超表达OsPR1A增强了Xa21介导的水稻对白叶枯病的抗性反应     

刘玉晴  燕高伟  张彤  兰金苹  郭亚璐  李莉云  刘国振  窦世娟 《中国农业科学》2021,54(23):4933-4942

【背景】前期研究发现,水稻病程相关蛋白质OsPR1A的表达受上游抗病基因Xa21调控,接菌后早期启动Xa21介导的OsPR1A较高水平表达对水稻抵抗白叶枯病菌至关重要。同时OsPR1A也受到水稻白叶枯病菌（Xanthomonas oryzae pv. oryzae,Xoo）的诱导表达。对于OsPR1A的研究绝大部分是作为抗性反应发生的标志基因佐证其他基因或途径在抗性中的作用,缺乏直接的证据证实OsPR1A本身的生物学功能。【目的】通过获得OsPR1a-OX超表达转基因植株,调查其表型及农艺性状,并明确OsPR1A蛋白质表达与抗性的关系,为鉴定OsPR1A功能提供依据。【方法】通过农杆菌介导法,将构建的OsPR1a-OX转化载体转入到水稻受体4021中,利用PCR和免疫印迹（western blot,WB）技术分别在基因水平和蛋白质水平上筛选并鉴定OsPR1A超表达阳性纯合株系。在成熟期,调查OsPR1A超表达转基因植株的表型及农艺性状（株高、穗长、分蘖数、结实率和籽粒大小等）。在31℃条件下,将生长2周的水稻幼苗TP309、4021和OsPR1A超表达转基因植株接种水稻白叶枯病菌,并在接菌0、2、4、6、8、10和12 d时测量病斑长度。在接菌0、4和6 d时,收集TP309、4021和OsPR1A超表达转基因植株的水稻叶片,提取蛋白质,利用WB技术检测OsPR1A的表达特征。【结果】构建了OsPR1a-OX转化载体,并转入到受体4021中,筛选并鉴定到2个OsPR1A超表达转基因纯合株系（#704和#709）。调查了OsPR1A超表达转基因植株在成熟期的表型及农艺性状,与对照4021相比,#704和#709的株高较矮、穗长较短、分蘖数减少、结实率降低,但籽粒稍大,可能与结实率低有关。在31℃条件下,OsPR1A超表达转基因植株的病斑长度与对照4021相比明显缩短,结果具有显著性差异（P<0.05）。在接菌0、4和6 d的材料中,超表达转基因植株#704和#709中OsPR1A始终有较高水平的表达丰度,从而提高了对白叶枯病菌的抗性。【结论】采用农杆菌介导法,获得OsPR1A超表达转基因植株;超表达OsPR1A影响到水稻的正常发育过程;超表达OsPR1A后增强了Xa21介导的水稻对白叶枯病的抗性。  相似文献   

Identification of immunodiagnostic antigens for cerebrospinal filariasis in horses by western blot analysis     

Masataka TAKESUE  Yuki OSAKA  Masanori MURANAKA  Yoshinari KATAYAMA  Hiromi IKADAI 《Journal of Equine Science》2016,27(1):17-18

  相似文献   

Comparison of dot blot and PCR diagnostic techniques for detection of white spot syndrome virus in different tissues of Penaeus monodon     

M.S. Shekhar  I.S. Azad  P. Ravichandran 《Aquaculture (Amsterdam, Netherlands)》2006,261(4):1122-1127

Comparison of PCR and dot blot diagnostic techniques for detection of white spot syndrome virus (WSSV) was made on different tissues of infected Penaeus monodon including eye stalk, eye stalk with eye, gills, cuticle, pleopod, periopods, uropods and telson. Dot blots of crude DNA extracted from infected tissue samples showed positive reactions with all the samples; however, the sensitivity of the dot blot was reduced with the purification of DNA samples extracted from pleopod, telson and uropod. PCR was found to be more sensitive when compared to dot blot. Both crude DNA and purified DNA samples extracted from all the tissues except for eye stalk with eye showed single step nested PCR positive reaction. The amplification of all or either of the three bands of 941 bp, 525 bp and 204 bp size varied with the tissues analysed. The severity of infection assessed by PCR amplification was found to be maximum in cuticle and telson followed by gill. Other tissues such as eye stalk, pleopod, periopods and uropod were observed to have mild infection. The maximum intensity of the PCR product was for the smallest amplified product of 204 bp followed by 525 bp and the weakest intensity was observed for the 941 bp size. The limitation of PCR due to inhibiting factors present in tissues could be overcome with the use of dot blot which gave positive reaction from the DNA extracted from eye stalk including the eye but yielded no amplification by PCR.  相似文献   

显微注射生长激素基因导入中国对虾（Penaeus chinensis）受精卵的研究   总被引：4，自引：0，他引：4  

刘萍  孔杰  王清印  李健  杨丛海  梁利群  孙孝文 《中国水产科学》1996,3(4):35-38

本实验采用显微微量注射方法，交羊生长激素基因导入中轻地虾受精卵，受精卵发育到蚤状幼体第三期后采样检测。ＰＣＲ检测结果表明：７个样品共９３尾幼体，有３个样品呈出阳性信号，基因转移比率至少在３％以上。同时斑点杂交结果也证明有两个明显阳性斑点。  相似文献   

Production of polyclonal antiserum against recombinant VP28 protein and its application for the detection of white spot syndrome virus in crustaceans     

Yoganandhan K  Syed Musthaq S  Narayanan RB  Sahul Hameed AS 《Journal of fish diseases》2004,27(9):517-522

The VP28 gene of white spot syndrome virus (WSSV) was cloned into pRSET B expression vector. The VP28 protein was expressed as a protein with a 6-histidine taq in Escherichia coli GJ1158 with NaCl induction. Antiserum was raised against this recombinant-VP28 protein in rabbits and it recognized VP28 protein in naturally and experimentally WSSV-infected shrimp, marine crabs, freshwater prawns and freshwater crabs. The antiserum did not recognize any of the other known WSSV structural proteins. Various organs such as eyestalks, head muscle, gill tissue, heart tissue, haemolymph, tail tissue and appendages were found to be good materials for detection of WSSV using the antiserum and detection of WSSV was successful in experimentally infected Penaeus monodon and P. indicus at 12 and 24 h post-infection (p.i.), respectively. The antiserum was capable of detecting WSSV in 5 ng of total haemolymph protein from WSSV-infected shrimp.  相似文献   

拟南芥Ran2小GTP结合蛋白抗血清的制备及其FITC荧光标记   总被引：1，自引：1，他引：0  

马立安  江涛  张忠明 《华中农业大学学报》2007,26(4):439-442

将原核表达、纯化的Ran2蛋白对新西兰兔进行免疫制备抗血清,ELISA测定其效价为1∶51 200。免疫印迹分析表明,原核表达、纯化的Ran2和拟南芥可溶性蛋白杂交带均在26 ku处。FITC荧光标记抗体检测Ran2绿色杂交带主要出现在细胞分裂间期的核膜上。  相似文献   

Generation of mouse monoclonal antibodies specific to tilapia immunoglobulin using fish immunoglobulin/BSA complex for monitoring of the immune response in Nile tilapia Oreochromis niloticus     

Tanapon Soonthonsrima  Pradit Wangman  Parin Chaivisuthangkura  Chalinan Pengsuk  Paisarn Sithigorngul  Siwaporn Longyant 《Aquaculture Research》2019,50(1):277-283

The simple immunoprecipitation method was used to isolate tilapia immunoglobulin (Ig) for immunization in order to produce monoclonal antibodies (MAbs) specific to tilapia Ig. First, the tilapia antiserum against bovine serum albumin (BSA) was prepared by peritoneal injection of BSA into tilapia, and the tilapia anti‐BSA antiserum was used to precipitate BSA to form the Ig/BSA immune complex. The Ig/BSA immune complex was then injected into Swiss mice for hybridoma production. After fusion, three hybridoma clones producing MAbs specific to the tilapia antibody were selected by dot blot and Western blot. All MAbs (101A, 59G, and 11A) were bound specifically to the heavy chain of immunoglobulin M (IgM). The MAbs 101A and 59G demonstrated twofold higher affinity than MAb 11A and the commercialized antibody. However, MAbs 11A could also bind to the heavy chain of IgM in Asian seabass, Lates calcarifer, as well. These MAbs can be used to monitor the immune responses of individual fish by indirect ELISA upon exposure to various antigens.  相似文献   

p5CS基因在蒙农杂种冰草植株中的表达及耐盐性研究   总被引：1，自引：0，他引：1  

王桂花  米福贵  刘娟  董淑君  霍秀文  杨宏雁 《华北农学报》2007,22(4):33-36

为尽快培育出适宜我国干旱荒漠地区大面积推广的耐盐冰草新品种,试验以经PCR和Southern blot杂交检测的含有p5CS基因的蒙农杂种冰草植株为材料,用Northern blot检测目的基因在转化植株中的表达;并用1.5%NaCl盐溶液进行胁迫处理确定转化植株的耐盐性。结果表明:转基因冰草植株与DIG标记探针杂交呈现明显的杂交带;盐胁迫下,游离脯氨酸含量增加较快,质膜透性、丙二醛含量增加较小,SOD活性较高。说明p5CS基因能够在冰草基因组的转录水平上表达,表达植株的耐盐性明显增加。  相似文献   

富含多糖的转基因石斛基因组DNA提取方法(英文)   总被引：3，自引：0，他引：3  

张妙彬  潘丽晶  范干群  陈继敏  程萍 《分子植物育种》2009,7(1)

在石斛兰转基因研究中,需通过PCR和Southern杂交等手段检测外源基因是否转入并整合到转化植株基因组.由于转化石斛植株生长缓慢,且含有多糖,因此从转化植株中提取高质量的基因组DNA以尽快对转基因苗进行分子检测存在较大困难.本研究旨在通过改良现有的DNA提取法(CTAB法或SDS法1,克服转化石斛植株基因组DNA提取中碰到的产量低,纯度不高而导致难以进行PCR或酶切等问题.在本研究所用的3种改良法中,方法Ⅱ能从少量的转化石斛苗中提取出高产量和高纯度的基因组DNA.研究结果表明方法Ⅱ提取的基因组DNA完全适用于转基因石斛的外源基因PCR扩增,限制性酶切和Southern杂交分析.  相似文献   

山葡萄VaCIPK18原核表达与多克隆抗体制备     

吴楠  张宁波  郑巧玲  陈卫平  徐伟荣 《核农学报》2020,34(7):1387-1396

类钙调磷酸酶B亚基蛋白(CBLs)互作蛋白(CIPKs) 作为类丝氨酸/苏氨酸蛋白激酶,在植物响应非生物胁迫信号转导中起着重要作用。基于前期山葡萄响应低温胁迫转录组测序结果,发现低温胁迫引发的早期伤害及感知阶段的激酶基因中涉及CBL-CIPK 信号通路的VaCIPK18表达显著上调。为进一步研究山葡萄(Vitis amurensis)VaCIPK18激酶参与低温胁迫的功能,采用同源克隆获得了VaCIPK18基因,其开放阅读框为1 320 bp,编码439个氨基酸。基于对VaCIPK18蛋白生物信息学分析,获取胞外结构域中抗原表位丰富的肽段,并将其C端调控结构域(230~439 aa)构建到原核表达载体pET28a-SUMO。将重组表达载体转化至大肠杆菌(E. coli Rosetta)中,经0.8 mmol·L^-1 IPTG、37℃诱导4 h表达出大小为42 kDa的包涵体蛋白。将重组蛋白作为抗原免疫日本大耳白兔,获得anti-VaCIPK18多克隆抗体,经检测具有高效价及特异性。Western Blot结果表明,该抗体可以与葡萄内源CIPK18特异性结合,且在50 kDa位置出现与预期一致的条带。同时,CIPK18在低温胁迫后葡萄叶片中蛋白表达水平与室温下相一致,但两种状态下均存在可能的磷酸化与泛素化修饰现象。本研究结果为进一步探究VaCIPK18的蛋白定位、表达及其功能奠定了基础。  相似文献