与大豆孢囊线虫病性相关的PAPD标记

利用ＲＡＰＤ－ＰＣＲ技术，对７个抗大豆孢囊线虫的大豆和１０个感病大豆品种的基因组ＤＮＡ进行了ＲＡＰＤ分析，供试的２００个随机引物均产生了清晰稳定的ＲＡＰＤ扩增产物，其中有２１个随机引物产生的ＲＡＰＤ扩增产物具有多态性，获得了５个与大豆孢囊线虫病抗性相关的ＤＮＡ片段，这些ＲＡＰＤ标记具有较高的重复性和稳定性，可辅助抗大豆孢囊线虫病大新品种的选育。     

小麦品种“Lee”中抗条锈病基因的RAPD标记

共用３８０个１０碱基随机引物，对抗条锈病基因ＹｒＬｅｅ的近等基因系进行了ＲＡＰＤ分析，其中有两个引物在抗病的近等基因系ＹｒＬｅｅ／６＊Ｔａｉｃｈｕｎｇ２９Ｆ３和基因供体亲本“Ｌｅｅ”中扩增出了特异的ＤＮＡ片段，而在轮回亲本“Ｔａｉｃｈｕｎｇ２９”中没有扩增出同样的ＤＮＡ片段，引物ＯＰＲ－７扩增出的ＤＮＡ片段长度约为４６５ｂｐ，引物ＯＰＲ－９扩增出的ＤＮＡ片段长度约国６６７ｂｐ。用基因供体亲本“Ｌｅ     

小麦品种“Lee”中抗条锈病基因的 PAPD 标记

共用３８０个１０碱基随机引物，对抗条锈病基因ＹｒＬｅｅ的近等基因系进行了ＲＡＰＤ分析，其中有两个引物在抗病的近等基因系ＹｒＬｅｅ／６＊Ｔａｉｃｈｕｎｇ２９Ｆ３和基因供体亲本“Ｌｅｅ”中扩增出了特异的ＤＮＡ片段，而在轮回亲本“Ｔａｉｃｈｕｎｇ２９”中没有扩增出同样的ＤＮＡ片段。引物ＯＰＲ－７扩增出的ＤＮＡ片段长度约为４６５ｂｐ，引物ＯＰＲ－９扩增出的ＤＮＡ片段长度约为６６７ｂｐ。用基因供体亲本“Ｌｅｅ”与感病品种“铭贤１６９”杂交的Ｆ２代分离群体（１８９株植株）进行的遗传连锁性分析表明，引物ＯＰＲ－７扩增出的特异ＤＮＡ片段与ＹｒＬｅｅ基因紧密连锁。     

单向锚定PCR富集扩增在克隆低丰度基因转录本中的应用

利用国际生物信息资源，通过计算机检索寻找与已知功能基因具有高度同源的ＥＳＴｓ，据此设计特异的ＰＣＲ扩增引物，并通过单侧特异引物搭配ｃＤＮＡ分子库载体引物（锚定引物），在低严紧条件下扩增各种组织的ｃＤＮＡ分子库以富集目标片段。继以单向锚定ＰＣＲ扩增产物为模板，用双侧特异引物再次扩增，获得的特异扩增ＰＣＲ产物经测序证实之后，将其用作探针杂交筛选相应的ｃＤＮＡ文库并进而克隆目的基因的全长ｃＤ－ＮＡ。此方法用于三个人类新基因的全长ｃＤＮＡ的分离与克隆，结果全部获得成功。这三个人类新基因分别是Ｈ－ＲａｌＧＤＳ基因、Ｈ－ＣＩＳ基因和Ｈ－Ｓ６ＰＫ基因，它们在ＧｅｎＢａｎｋ数据库的登录号分别是：ＡＦ０２７１６９、ＡＦ０３５９４６及ＡＦ０３７４４７。本文结果提示这一方法对于应用现有的人类基因组生物信息资源，克隆具有重要生物学功能的人类新基因，尤其是克隆呈低丰度的基因转录本全长序列具有较高的实用价值。     

运用一步化体外转录—翻译系统筛查基因突变的新技术

报道了一种筛查基因突变的新技术－ＰＣＲ结合一步化体外转转录－翻译系统。该技术包括：（１）ＰＣＲ扩增靶ＤＮＡ片段，并使ＤＮＡ产物的一端或两端带上Ｔ７Ｔ１序列；（Ｔ１：翻译起始信号，ＣＣＡ ＣＣＡ ＴＧ）；（２）以核的为模板，利用一步化体外转录－翻译系统合成相应肽链；（３）用ＳＤＳ－ＰＡＧＥ及高分辨ｐＨ梯度ＰＡＧＥ（聚丙烯酰胺凝胶电泳）分离分析多肽产物，通过异常电泳图谱而发现基因突变，本文以已知突变的     

大豆灰斑病1号生理小种抗性基因的RAPD标记

以高抗品种东农９６７４为父本,以高感品种东农８７－１０４为母本,杂交得到Ｆ２代群体。该群体经人工接种灰斑病１号生理小种后,分别从抗、感材料中取１５株的叶片提取ＤＮＡ组成抗,感池,用８２０个１０ｍｅｒ随机引物进行ＲＡＰＤ多态性分析,其中３个引物在抗,感池间出现稳定的多态性标记ＯＰＫ０３８４０,ＯＰＭ１７１７００,ＯＰＯ１０９５０,并在Ｆ２代个体中表现出抗性与多态性标记协同分离的趋势,这三个标记与抗性     

DNA指纹图谱在农作物品种鉴定中的应用

ＤＮＡ(脱氧核糖核酸 )是细胞染色体中的遗传基因 ,其分子各个片段就代表各个遗传信息。由于ＤＮＡ指纹图谱直接反映ＤＮＡ水平上的差异 ,它成为当今最先进的遗传标记系统。ＤＮＡ指纹图谱技术主要有 :限制性片段长度多态性(Ｒｅｓｔｒｉｃｔｉｏｎｆｒａｇｍｅｎｔｌｅｎｇｔｈｐｏｌｙｍｏｒ ｐｈｉｓｍ ,简称ＲＦＬＰ)、随机扩增多态性 (ＲａｎｄｏｍａｍｐｌｉｆｉｅｄｐｏｌｙｍｏｒｐｈｉｃＤＮＡ ,简称ＲＡＰＤ )、小卫星ＤＮＡ(Ｍｉｎｉ ｓａｔｅｌｌｉｔｅＤＮＡ)、微卫星ＤＮＡ(ＭｉｃｒｏｓａｔｅｌｌｉｔｅＤＮＡ)、内部简单重…     

玉米自交系8112特异SCAR标记的获得

通过对３个玉米骨干自交系特异ＲＡＰＤ标记的克隆、测序、合成特异引物及ＳＣＡＲ－ＰＣＲ反应条件优化,成功地把其中一个自交系８１１２的特异ＲＡＰＤ标记（０．９ｋｂ）转换成了稳定的ＳＣＡＲ标记。为把局限于实验室水平的ＲＡＰＤ标记推向作物种质鉴定和种子纯度检验的实验应用开辟了一条途径。     

用Bubble PCR法对一个新的锌指基因进行外显子划界

介绍了一种确定基因外显子－内含子交界点的快速、准确的方法，应用该方法对本室克隆的一个新的锌指基因ＺＮＦ１９１进行了外显子划界。将含有ＺＮＦ１９１全长ｃＤＮＡ的基因组ＤＮＡ经限制酶酶切后与退火的Ｂｕｂｂｌｅ ｌｉｎｋｅｒ连接，以该连接产物为模板，用依据ｃＤＮＡ序列设计的引物和Ｂｕｂｂｌｅ ｌｉｎｋｅｒ上的特异引物进行ＰＣＲ扩增，继以相同的扩增条件对扩增产物进行测序，从而确定了外显子－内含子的交界点。     

快速一步法（ROSE法）提取DNA应用于RAPD—PCR扩增

对ＲＯＳＥ法的操作程序进行了进一步的简化，“一管一步”即可完成ＤＮＡ提取全过程，系统地与ＣＴＡＢ和ＳＤＳ法进行了比较，其提取过程明显比ＣＴＡＢ法、ＳＤＳ法简单，提取效率分别比ＣＴＡＢ、ＳＤＳ提高４倍和９倍，无污染物产生；与ＣＴＡＢ和ＳＤＳ法比分别节省费用约２００％和７００％。提取的ＤＮＡ能满足ＲＡＰＤ－ＰＣＲ研究的需要。因此，改良后的ＲＯＳＥ法可能是一种适合于ＤＮＡ提取自动化的方法，同时可能成为植     

抗 SMV 栽培大豆种质资源的 SCAR 标记指纹图谱分析

依据与ＳＭＶ（ＳｏｙｂｅａｎＭｏｓａｉｃＶｉｒｕｓ）抗性基因紧密连锁的共显性ＳＣＡＲ标记ＳＣＷ—０５６６０的序列测定结果，合成了两对ＳＣＡＲ（ＳｅｑｕｅｎｃｅＣｈａｒａｃｔｅｒｉｚｅｄＡｍｐｌｉｆｉｅｄＲｅ－ｇｉｏｎｓ）标记特异引物，对３０个栽培大豆品种进行了指纹图谱分析。结果表明，较短的片段Ｓ—５６００和Ｓ—０５６６０是抗病品种的特征性条带；而较长的片段Ｓ—５１０００和Ｓ—５１６００是感病品种的特征性条带。Ｓｏｕｔｈｅｒｎ杂交结果表明，这两对引物扩增出的条带具有同源性。     

紫菜无性系特异分子标记的获得

用RAPD技术对我国紫菜生产中广泛应用的15个无性系丝状进行了遗传多样性分析,用120个Operon引物进行了筛选,从中挑选可以扩增出清晰,稳定,重复性好和多态性高的8个引物,并用这8个引物从15个无性系中找到了8个特异的RAPD分子标记,而且分别来自8个不同的无性系,利用这些标记可以有效地对这8个无性系进行特异性鉴定,现已完成了对这些RAPD特异产物的回收和克隆,把它们转换成SCAR标记或STS标记的工作正在进行中,这些特异标记的发现对紫菜无性系种质鉴定,持久合理利用以及产权保护具有重要意义。     

Risk assessment of cadmium-contaminated soil on plant DNA damage using RAPD and physiological indices

Impact assessment of contaminants in soil is an important issue in environmental quality study and remediation of contaminated land. A random amplified polymorphic DNA (RAPD) 'fingerprinting' technique was exhibited to detect genotoxin-induced DNA damage of plants from heavy metal contaminated soil. This study compared the effects occurring at molecular and population levels in barley seedlings exposed to cadmium (Cd) contamination in soil. Results indicate that reduction of root growth and increase of total soluble protein level in the root tips of barley seedlings occurred with the ascending Cd concentrations. For the RAPD analyses, nine 10-base pair (bp) random RAPD primers (decamers) with 60-70% GC content were found to produce unique polymorphic band patterns and subsequently were used to produce a total of 129 RAPD fragments of 144-2639 base pair in molecular size in the root tips of control seedlings. Results produced from nine primers indicate that the changes occurring in RAPD profiles of the root tips following Cd treatment included alterations in band intensity as well as gain or loss of bands compared with the control seedlings. New amplified fragments at molecular size from approximately 154 to 2245 bp appeared almost for 10, 20 and 40 mg L(-1) Cd with 9 primers (one-four new polymerase chain reaction, (PCR) products), and the number of missing bands enhanced with the increasing Cd concentration for nine primers. These results suggest that genomic template stability reflecting changes in RAPD profiles were significantly affected and it compared favourably with the traditional indices such as growth and soluble protein level at the above Cd concentrations. The DNA polymorphisms detected by RAPD can be applied as a suitable biomarker assay for detection of the genotoxic effects of Cd stress in soil on plants. As a tool in risk assessment the RAPD assay can be used in characterisation of Cd hazard in soil.     

Current Research Status of Heterodera glycines Resistance and Its Implication on Soybean Breeding

Heterodera glycines (i.e., soybean cyst nematode, SCN) is the most damaging nematode pest affecting soybean crop worldwide. This nematode is managed by means of crop rotation with selected resistant sources. With increasing reports of virulent SCN populations that are able to break the resistance within commonly used sources, there is an increasing need to find new sources of resistance or to broaden the resistance background. This review summarizes recent findings about the genes controlling SCN resistance in soybean, and about how these genes interact to confer resistance against SCN in soybean. It also provides an update on molecular mapping and molecular markers that can be used for the mass selection and differentiation of different resistance lines and cultivars in order to expedite conventional breeding programs. In-depth knowledge of SCN parasitism proteins and soybean resistance responses to the pathogen is critical for the diversification of resistant sources through gene modification, gene stacking, or incorporation of novel sources of resistance through backcrossing or genetic engineering.     

中国不同纬度野生大豆和栽培大豆AFLP分析

采用ＡＦＬＰ分子标记技术,对我国不同纬度的２０份野生大豆和载培大豆（Ｇ．ｍａｘ）进行了多样性分析,结果表明：（１）我国野生大豆的遗传多样性较栽培大豆更为丰富;（２）根据ＡＦＬＰ分析结果,将野生大豆和栽培大豆完全划分为２类,并发现野生大豆栽培大豆的种特异谱带,说明野生大豆和栽培大豆作为２个种有遗传基因的;（３）野生大啼和栽培大豆不同纬度品种间纬度相傧首先聚在一起,表明不同进化类型的大豆其遗传距离与纬     

紫菜自由丝状体SCAR标记的获得

应用RAPD技术对27个紫菜自由丝状体材料进行遗传多样性分析，获得7个紫菜材料的7个特异标记。对这7个特异片段进行了回收、克隆，对其中5个片段已完成了序列测定，根据这5个序列的两端顺序合成了5对SCAR—PCR引物，经过优化PCR反应条件成功地将紫菜材料Porphyra tenuipedalis的RAPD扩增片段OPZ-19140和P．yezoensis qd-8的RAPD扩增片段OPJ-18488转换成两个SCAR标记。因为这两个标记的序列已全部测通，故又称为STS标记。这两个SCAR标记(STS标记)可以作为紫菜P．tenuipedalis和P.yezoensis qd-8自由丝状体材料种质鉴定、优良品系选育和产权保护的分子标记。     

五个紫菜品系间遗传差异的RAPD分析

应用随机引物扩增片段多态性（RAPD）技术对2种紫菜的5个品系进行遗传多样性分析。共筛选出21条随机引物,PCR反应得到147条扩增片段。根据共享扩增片段计算遗传相似性指数（F）和相对遗传距离,利用NJ法构建系统树。结果表明,条斑紫菜或坛紫菜的养殖品系首先聚类在一起,两个条斑紫菜养殖品系之间的遗传距离是0．32,两个坛紫菜养殖品系之间的遗传距离是0．31。条斑紫菜养殖品系CPY1－08A与坛紫菜养殖品系CPH8－83之间的遗传距离最大,达0．42。本文结果显示RAPD可以作为简便有效的分子工具应用于紫菜的遗传多样性和种质鉴定研究中。     

离子注入拟南芥种子引起M1和M2代变异的遗传分析

采用改良PAPD技术,对不同离子注入的拟南芥种子M1代基因池DNA,M1和M2代株植物DNA进行变异分析及遗传稳定性分析。M1代基因池DNA分析表明：178个随机引物中有53个随机引物扩增出差异带。重复实验证明一些引物的PCR产物的条带具有相当的稳定性。实验结果表明,变异频率在一定范围内与注入剂量有关。特别是M2代的遗传稳定性分析证明：利用与M1代相同的引物进行PCR分析,其变异带与M1吻合,故可能离子注入引起的变异是真实遗传的。     

Freshwater toxic cyanobacteria induced DNA damage in apple (Malus pumila), rape (Brassica napus) and rice (Oryza sativa)

Cyanobacteria in freshwater ecosystems can present a harmful effect on growth and development of plants through irrigation with contaminated water. In this study, the effects of microcystins (MCs)-containing cyanobacteria extract (CE) on DNA damage of apple, rape and rice were investigated to explore the phytotoxic mechanism of MCs through DNA fragmentation and RAPD analysis. Determination of DNA fragmentation by fluorescent dye DAPI showed that significant DNA damage was observed in rice seedlings after exposure to CE while DNA fragmentation in rape seedlings and apple cultures did not differ significantly between treatment and control groups. Qualitative characterization of genomic DNA fragmentation by agarose gel electrophoresis supported the quantitative determination using DAPI. The main changes in RAPD profiles of rape seedlings following exposure of lower doses of CE were variation in band intensity for the primers F03 and S01, while higher doses of CE caused loss of normal bands and appearance of new bands except band intensity changes. The data presented here demonstrate that DNA damage in plants occurs following exposure of microcystins, and the polymorphic RAPDs may be used as an investigation tool for environmental toxicology and as a useful biomarker for the detection of genotoxic effects of microcystins on plants.