大白菜中保护酶活性及H2O2含量变化与TuMV抗性关系的研究

选取抗TuMV的8407、河304和感TuMV的冠291和春月黄为试验材料,于苗期接种TuMV-C4,接种后测定24 d内叶片中超氧化物岐化酶（SOD）、过氧化物酶（POD）、过氧化氢酶（CAT）这3种过氧化氢代谢相关酶的活性以及过氧化氢H2 O2的含量。结果表明：接种TuMV后, POD、CAT的活性及H2 O2含量的变化在不同材料之间存在明显差异。抗病材料在接种后,POD、CAT的活性及H2 O2含量虽有变化,但均能逐渐恢复正常;感病材料在接种后,POD、CAT的活性及H2 O2含量均有较大变化,且始终无法恢复正常。总体而言,叶片中的H2 O2和CAT与大白菜的TuMV抗性关系较为紧密,其次是POD,而SOD与TuMV抗性的关系不大。     

HC-Pro基因片段介导的高抗TuMV

芜菁花叶病毒(turnip mosaic viruses,TuMV)是侵染重要经济作物的主要病毒之一,寄主范围十分广泛,尤其是对十字花科作物的危害最为严重。为了获得对TuMV持久稳定的高度抗性,本研究以TuMV HC-Pro基因的453 bp保守序列为靶标,构建了植物表达RNAi载体pBBBTu-HC-Pro,并转化了TuMV的天然宿主拟南芥。对转基因拟南芥用北京地区流行的TuMV强致病株BJ-C4进行了抗病接种鉴定。鉴定的13个单拷贝转基因株系中有4个对TuMV的BJ-C4株表现出高度抗性,抗性比对照提高约80%以上,且抗性可以稳定遗传。经半定量和荧光定量PCR方法检测,在高抗转基因植株体内几乎检测不到病毒的累积,抗病效果明显。该载体在利用基因工程抗TuMV育种中具有广阔的应用前景。     

甘蓝抗芜菁花叶病毒育种研究进展

摘要：本文简述了甘蓝TuMV抗性种质资源的现状和各种分子生物技术应用发展的前景,从TuMV的发生、甘蓝抗TuMV种质资源的挖掘,甘蓝对TuMV的抗性规律,植-病互作分子机制,甘蓝抗TuMV分子标记的开发与应用和基因工程技术在甘蓝抗TuMV育种中的应用等方面综述了近年来的研究进展,为今后甘蓝抗性育种提供参考和借鉴。     

黄瓜绿斑驳花叶病毒的鉴定及分子检测

【研究目的】黄瓜绿斑驳花叶病毒（Cucumber green mottle mosaic virus,CGMMV）的快速鉴定检测是控制该潜在危险性有害生物蔓延的有效途径。【方法】通过汁液摩擦接种、透射电镜观察、双抗夹心酶联免疫吸附测定法（DAS-ELISA）和RT-PCR方法对该病毒进行鉴定;同时对RT-PCR反应体系及扩增程序进行优化,建立CGMMV免疫捕获RT-PCR（IC-RT-PCR）检测方法。【结果】血清学、生物学、电镜观察和分子生物学方法证明供试样本为黄瓜绿斑驳花叶病毒引致。IC-RT-PCR方法可以简化RNA的提取,降低对试验材料要求。【结论】IC-RT-PCR的建立为CGMMV的检测提供了操作更为简便、特异性更强的快速、准确的检测方法。     

道地中药材川贝母特异性PCR鉴别方法研究

为建立快速检测中药材川贝母成分的特异性PCR法,从NCBI数据库下载植物HMGR基因序列,根据同源性区域设计兼并引物,对川贝母及其易混品鳞茎基因组进行扩增。通过比对川贝母特异性DNA片段区域,设计引物扩增获得川贝母基原物种特异核酸片段。从川贝母基原鳞茎中提取DNA,设计特异引物对BMH-TF/BMH-TR,优化PCR扩增条件,通过特异性、灵敏度实验验证,建立特异性PCR检测体系。采用该方法从川贝母基原中扩增出一条120 bp左右条带,而非川贝母样品、空白对照在相同条件下无扩增条带,检测灵敏度为2 ng/μL,检测下限为4 ng。对10个市售药材的检测结果证明,该方法简便可行、重现性好。该PCR法可快速、准确鉴别川贝母基原,具有操作简便、快速、特异性高的优点。     

化学发光免疫方法的研究进展

化学发光免疫分析方法是将化学发光和免疫融为一体,是继放射免疫分析和酶联免疫分析的一种新型免疫分析技术。化学发光免疫分析以化学发光物为指示物,通过免疫反应和发光反应,对目标物进行检测,具有高灵敏度、高特异性、简便、快速和发展迅速的特点,并逐渐运用到临床检验、药物分析、环境监测和食品安全等领域。     

大白菜芜菁花叶病毒病抗性遗传分析

为了明确不同感病亲本对大白菜芜菁花叶病毒病抗性遗传规律的影响,以大白菜抗TuMV的国家级抗源材料8407和高抗TuMV的高代自交系材料73为亲本之一,分别与2个感病材料冠291和06-247构建F1杂交组合,并制备F2群体。采用摩擦接种法对上述亲本及其F1、F2群体接种TuMV-C4,采用生物学观察的方法,根据病情分级和归类标准对大白菜TuMV抗性进行鉴定。结果表明,以8407为抗病亲本,当感病亲本为冠291时,表现为由1对显性基因控制;当感病亲本为06-247时,表现为由1对隐性基因控制。以73为抗病亲本,当感病亲本为06-247时,TuMV抗性由1对隐性基因控制;同一个抗病亲本,当感病亲本为冠291时,又表现为由两对隐性基因控制。因此,大白菜TuMV的抗性遗传规律十分复杂,同一个抗病材料,当感病亲本不同时,所配制的组合间TuMV抗性表现不同。     

鸡柔嫩艾美耳球虫特异性抗体EtMIC4-N蛋白BAS-ELISA检测方法的建立

为了检测柔嫩艾美耳球虫感染鸡血清中特异性抗体水平,原核表达纯化EtMIC4-N蛋白,采用免疫印迹法确定其反应原性,再以纯化后的蛋白为包被抗原,以生物素标记的羊抗鸡IgG为二抗,以HRP结合链霉亲和素为酶标抗体,建立了检测柔嫩艾美耳球虫抗体的BAS-ELISA方法。经检测,EtMIC4-N蛋白最佳包被浓度为7μg/mL,最适封闭液为1%BSA,最佳血清稀释度为1:80,二抗稀释度为1:100000,酶标抗体稀释度为1:100000,最适显色时间为15min,组内和组间变异系数均小于10%。通过动物感染试验验证,结果表明建立的BAS-ELISA可以用于检测柔嫩艾美耳球虫特异性抗体的检测,并且具有简便,快速,灵敏,成本低等特点。     

S1核酸酶突变检测法的优化

功能基因组学研究需要高通量的突变检测方法,S1核酸酶法就是其中一种,具有简单、费用低等优点,但由于该酶酶反应条件难以控制,很容易发生非特异性切割。本文以已知插入突变的玉米叶绿素a/b结合蛋白基因的启动子为对象,优化了该突变检测体系,基本研究清楚了导致S1核酸酶非特异性降解双链DNA的原因。     

Quantitative trait loci analysis for resistance against Turnip mosaic virus based on a doubled-haploid population in Chinese cabbage

Turnip mosaic virus (TuMV) as the major virus infecting Brassica crops, often cause severe yield and quality losses in Chinese cabbage production in China. In this study, quantitative trait loci (QTL) analysis for TuMV resistance was conducted with a population of 100 doubled-haploid lines derived from the F₁ between 91-112 (resistant) and T12-19 (susceptible) through microspore culture. A total of 376 molecular markers including 235 amplified fragment length polymorphisms, 129 random amplified polymorphic DNAs, 10 simple sequence repeats, one SCAR and one morphological marker were employed to construct a linkage map with 10 linkage groups covering 809.1 cM with an average distance of 2.2 cM between loci. Resistance was assessed by artificial inoculation at the seedling stage and at the adult stage in field conditions, respectively. Four QTLs controlling TuMV resistance were identified with JoinMap QTL 4.0 and interval mapping method. Two QTLs ( Tu1 , Tu2 ) were associated with resistance at the seedling stage, each accounting for 58.2% and 14.7% of the phenotypic variation, respectively. Two QTLs ( Tu3 , Tu4 ) were found corresponding to the disease resistance at the adult plant stage, explaining 48.5% and 32.0% of the phenotypic variation, respectively. Marker-assisted selection for these major QTLs involved in TuMV resistance could be useful in Chinese-cabbage breeding programmes.     

Development of gene‐based markers for the Turnip mosaic virus resistance gene retr02 in Brassica rapa

Turnip mosaic virus (TuMV) is responsible for a serious disease that affects the production of Chinese cabbage. Previous studies have cloned a series of TuMV resistance genes and developed molecular markers. In this study, a derived cleaved amplified polymorphism sequence (dCAPS) marker and a Kompetitive Allele Specific PCR (KASP) marker were developed based on a single recessive gene, retr02, which confers broad‐spectrum TuMV resistance in Chinese cabbage by means of an additional G at the junction of exon 1 and intron 1. The two markers were able to detect the retr02 allele in Chinese cabbage accessions used in breeding programmes. Compared with the dCAPS marker, the KASP marker was flexible, cost‐effective and quick to process, which is likely to be beneficial in establishing high‐throughput assays for marker‐assisted selection.     

Identification and mapping of a novel Turnip mosaic virus resistance gene TuRBCS01 in Chinese cabbage (Brassica rapa L.)

We aimed to identify Turnip mosaic virus (TuMV) resistance genes in Chinese cabbage by analysing the TuMV resistance of 43 P₁ (resistant), 88 P₂ (susceptible), 26 F₁, 104 B₁ (F₁ × P₁), 108 B₂ (F₁ × P₂) and 509 F₂ individuals. All parents and progeny populations were mechanically inoculated with TuMV‐C4. Both F₁ and B₁ populations showed TuMV resistance. Resistant: susceptible ratios in the B₂ and F₂ populations were 1 : 1 and 3 : 1, respectively. TuMV resistance in P₁ was controlled by a dominant gene, TuRBCS01. Bulked segregation analysis was performed to identify simple sequence repeat or insertion or deletion markers linked to TuRBCS01. Data from 108 B₂ individuals with resistant or susceptible phenotypes were analysed using mapmake r/exp 3.0. Polymorphic marker sequences were blast searched on http://brassicadb.org/brad/ . TuRBCS01 was found to be linked to eight markers: SAAS_mDN192117a_159 (3.3 cM), SAAS_mDN192117b_196 (4.0 cM), SAAS_mDN192403_148 (13.0 cM), SAAS_mGT084561_233 (6.8 cM), BrID10723 (3.3 cM), mBr4041 (3.3 cM), SAAS_mBr4055_194 (2.6 cM) and mBr4068 (4.0 cM). Further, TuRBCS01 was mapped to a 1.98‐Mb region on chromosome A04 between markers BrID10723 and SAAS_mBr4055_194.     

大白菜抗TuMV抗源的选育

大白菜抗源“8407”是河北省农林科学院蔬菜研究所选育的高抗芜菁花叶病毒(TuMV)的抗源材料.1985年经国家专家组验收为国家大白菜抗源.1987年鉴定,“8407”对全国十个省市19个TuMV分离物中的17个表现免疫,两个有轻微症状,因此被确认为我国大白菜对TuMV垂直抗性最好的抗源材料.该材料是1978至1983年从青麻叶系统内蒙古长炮弹品种,经连续6代自交选育而成.在逐年自交的同时,进行了亲和指数和配合力的测定.1983年用“8407”抗源和另一自交系,育成高抗TuMV兼抗霜霉病和软腐病的多抗型“8361”新品种.     

Inheritance of resistance to turnip mosaic virus in Chinese cabbage

Summary Turnip mosaic virus (TuMV) is the most important virus of commercially grown cole crops in many Asian countries, affecting both yield and quality. TuMV-infected Chinese cabbage becomes unmarketable because of the presence of black spots and necrosis often induced by the virus. Resistance breeding is complicated by the existence of five strains of the virus, one of which was discovered in 1985 for the first time in Taiwan. Resistance to strains C1 to C3 is readily available among the Chinese cabbage germplasm at AVRDC, whereas resistance to strains C4 and C5 is rarely found. To elucidate the inheritance of resistance to TuMV, P₁, P₂, F₁, F₂ and BC₁ generations of crosses between the resistant line 0–2 and three susceptible lines, E-7, E-9 and FL-9, were inoculated with strains C4 and C5. Segregation ratios obtained by visual observation and enzyme-linked immunosorbent assay (ELISA) indicate that two recessive genes confer resistance to both TuMV-C4 and TuMV-C5.     

大白菜抗芜菁花叶病毒(TuMV)EST-SSR信息分析与引物筛选

为了研究大白菜抗芜菁花叶病毒(TuMV),对大白菜及芸薹属中与抗性有关的EST序列进行了搜索和SSR筛选,并根据SSR两端的序列设计引物,以1对大白菜TuMV抗、感材料的基因组DNA为模板,对上述引物进行了筛选。结果表明,在132条大白菜EST和895条芸薹属抗性EST序列中,共筛选出166个符合条件的SSR(16.2%)。其中包含6种核苷酸重复基元,占主导地位的分别是二核苷酸重复(33.7%)和三核苷酸重复(50.0%),其余重复类型所占比例较小。根据上述SSR两翼的序列,共设计引物196对。引物筛选结果表明,能够进行成功扩增的有156对,占79.6%,其中35对引物在2份材料中具有多态性。因此,利用芸薹属中抗性有关的EST序列设计SSR引物,可用于大白菜TuMV抗性有关基因的标记。     

天津地区十字花科蔬菜病毒类型及其消长规律的研究

本文论述了天津地区十字花科蔬菜的病毒类型.主要分为四个类型:第一类芜菁花叶病毒(TuMV),第二类烟草花叶病毒(TMV),第三类黄瓜花叶病毒(CMV),第四类称为甘兰僵矮病毒,其中以芜菁花叶病毒为主导类型.调查鉴定结果表明,本地区夏季十字花科蔬菜病毒,主要来自种株白菜.二秋小白菜,小油菜以及秋甘兰,秋萝卜可为秋季大白菜直接提供毒源和传播介体.夏季栽培的或野生的十字花科植物是秋白菜病毒病的桥梁寄主.根据病毒消长规律的研究结果提出了秋白菜病毒病的防治途径.     

大白菜eIF(iso)4E.a假基因突变体的鉴别及相关检测标记的开发

马铃薯Y病毒在侵染寄主时与eIF4E或eIF(iso)4E的相互作用是一种普遍机制。大白菜病毒病主要病源是TuMV,TuMV在侵染大白菜时能同时与eIF4E和eIF(iso)4E发生作用,且在大白菜基因组中上述两基因各有三个拷贝。因而鉴定大白菜资源在上述两个位点的多样性、发现各位点的功能缺失突变体至关重要。采用同源克隆和Tail-PCR技术,鉴定了12份大白菜抗/感TuMV材料中eIF(iso)4E.a位点的多样性。结果表明：5份材料的eIF(iso)4E.a基因组序列完整,但与已知序列相比均存在显著差异,包含3种新的单倍型;7份材料的eIF(iso)4E.a缺失了第四、五外显子及3'端约1 kb的大片段,是个假基因。同时开发了鉴别完整基因与假基因的共显性标记。该策略为鉴定大白菜中eIF4E和eIF(iso)4E其他位点的多样性提供了借鉴,开发的相关检测标记可以作为标记辅助选择的工具用于创新抗病毒大白菜新种质及培育抗病毒大白菜新品种。