应用农杆菌转化水稻的研究

利用农杆菌ＬＢＡ４４０４（ＰＴＯＫ２３３）转化水稻成熟胚愈伤组织，通过筛选获得抗性伤组织并再生出转化植株，转化频率为８．９％，转基因分子生物学鉴定的阳性，外源基因ｇｕｓ在转化植株表达亦提供了转化成功的证据。转基因植株的当代及Ｔ１代植株获得了种子。摸索了应用农杆菌转化水稻的方法，条件。利用干燥处理法抑菌并提高转化植株再生频率。     

Tid基因对水稻的转化及转基因植株的抗虫性

利用基因枪转化法将大豆Kunitz型胰蛋白酶抑制剂(SKTI)基因Ti^d转入北方推广的水稻(Oryza sativa L)品种丰优301和通887，所获得的潮霉素抗性植株通过GUS组织化学分析、PCR检测、Southern blot分子检测，证实Ti^d基因已经转入水稻基因组中，为转基因植株。用转基因水稻植株叶片进行了室内饲喂水稻二化螟(Chilo suppressalis)实验，抗虫性分析结果表明，与对照比较，部分转基因水稻植株明显地增强了对水稻二化螟虫的抗性。对转基因R1代植株进行PCR和PCR Southern分析，表明外源基因在转基因植株后代今稳定遗传。     

天花粉蛋白基因在水稻中转化及抗虫研究

利用基因枪转化法将天花粉蛋白基因导入水稻。经ＰＣＲ、Ｓｏｕｔｈｅｒｎ和Ｎｏｒｔｈ－ｅｒｎ杂交分分析证明外源基因已整合到水稻基因组中并有转录。抗虫试验表明,与对照比较转基因水稻明显地增强了对螟虫、稻飞虱的抗性。     

水稻白叶枯病菌受寄主诱导基因的鉴定

用一个具链霉素（Ｓｍ）抗性并含无启动子ＣＡＴ基因的广宿主启动子探针质粒ＰＩＪ３１００，用鸟枪法在ＣＡＴ基因上游的ＢａｍＨ１克隆位，或插入水稻白叶枯病菌（Ｘａｎｔｈｏｍｏｎａｓｏｒｙｚａｅｐｖ．ｏｒｙｚａｅ），用ＢａｍＨ１完全酶切的染色体ＤＮＡ片段，将重组质粒转化大肠杆菌ＥＤ８７６７在含链霉素的ＬＢ单板上筛选转化子。得到容量为６０００个转化子的克隆群体，其中２％的克隆含有水稻白叶枯病菌启动子活性片段，在平板上表现氯霉素（Ｃｍ）抗性。在帮助质粒ＰＲＫ２０１３的帮助下，通过三亲支配，将含有水稻白叶枯病菌启动子片段的重组质粒转移进野生型的水稻白叶枯病菌中去，在含链霉素的ＰＳＡ平板上筛选１６００个接合子，其中一个在平板上表现氯霉素抗性及含有组成表达的水稻白叶枯病菌启动子。随机选取２００个平板上对氯霉素敏感的接合子，接种用氯霉素处理的水稻感病品种金南风，得到１５个比对照明显致病的克隆。用其中一个含受水稻特异诱导启动子的重组质粒为探针，在水稻白叶枯病菌野生菌基因文库中筛选到２７个阳性克隆。     

转PSAG12-ipt基因水稻植株的获得及其延衰性的初步研究

运用农杆菌转化法将叶片衰老抑制基因PSAG12-ipt转入南方推广的优质籼型不育系中A、恢复系明恢63、南胜10号,所获得的潮霉素抗性植株通过GUS组织化学分析、PCR检测、Southern blot分子检测证实,PSAG12-ipt已经转入水稻基因组中.对转基因植株进行了延衰性考察,结果表明,与对照组相比,部分转基因水稻植株明显表现出叶片推迟衰老,提高了单株有效穗数、千粒重.对转基因R1代植株进行了PCR和PCR Southern分析,表明外源基因在转基因植株后代中稳定遗传.     

基因枪法转化水稻(Oryza sativa L.)获得可育的转抗虫基因水稻再生植株

应用基因枪（particle bombardment)转化技术成功地将豇豆胰蛋白酶抑制剂（cowpea trypsin inhibitor,CpTI)基因分别导入三个水稻粳稻栽培品种（Oryza sativa L.,japonica)中花8、中花10和中作321的幼胚和胚性愈伤组织中,并由此获得了可育的再生植株,抗性植株的筛选频率为6．1％－12．24％。经PCR、Southern blot分子检测和ELISA检测等证实所获得的潮霉素抗体植株为转基因植株。抗虫性分析结果表明,部分转基因水稻植株对玉米螟虫（Ostrinia nubilalis)有较好的抗性。     

水稻白叶枯病菌无毒基因的研究初报

报道了用基因标签法克隆水稻白叶枯病菌无毒基因的研究过程。通过接合实验，将Ｔｎ５引入水稻白叶枯病菌日本系统５号小种中去，在ＬＢ＋Ｋｍ平板上筛选了２５００个接合子；用剪叶接种法，将接合子接种水稻品种ＩＲ２６，得到了４个无毒基因突变体，分别用粘粒载体ｐＳａ７４７和质粒载体ｐＵＣ１９构建了ＪＸＯＶ核基因文库和无毒基因探针，采用菌落原位杂交法在基因库中钓取了１３个无毒基因克隆。该无毒基因片断在此病原的不同小     

Patatin启动子驱动的乙肝表面抗原基因在马铃薯中的表达

按常规分子克隆方法将马铃薯中高表达的ｐａｔａｔｉｎ启动子和乙肝表面抗原基因分别插入双元质粒ｐＢＩ１０１成为植物表达载体ｐＰＨＧ９,由前者驱动后者表达;通过农杆菌转化法将乙肝表面抗原基因导入马铃薯并获得再生植株;用ＥＬＩＳＡ方法检测经ＰＣＲ鉴定的转基因植株中乙肝表面抗原的含量,对照标准曲线得知转基因马铃薯植株中的乙肝表面朱含量高达１００￣１７４ｎｇ／ｍｇ可溶性蛋白。     

牙鲆抗菌肽hepcidin基因在大肠杆菌中的融合表达

根据从实验室克隆的牙鲆抗菌肽hepcidin基因(GenBank登陆号DQ129693)的序列,设计了特异性引物,PCR扩增其成熟肽片段.重组至融合表达载体pGEX-4T-1中,构建成牙鲆抗菌肽hepcidin基因融合表达载体pGEX-fhep,转化至大肠杆菌BL21(DE3)plyS中,筛选得到的阳性克隆用IPTG进行诱导.SDS-PAGE电泳显示在29kD处有特异性的蛋白条带出现,Western-blotting 检测表明已经成功表达了融合蛋白,表达的融合蛋白最高占总蛋白的21%.纯化得到的GST-fhep用凝血酶切割后,用亲合层析得到牙鲆的抗菌肽hepcidin.抑菌实验结果表明抗菌肽对大肠杆菌(ATCC25922)有一定程度的抑制作用.     

中科院等离子体物理所用离子束介导法培育出转基因水稻

中科院等离子体物理所用离子束介导法培育出转基因水稻中科院等离子体物理研究所用离子束介导的方法，将潮霉素抗性基因整合进入水稻ＤＮＡ中，成功地培育出抗潮霉素的转基因水稻。用离子束介导法培育转基因水稻这在国际上属首次。这项研究成果为水稻等农作物的定向育种及...     

抗稻瘟病新基因pi-hit-1的克隆与功能研究

采用差异显示法(DD-PCR)寻找易感稻瘟病的突变品系与野生型对照品系中的差异表达基因。结果发现与野生型对照相比,Rubisco小亚基、pi-hit．1(AF450251)和pi-hit-2(AF448491)基因在易感稻瘟病的突变品系中高表达,pi-hit-1和pi-hit-2是功能未知的新基因。进一步采用电子克隆和RT-PCR方法克隆了pi-hit-1基因的全长eDNA(AF514859),该基因编码的蛋白质属于ATP依赖的Clp蛋白酶家族。分析pi-hit-1基因的组织特异性表达发现,此基因在叶片组织特异表达。进一步设计接种诱导实验研究pi-hit-1基因表达与水稻稻瘟病易感性的关系。在易感稻瘟病的突变品系中pi-hit-1受稻瘟病菌诱导,接种后其表达量明显升高,而野生型对照品系pi-hit-1基因表达在接种前后无明显变化。比较易感稻瘟病的突变品系与对照品系pi-hit-1基因序列差异发现,稻瘟病敏感品系中pi-hit-1基因第一外显子发生缺失突变使pi-hit-1蛋白功能缺失,导致突变品系易感稻瘟病。这些实验结果提示野生型pi-hit-1是稻瘟病抗性基因。     

表达harpin基因的大肠杆菌DH5（pCPP430）诱导植物抗病性的研究

用表达ｈａｒｐｉｎ基因的大肠杆菌ＤＨ５进行了诱导植物抗病性的研究，发现表达ｈａｒｐｉｎ基因的重组菌除可明显诱发烟草、番茄叶片的过敏反应外，对玉米、水稻叶片也可诱发过敏反应，反应比相应阳性对照病菌速度快，坏死斑也更典型。用ＤＨ５菌悬液喷雾或注射诱导后，番茄对早疫病、水稻对稻瘟病、玉米对大、小斑病、马铃薯对软腐病的抗性均有不同程度的提高，与水杨酸诱导的抗性效果相当或比其略高。     

Plant Disease Diagnosis and Image Classification Using Deep Learning

Indian agriculture is striving to achieve sustainable intensification, the system aiming to increase agricultural yield per unit area without harming natural resources and the ecosystem. Modern farming employs technology to improve productivity. Early and accurate analysis and diagnosis of plant disease is very helpful in reducing plant diseases and improving plant health and food crop productivity. Plant disease experts are not available in remote areas thus there is a requirement of automatic low-cost, approachable and reliable solutions to identify the plant diseases without the laboratory inspection and expert's opinion. Deep learning-based computer vision techniques like Convolutional Neural Network (CNN) and traditional machine learning-based image classification approaches are being applied to identify plant diseases. In this paper, the CNN model is proposed for the classification of rice and potato plant leaf diseases. Rice leaves are diagnosed with bacterial blight, blast, brown spot and tungro diseases. Potato leaf images are classified into three classes: healthy leaves, early blight and late blight diseases. Rice leaf dataset with 5932 images and 1500 potato leaf images are used in the study. The proposed CNN model was able to learn hidden patterns from the raw images and classify rice images with 99.58% accuracy and potato leaves with 97.66% accuracy. The results demonstrate that the proposed CNN model performed better when compared with other machine learning image classifiers such as Support Vector Machine (SVM), K-Nearest Neighbors (KNN), Decision Tree and Random Forest.     

Studies on the sources of benzo[a]pyrene in grain and aboveground tissues of rice plants

Rice plant pot experiments designed to identify benzo[a]pyrene (B[a]P) sources in plant tissues were conducted in an air-quality controlled greenhouse built to prevent contamination from B[a]P air pollution. Results from quartz sand cultures with control and 50, 100 and 500 microgkg(-1) of B[a]P treatments were compared with those from outdoor field experiments, in which rice plants were exposed to polluted air in the urban area of Shenyang, China. When B[a]P was strictly controlled in both air and quartz sand culture medium, the background values of B[a]P in rice plant tissues were uniformly very low. There was no significant difference of B[a]P contents of rice grain between control and treatments of B[a]P in controlled air quality trials. This indicated that the source of B[a]P in the rice grains is not from any B[a]P in the root culture media. The B[a]P content of rice grain, husk, and stem with leaf sampled from outdoor field was up to 7.33-, 9.21- and 27.10-fold higher than corresponding tissues from air-quality controlled conditions. This indicated that polluted air is the main source of B[a]P in aboveground tissues. Therefore control of B[a]P pollution in ambient air is of prime importance for improving the quality of cereal crops.     

转水稻巯基蛋白酶抑制剂基因毛白杨的获得

通过农杆菌介导法将水稻巯基蛋白酶抑制剂基因导入毛白杨。毛白杨叶片到农杆菌共培养，采用卡那霉素３０ｍｇ／Ｌ筛选转化子获得抗性芽，然后转到卡那霉素５０ｍｇ／Ｌ的培养基上继续筛选，待转化芽长至１～２ｃｍ时切下移至卡那霉素５０ｍｇ／Ｌ的生根培养基上获得完整再生植株。通过ＰＣＲ检测和ＰＣＲ－Ｓｏｕｔｈｅｒｎ杂交证明水稻巯基蛋白酶抑制剂基因已整合到杨树基因组中。     

Genotypic dependent effect of exogenous glutathione on Cd-induced changes in proteins, ultrastructure and antioxidant defense enzymes in rice seedlings

Greenhouse hydroponic experiments were conducted using Cd-sensitive (cv. Xiushui63) and tolerant (Bing97252) rice genotypes to evaluate how different genotypes responded to Cd toxicity in presence of glutathione (GSH). Results showed that GSH alleviates Cd-toxicity, ameliorates Cd-induced damages on leaf/root ultrastructures. Nine proteins in roots were identified, using 2-DE coupled with mass spectrometry, whose expression were down-regulated in Xiushui63, up-regulated/unchanged in Bing97252 by Cd; coinstantaneously enhanced/unchanged in Cd + GSH over Cd alone treatment in both genotypes. They are l-ascorbate peroxidase, putative short-chain dehydrogenase/reductase, Glycolipid transfer protein, elongation factor, Os04g0652700, carbonic anhydrase, Os08g0374000, chitinase, and putative disease resistance response protein. Eight proteins in leaves with expression of increase in Bing97252 but down-regulate/unchange in Xiushui63, categorized as four groups of their functions: carbon metabolism, TCA cycle, photorespiration and RNA processing. Furthermore, we identified eight proteins with repressed expression in Cd-treated and up-regulated in Cd + GSH-treated rice leaves of Xiushui63.     

重组家蚕抗菌肽CM4对癌细胞骨架及核骨架破坏作用的观察

报道了重组家蚕抗菌肽ＣＭ４对离体Ｕ９３７,Ｈｅｌａ癌细胞骨架及核骨架损伤作用的扫描电镜观察。随着时间的延长,经重组家蚕抗菌肽ＣＭ４作用后的癌细胞骨架散乱,断裂,萎缩成团状;癌细胞呆断裂,部分凝聚成团,结构不完整。重组家蚕抗菌肽ＣＭ４与正常人白细胞作用后细胞骨架及核骨架未见损伤现象。