四甲基氯化铵在PCR扩增小麦基因中的关键作用

利用高简并性引物,用PCR法从小麦DNA或cDNA中合成小麦几丁质酶基因、葡 聚糖酶基因和苯丙氨酸解氨酶基因片段。在PCR反应中添加四甲基氯化铵(TMACl)是合成这些特异基因片段的关键。合成的PCR片段都经末端补齐和磷酸化后用于克隆。核酸序列分析证实,这些PCR产物分别与用于设计PCR引物的基因具有高度的同源性。 Abstract:In the presence of tetramethy1 ammonium chloride(TMAC1),a chitinase gene sequence,a phenylalanine ammonia-lyase gene sequence and a glucanase cDNA sequence of wheat were amplified with highly degenerate primers by PCR.The inclusion of TMAC1 in the PCR reactions was essential for successful amplification of the desired sequences from genomic DNA or cDNA in wheat.The ends of the PCR fragments were made flush and phosphorylated prior to cloning.Sequence analyses of the above PCR fragments confirmed their identities,showing high sequence similarities to the genes used for the design of PCR primers.     

Characterization of Wheat Random Amplified Polymorphic DNA Markers Associated with the H11 Hessian Fly Resistance Gene

In Tunisia, the Hessian fly Mayetiola destructor Say is a major pest of durum wheat （Triticum durum Desf.） and bread wheat （T. aestivum L.）. Genetic resistance is the most efficient and economical method of control of this pest. To date, 31 resistance genes, designated H1-H31, have been identified in wheat. These genes condition resistance to the insect genes responsible for virulence. Using wheat cultivars differing for the presence of an individual Hessian fly resistance gene and random amplified polymorphic DNA （RAPD） analysis, we have identified a polymorphic 386-bp DNA marker （Xgmib1-1A.1） associated with the H11 Hessian fly resistance gene. Blast analysis showed a high identity with a short region in the wild wheat （T. monococcum） genome, adjacent to the leaf rust resistance Lr10 gene. A genetic linkage was reported between this gene （Lr10） and Hessian fly response in wheat. These data were used for screening Hessian fly resistance in Tunisian wheat germplasm. Xgmib1-1A.1-like fragments were detected in four Tunisian durum and bread wheat varieties. Using these varieties in Hessian fly breeding programs in Tunisia would be of benefit in reducing the damage caused by this fly.     

Recycling Isolation of Plant DNA,A Novel Method

DNA is one of the most basic and essential genetic materials in the field of molecular biology.To date,isolation of sufficient and good-quality DNA is still a challenge for many plant species,though various DNA extraction methods have been published.In the present paper,a recycling DNA extraction method was proposed.The key step of this method was that a single plant tissue sample was recycled for DNA extraction for up to four times,and correspondingly four DNA precipitations(termed as the 1st,2nd,3rd and 4th DNA sample, respectively) were conducted.This recycling step was integrated into the conventional CTAB DNA extraction method to establish a recycling CTAB method.This modified CTAB method was tested in eight plant species,wheat,sorghum,barley,corn,rice,Brachypodium distachyon,Miscanthus sinensis and tung tree.The results showed that high-yield and good-quality DNA samples could be obtained by using this new method in all the eight plant species.The DNA samples were good templates for PCR amplification of both ISSR and SSR markers.The recycling method can be used in multiple plant species and can be integrated with multiple conventional DNA isolation methods,and thus is an effective and universal DNA isolation method.     

一种提高RAPD技术扩增效率的有效方法

RAPD技术是由Williams等于1990年首先创立的一种DNA分子标记技术。但是由于低的退火温度和短的引物序列等原因,常常使得RAPD技术的分辨率和可重复性低。本文介绍一种通过延长从退火到延伸的ramp时间,提高RAPD产物的分辨率和产量,由此提高RAPD技术的扩增效率的有效方法。 Abstract:The random amplified polymorphic DNA(RAPD)is a technique of DNA molecular marker,which was first established by Williams in 1990.The resolution and repetition is low because of the low annealed tempture and short primers in the RAPD.In this paper we introduce an improved method for increasing the efficiency of the technique of RAPD by prolonging the ramp time from annealing to extension and increasing the resolution and production.     

小麦及其近缘种中基因组特异性DNA重复序列的研究进展

本文对小麦族植物中基因组特异性DNA重复序列的分类、基本特征、分离和鉴定方法、在小麦遗传改良中的应用以及未来研究的发展趋势进行了简述。综合已有的研究结果可以看出基因组特异性DNA重复序列是小麦族植物基因组特异性形成的重要构成部分。对基因组特异性DNA重复序列的研究是认识小麦族植物基因组的有效途径之一,基因组特异性DNA重复序列的应用将进一步促进小麦族植物分子细胞遗传学和普通小麦遗传改良研究的进展。 Advances in Studies of Genome-Specific Repetitive DNA Sequences in Wheat and Related Species BAI Jian-rong1,2,JIA Xu1,WANG Dao-wen1 1.The State Key Laboratory of Plant Cell and Chromosome Engineering,Institute of Genetics and Developmental Biology,The Chinese Academy of Sciences,Beijing 100101,China; 2.Crop Genetics Institute,Shanxi Academy of Agricultural Sciences,Taiyuan 030031,China Abstract:In this paper we review recent advances in studies of several aspects of genome specific repetitive DNA sequences in wheat and related species.The available results demonstrate that genome specific repetitive DNA sequences are important components of genome specificity in wheat and related species.Research on genome specific repetitive DNA sequences is essential to the elucidation of genome function.The application of genome specific repetitive DNA sequences will aid molecular cytogenetic studies in wheat and related species and contributes to genetic improvement of common wheat. Key words：wheat;genome specific repetitive DNA sequence;chromosome     

Retinal ganglion cells of high cytochrome oxidase activity in the rat

Retinal ganglion cells in the rat were studied using the heavy metal intensified cytochrome oxidase and horseradish peroxidase histochemical methods.The results show that a population of large retinal ganglion cells was consistently observed with the cytochrome oxidase staining method in retinas of normal rats or rats which received unilateral thalamotomy at birth.These cytochrome oxidase rich ganglion cells appeared to have large somata,3-6 primary dendrites and extensive dendritic arbors,and are comparable to ganglion cells labeled by the wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP).However,the morphological details of some of the cells revealed by the cytochrome oxidase staining method are frequently better than those shown by the HRP histochemical method.These results suggest that the mitochondrial enzyme cytochrome oxidase can be used as a simple but reliable marker for identifying and studying a population of retinal genglion cells with high metabolic rate in the rat.     

The Comparative and Functional Study between Two Construction Methods of shRNA Expression Vector Targeted LMP1 Gene Encoded by EBV

To look for a more stable and convenient way of constructing short hairpin RNA expression vectors targeting the latent membrane protein-1(LMP-1)encoded by Epstein-Barr virus(pshLMP1),and to study the inhibition function of pshLMP1 expression vectors in HNE1 cells,we designed the pshLMP1 expression cassette and pshLMP1 expression vectors by both the annealing method and PCR method and then co-transfected with pEGFP-N1-1158 into HNE1 cells to observe the mRNA and protein levels of LMP-1 genes by green fluorescence analysis,RT-PCR and western blot.pshLMP1 expression vectors were successfully obtained by both methods but better cloning efficiency was achieved and fewer deletions and mutations of nucleotides were achieved with the PCR method.Furthermore,the mRNA and protein levels of LMP-1 genes were down-regulated by pshLMP1 expression vectors.According to our research,we found that the PCR method provides a more efficient way to construct pshLMP1 expression vectors which have the ability to inhibit the function of LMP-1 genes expressed in HNE1 cells,and also provides a novel application of RNA interference technology against-EBV.     

Developmental pathways for shaping spike inflorescence architecture in barley and wheat

Grass species display a wide array of inflorescences ranging from highly branched compound/panicle inflorescences to unbranched spike inflorescences. The unbranched spike is a characteristic feature of the species of tribe Triticeae, including economically important crops,such as wheat and barley. In this review, we describe two important developmental genetic mechanisms regulating spike inflorescence architecture in barley and wheat.These include genetic regulation of(i) row-type pathway specific to Hordeum species and(ii) unbranched spike development in barley and wheat. For a comparative understanding, we describe the branched inflorescence phenotypes of rice and maize along with unbranched Triticeae inflorescences. In the end, we propose a simplified model describing a probable mechanism leading to unbranched spike formation in Triticeae species.     

Use of Random Amplified Polymorphic DNA Analysis for Economically Important Food Crops

The objective of this review is to summarize numerous studies on the use of the random amplified polymorphic DNA （RAPD） technique on rice, corn, wheat, sorghum, barley, rye, and oats to examine its feasibility and validity for assessment of genetic variation, population genetics, mapping, linkage and marker assisted selection, phylogenetic analysis, and the detection of somaclonal variation. Also we discuss the advantages and limitations of RAPD. Molecular markers have entered the scene of genetic improvement in different fields of agricultural research. The simplicity of the RAPD technique made it ideal for genetic mapping, plant and animal breeding programs, and DNA fingerprinting, with particular utility in the field of population genetics.     

Alien DNA introgression and wheat DNA rearrangements in a stable wheat line derived from the early generation of distant hybridization

In general, it requires five or more generations to develop a stable line by the traditional breeding method of cultivar hybrids in self-pollinated crops, such as wheat, barley, and rice. No doubt, the breeding period will be shortened and thus the breeding effi-ciency will be improved if the hybrids can stabilize in early generation. The method of haploid breeding, that is, obtaining stable pure lines (DH lines) of cultivar hybrids with the aid of anther culture and then chro-mosome doubling,…     

小麦抗茎腐病种质筛选及鉴定新方法的建立

为筛选针对我国黄淮麦区小麦茎腐病抗病新种质,建立可区分小麦其他茎基部病害的茎腐病抗性鉴定方法,本文采用室内苗期鉴定方法对主要来自黄淮麦区的108份小麦品种和高代品系进行茎腐病抗性评价,对其中45份小麦材料同时采用荧光定量PCR方法测定基部茎秆的禾谷镰刀菌DNA含量并与其茎腐病平均病级进行相关分析。共筛选到中抗茎腐病材料22份,未发现高抗和免疫品种(系);相关分析结果表明,小麦基部茎秆禾谷镰刀菌DNA含量与其茎腐病平均病级呈极显著正相关(r=0.73**),小麦基部茎秆禾谷镰刀菌DNA含量可以作为小麦茎腐病抗性的重要参考。抗病新种质的筛选和荧光定量PCR抗性评价方法的建立将为今后黄淮麦区小麦抗茎腐病品种的培育提供帮助。     

一种适于PCR扩增的小麦基因组DNA快速提取法

许多小麦分子生物学研究需要对大量的小麦样品进行PCR检测,因此,建立一种快速提取小麦基因组DNA的方法十分必要。根据国外报道的一种快速提取水稻和玉米基因组DNA的方法,我们对部分提取步骤进行变动后,在小麦上进行了尝试,长度为1．5kb的片段能得到稳定的扩增。该方法样品研磨在1.5ml的离心管内进行,后续操作不用酚、氯仿、CTAB、SDS和巯基乙醇,整个提取过程不需要使用通风橱,操作步骤简单,花费时间少,而且提取的小麦基因组DNA完整性好,量也较可观。一个DNA样品可供50～100次PCR反应使用,适用于小麦遗传多样性、分子标记辅助选择、转基因后代检测以及引物筛选、分子标记定位等多种研究。     

Specific detection of wheat residues in processed foods by polymerase chain reaction

A sensitive qualitative detection method for wheat in foods using polymerase chain reaction (PCR) was developed. Trace amounts of wheat in commercial food products could be qualitatively detected by this method. The sensitivity of the proposed PCR method appears to be similar to that of ELISA. The present method should be very useful for detecting wheat residues in processed foods.     

HMW-GS的SDS-PAGE图谱在小麦品质评价中的应用

采用SDS—PAGE技术对陕西关中地区各时期大面积推广小麦品种、品种资源和新品系的HMW—GS组成进行了分析。在该地区50多年大面积推广的33个品种中,检测出9种HMW亚基(对)及其组成;品种HMW—GS的评分在5～10分之间,平均6．9分;4个时期品种HMW-GS的平均评分有升有降,优质亚基出现的频率普遍偏低;向小麦品种中聚合多种优质HMW—GS将成为陕西关中未来小麦育种的主要目标之一。53种小麦品种资源的亚基或亚基对组合类型比较丰富,具有一批携带优质亚基5 10、1、2*、7 8、14 15或17 18等资源。8个新选品系中,有4个品系携带了多种优质亚基,其中3个品系HMW—GS的评分为10分;Q1043已被审定通过,目前正在陕西关中推广种植。实践证明,采用SDS—PAGE方法对生产上推广品种、品种资源和新选品系的HMW—GS变异研究,有助于在短时间内了解生产上推广小麦品质生产现状、制订育种目标、选配亲本和品系品质性状筛选,是一种非常实用的品质快速检测方法。     

适合流式细胞仪分析的小麦细胞核提取方法比较

为制备供流式细胞仪分析的高纯度小麦细胞核悬液,以冬小麦"临优2018"为材料,分别采用酶解法和直接剪切法对其幼苗的细胞核进行提取,对所得到的细胞核在形态结构和数量等方面进行了分析和比较,根据其优缺点优化出最适合流式细胞仪分析的小麦幼苗细胞核的提取方法。结果显示:在直接剪切法所用的3种细胞核提取缓冲液中,MgSO4缓冲液的提取效果最好,细胞核形态及内部结构完整,且得到的细胞核量多;OttoⅠ对细胞核的提取效果显著,但是在加入OttoⅡ后细胞核破裂明显;Tris.MgCl2缓冲液提取的细胞核数量较少;酶解法制备的细胞核悬浮液中杂质较多,且需时较长。结果表明采用MgSO4提取缓冲液的直接剪切法是适合流式细胞仪对小麦幼苗DNA含量的分析。     

定位转基因所在的小麦基因组的细胞遗传学方法

本文设计了一种利用细胞遗传学原理定位转基因小麦部转基因所在的基因组的系统方法,并对该方法的有效性和可靠性进行了验证。该方法利用分别含有AABB、AADD和BBDD基因组的小麦近缘四倍体与等测转基因小麦杂交,F1一倍体与同源非转化品种回交,根据回交群体中在抗性基因控制的性状上的分离比例以及各个体的染色体数目分析,从而对转基因小麦中转基因所在的基因组进行定位,该方法简单、可靠,实用性强,可供相关研究采用。     

The production of hypoallergenic wheat flour and the analysis of its allergy suppressive effects

We propose a novel method to produce hypoallergenic wheat flour suitable for patients allergic to wheat by using enzymatic fragmentation with cellulase and actinase. The hypoallergenic flour displayed potent inhibitory activity against allergen absorption and actively suppressed allergic reactions, probably inducing oral tolerance. The results suggest that hypoallergenic wheat flour has allergy-suppressive effects without inducing side effects.     

Evaluation and Elimination of the Interference by Starch in the Biuret Determination of Wheat Protein

In the biuret determination of wheat protein, starch, color substances and lipids extractable with an alkaline solution such as biuret reagent were found to interfere with the biuret method. Comparative evaluation of their effects revealed that starch, a major component of wheat, has the most significant effect on the biuret method. In the presence of starch, the correlation between Kjeldahl protein and the optical density of biuret was poor.

It was found that several organic solvents effectively retarded solubilization of wheat starch in the biuret reaction mixture. By treatment of the wheat samples with these solvents prior to the biuret procedure, the effect of starch was completely eliminated and the optical density of biuret became closely correlated with Kjeldahl protein.     

小麦根蛋白提取与双向电泳方法的优化与应用

为了建立一套适合小麦根蛋白质组分析的双向电泳系统（2-DE）,得到更加清晰的电泳图谱,本研究以小麦幼苗根系为材料,对根蛋白的提取方法、上样量等进行了优化。研究发现,上样量为1200μg时,Trizol抽提法提取的蛋白能够获得图像清晰、分辨率高、重复性好的双向电泳图谱,基本满足小麦根系蛋白质组学的分析和研究。     

安徽省冬小麦施肥现状与肥料利用效率

冬小麦是安徽省重要的粮食作物。合理施肥是冬小麦获得高产的重要措施,明确当前安徽省冬小麦施肥现状和存在的问题对于指导冬小麦科学施肥具有重要意义。本研究以安徽省冬小麦为对象,于2018年对全省冬小麦主产区1591户农户进行调查,调查内容包括肥料品种、肥料用量、施肥方式、种植面积和产量水平。根据调查结果统计分析安徽省冬小麦种植过程中的施肥现状及存在问题,并以全省冬小麦平均产量和平均施肥量为基准,采用Cate-Nelson方法(十字交叉法)评估安徽省冬小麦产量与氮、磷和钾肥施用的关系,以探索安徽省冬小麦进一步增产增效的主要施肥途径。结果表明: 安徽省冬小麦平均产量为5185 kg·hm^-2,氮、磷和钾肥平均施用量分别为206、80和78 kg·hm^-2。其中旱茬麦氮、磷和钾肥平均施用水平比稻茬麦分别高14、16和3 kg·hm^-2。总体上,安徽省冬小麦种植中化肥平均用量趋于合理化,但是在施肥方式、养分运筹和肥料品种上还存在不合理现象。Cate-Nelson方法结果表明,全省冬小麦氮、磷、钾肥施用量中,仅有23.8%、21.2%和25.7%低于全省平均水平却获得较高的产量,此时氮、磷、钾肥偏生产力最高;有26.3%、19.3%和22.5%的施用量低于全省平均水平但没有实现高产;有26.2%、29.6%和25.0%的施用量虽然获得了高产,但施用量较高,氮磷钾肥偏生产力较低。这说明安徽省冬小麦增产增效还有很大的空间。全省冬小麦基肥和追肥机械化比例分别为62.7%和10.0%。虽然氮肥普遍实现了分次施用,但氮肥作基肥的比例仍然占到全生育期氮肥总量的69.0%,应适当降低。此外,在肥料品种选择上存在偏施化肥、不重视有机肥等问题。