隆了山黧豆CASase基因DNA全长,成功构建了该基因上4个sgRNA靶位点的CRISPR/Cas9敲除载体。     

向量优化中E超有效解的最优性条件

基于改进集而提出的向量优化问题的E-超有效性是对经典的超有效性概念的重要推广.在实局部凸拓扑线性空间中,利用邻近E-次似凸性建立了向量优化问题E-超有效解的一些最优性必要与充分条件,推广了一些已有结果到近似解.     

基于Variance–SFFS的小麦叶部病害图像识别

利用中值滤波结合k均值聚类的方法分割出小麦白粉病、条锈病和叶锈病叶部病斑,分别采用颜色矩和灰度共生矩阵的方法提取病斑的颜色特征和纹理特征参数,设计了一种基于Variance算法初选与序列浮动前向选择搜索算法(SFFS)相结合的特征选择方法,选择出优良的特征子集,实现对小麦3种叶部病害的识别。试验以SVM为分类器,利用特征选择方法获得的特征子集识别准确率为99%,与采用主成分分析(PCA)方法进行特征降维获得的子集的识别准确率比较,能有效降低特征维度,提高识别准确率。