外源DNA导入糯玉米自交系D0代变异植株过氧化物酶同工酶分析

采用改良浸种法分别将甘蔗总DNA与孟加拉超甜玉米自交系总DNA导入糯玉米自交系12-9-10,在D0代分别获得变异植株,采用聚丙烯酰胺垂直电泳法对该植株及供受体进行了过氧化物酶同工酶分析,其结果表明植株D0(糯×甘300)的过氧化物酶同工酶在迁移率Rf5=0.33和Rf6=0.40分别出现一条供体特有而受体没有的酶带,其酶带强弱与供体相同;在迁移率Rf8=0.61出现一条供受体都没有的弱带。植株D0(糯×孟甜300)-2在迁移率Rf6=0.59增加了一条供受体都没有的弱带。说明了外源DNA(或DNA片段)已转移到糯玉米自交系12-9-10中,并得到了表达。     

离子注入对萝卜过氧化物酶、淀粉酶和蛋白酶同工酶的影响

研究了氮离子和氩离子注入萝卜种子对萝卜幼苗蛋白含量、过氧化物酶活性及过氧化物酶、淀粉酶和蛋白酶同工酶的影响。结果表明 :离子注入后 ,减低萝卜过氧化物酶活性和蛋白含量。萝卜不同生长时期同工酶变化不一样。在子叶时期 ,过氧化物酶同工酶谱带无明显变化 ,淀粉酶同工酶有酶带的消失 ;而在真叶时期 ,过氧化物酶在负极区减少一条酶带 ,Rf为 0 .2 2 ,正极区增加一条酶带 ,Rf为 0 .6 ,且随剂量增加 ,酶带着色增强 ;淀粉酶同工酶在注入剂量为 5× 10 5N+ / cm2 )时 ,有同工酶带增加 ,Rf为 0 .6 1。低剂量时蛋白酶活性增强 ,谱带增多 ,大剂量则减弱。因此 ,N+和 Ar+注入后 ,可影响萝卜过氧化物酶和淀粉酶的表达及蛋白质的合成或降解。     

辐射对大麦过氧化物酶同工酶谱影响的初步研究*

γ射线处理大麦种子后其幼苗组织中过氧化物酶同工酶谱发生较大的变化。在偏负极端RfO.29-0.38出现新的酶带区,酶带数目随辐射剂量的增加而增加。低剂量辐射后在该区出现1-2条新同工酶带,以RfO.29 和Rf0.34酶带为主;较高剂量辐射后在该区出现2-3条新酶带,Rf为0.29、0.34或Rf0.29、0.34、0.38。研究结果表明这一带区的过氧化物酶同工酶谱与辐射损伤有一定的关系,其变化特点能反映出供试品种的辐射敏感性。     

几种同工酶与猕猴桃品种对溃疡病抗性关系的研究

应用聚丙烯酰胺凝胶电泳、同工酶分析技术分别研究了猕猴桃植株体内过氧化物酶(POD)、多酚氧化酶(PPO)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、酯酶(EST)同工酶谱带的变化,结果表明:自然感染溃疡病前后,此6种同工酶谱带特征在不同抗感品种中表现出一定的差异.未感染溃疡病菌前,抗(感)品系枝条、叶片POD同工酶均有2条酶带,PPO同工酶有3条酶带,但感病品种酶带颜色深且粗,而抗病品种酶带颜色浅且细,叶片酶带颜色深于枝条;SOD、CAT同工酶谱带均为1条,Rf值分别为0.38、0.28,感性品种较抗耐品种谱带亮度高活性强;自然发病后,抗(感)品系POD、PPO同工酶谱带数都增加,分别为4、3条和5、4条,且抗病品种新酶带出现较感病品种早且酶带粗颜色深活性强,感病品系虽也有新酶带出现,但酶带少活性弱,抗病品系枝条、叶片POD、PPO同工酶新谱带的Rf值分别为0.63、0.67和0.85、0.87;抗感病品种SOD、CAT同工酶都被诱导产生了1条新的同工酶谱带,Rf分别为0.32和0.27,新酶带现色时间迟,且酶带颜色浅活性弱,但抗耐品种较感性品种谱带亮且活性强;EST同工酶于自然发病前后变化不大,与抗病性关系不很明显.     

辐照外源DNA导入番茄两种同工酶的研究

采用聚丙烯酰胺梯度凝胶电泳对辐照外源DNA导入番茄的受体后代进行过氧化物酶和细胞色素氧化酶同工酶研究,并对供体叶形在受体后代中的表达和后代株高进行分析。研究表明,多数受体后代与受体对照的两种同工酶图谱差异较大而与供体对照的两种同工酶图谱较为相似,这与它们在外观性状（叶形和株高）上的差异是吻合的。受体后代与体供在同工酶图谱上的相似性以及供体外观性状在受体后代的表达表明了外源DNA已经导入受体,并得到整合表达,说明辐照外源DNA导入技术是改良番茄品种,丰富育种材料的一条有效途径,在番茄选育新品种方面有良好的应用前景。     

不同生育期菊芋叶片过氧化物酶同工酶表达特点的研究

用聚丙烯酰胺凝胶电泳(PAGE)法,分离了不同生育期菊芋叶片过氧化物酶(Helianthus tuberosus.1eves peroxidase,HLP)同工酶,并测定了HLP的酶活性;比较了菊芋叶及其他4种植物POD的特点。结果表明:(1)同一植株不同部位的HLP酶活性大小顺序为,中部成熟叶(6.89×103U/g)>底部衰老叶(5.38×103U/g)>上部嫩叶(2.82×103U/g);由嫩叶→成熟叶→衰老叶的发育过程中,HLP同工酶酶带共有9条,出现的方式有稳定型(Rf 0.81带)、酶活性先弱后强型(Rf0.52、0.67、0.74、0.85带)、酶带先有后无型(Rf 0.70、0.72带)、酶带先无后有型(Rf 0.77)4种。(2)几种植物POD的酶活性的大小顺序是:甘蔗叶>竹笋壳>菊芋叶>大豆壳;POD同工酶电泳最大迁移率(Rf)大小顺序是:菊芋叶(0.85)>甘蔗叶(0.70)>竹笋壳(0.61)>大豆壳(0.57)>辣根(0.55),HLP同工酶属阴离子型。探讨了菊芋POD的表达特点。     

外源DNA导入大豆后代种皮颜色变异的过氧化物酶分析

本实验用聚丙烯酰胺凝胶电泳的方法,对外源DNA导入大豆引起种皮颜色变异的后代进行了过氧化物酶同工酶的酶谱分析.结果表明:不同种皮颜色的后代含有供体的谱带.这说明了外源DNA片断已整合到受体基因组中并得到表达.     

张杂谷苹果酸脱氢酶和过氧化物酶同工酶研究

以张杂谷‘3号’、‘5号’、‘6号’及其亲本为材料,通过聚丙烯酰胺垂直板凝胶电泳技术研究不同材料不同生育期苹果酸脱氢酶和过氧化物酶同工酶酶谱特性,同时对总酶活进行了测定,探讨谷子杂种优势的形成机理。结果表明:(1)张杂谷及其亲本叶片苹果酸脱氢酶共有5条酶带,有共同酶带和偏母本型酶带;过氧化物酶同工酶共有14条,属于共同酶带;从负极到正极苹果酸脱氢酶同工酶Rf分别为0.611、0.626、0.642、0.684和0.716,而过氧化物酶同工酶Rf分别为0.08、0.21、0.24、0.30、0.36、0.44、0.49、0.58、0.66、0.69、0.72、0.75、0.85和0.89。(2)杂种苹果酸脱氢酶同工酶和母本酶谱同型,过氧化物酶同工酶酶谱和父母本一致;供试品种苹果酸脱氢酶酶谱相对简单,酶带集中,而过氧化物酶同工酶酶带数量、活性和宽度差异性较大,酶谱比较复杂。(3)所有品种苹果酸脱氢酶总活性在抽穗期最高,过氧化物酶总活性在灌浆初期最高。杂种苹果酸脱氢酶活性在苗期和抽穗期表现出不同程度的超亲优势,而过氧化物酶在抽穗和灌浆初期均表现出不同程度的超亲优势,这可能与杂种优势有关。     

离子注入哈茨木霉筛选高产促植物生长物质菌株的研究

用 N+注入哈茨木霉所获得的突变型的发酵液浇灌水稻种子 ,结果发现 :5× 10 - 6、5× 10 - 5、2× 10 - 4木霉突变株发酵稀释液处理的种子幼苗长势优于对照组和木霉原种组。其 5 0倍、15 0倍木霉发酵稀释液浇灌的种子幼苗过氧化物酶同工酶与对照和原种相比均出现了新的谱带 A- 3、C- 3带。各突变株 5 0倍发酵稀释液和原种发酵液浇灌的水稻幼苗过氧化物酶同工酶出现了 B- 3带。而四种浓度的木霉发酵稀释液处理的水稻幼苗 ,其酯酶同工酶与对照组和原种组相比 ,无明显差异     

湖北海棠阶段转变过程中几种同工酶变化研究

采用聚丙烯酰胺凝胶电泳法,研究了湖北海棠1年生实生苗(S1)、4年生(S4)、14年生(S14)实生树及4年生成年芽嫁接树(G4)不同高度叶片的过氧化物酶(POX)、细胞色素氧化酶(COD)、酯酶(EST)、乙醇脱氢酶(ADH)同工酶的变化。结果表明：多年生实生树随着高度的增加,POX Rf 0.56、060,COD Rf 0.50,EST Rf 0.09、015、075、077,ADH Rf 0.76等几条酶带呈现差异,出现了童区和成年区各自特有的同工酶带,随高度变化童区特征酶带消失或成年区特征酶带出现的高度均保持在150～200 cm之间,同当年植株开花最低位置相同,S4、S14童区与S1同工酶谱带完全一致,S4、S14成年区与G4 同工酶谱基本保持一致,G4同工酶谱并未随高度增加而发生变化。四种同工酶可以作为阶段转变的指标。S4、S14童区与成年区谱带的差异是高度的结果,高度是湖北海棠阶段转变的重要因子。     

玉米/大豆间作条件下的作物根系生长及水分吸收

通过田间试验研究了玉米/大豆条带间作群体的根系分布及土壤水分吸收规律.结果表明：水分充足条件下,土壤剖面内玉米和大豆根系的分布模式近似于三角形;玉米根系水平分布范围较大,侧向伸展长度约为58 cm,16～22 cm土层的玉米根系侧向伸展最远,玉米根系不仅分布于间作条带行间,而且生长到大豆条带的行间;大豆根系水平分布于相对有限的区域内,侧向伸展长度约为26 cm.作物根质量密度随着距作物行（玉米或大豆）距离的增加而减少,玉米行和边行大豆根质量密度的90%分布于0～30 cm土层.距玉米行10 cm处玉米的根质量密度高于大豆,距玉米行20 cm处大豆的根质量密度大于玉米,两种作物根质量密度的85%都分布于0～30 cm土层内.间作条带内水分变化主要集中在0～30 cm土层,水分变化量依次为：玉米区域>大豆区域>条带行间.表明在水分充足条件下,间作作物优先在自己的区域吸水,根系混合区吸水滞后发生.     

不同杂种优势类群玉米幼胚愈伤组织的诱导及植株再生特性的研究

以Reid、唐四平头和其它种质等3个杂种优势类群共19份玉米自交系为试验材料,以玉米幼胚作为外植体,研究了基因型、培养基和激素对玉米幼胚愈伤组织的诱导及植株再生的影响,结果表明供试材料均能进行愈伤组织的诱导,但是仅有12个自交系能再生植株。N6和改良N6培养基有助于提高愈伤组织的质量及其生长速度,2,4-D在愈伤组织的诱导中起着关键性作用。在诱导培养基中添加0.2mg／L的6-BA或KT会使胚性愈伤组织的诱导频率下降以及降低愈伤组织的质量。在胚状体诱导培养基中添加1mg／L的KT能促进绿苗的分化,但是浓度过高会使丛生苗分化过多。此外,通过对不同杂种优势类群自交系玉米幼胚培养特性的分析,发现在唐四平头类群的4个自交系中,黄早四的绿苗分化率仅为0.5％,其它3个自交系不能再生植株。但是,从Reid和其它种质类群的供试自交系中筛选出了胚性愈伤组织的诱导频率和绿苗分化率均较高的、适合于遗传转化的受体材料,如3189／4380、4380／陕综5、8103、先早17、18-599红、18-599白、501、178和冀53。     

Influence of Helminthosporium maydis, Race T, Toxin on Potassium Uptake in Maize Roots: II. Sensitivity of Development of the Augmented Uptake Potential to Toxin and Inhibitors of Protein Synthesis

Basal K⁺ uptake in the root midzone region (cm 2 + 3 + 4) of N and T cytoplasmic versions of each of four maize inbreds was equally sensitive to the toxin(s) of Helminthosporium maydis, race T. Basal K⁺ uptake in the root apex (0-1 cm) and augmented K⁺ uptake in the root midzone were more toxin-sensitive in inbreds W64A(T) and Mo17(T) than in inbreds W64A(N) and Mo17(N). This differential response of N and T cytoplasms to toxins was not found for corresponding cytoplasms of inbreds WF9 and B37.     

玉米自交系幼胚培养能力与遗传关系研究(简报)

玉米幼胚培养获得的胚性愈伤组织具有较强的长期继代能力和再生植株能力,常被用于构建转基因受体系统。然而基因型是制约玉米组织培养植株再生的重要因素之一,不同玉米基因型的幼胚培养能力关系到其遗传转化研究的结果[1]。     

Distribution of Unlinked Receptor Sites for Transposed Ac Elements from the Bz-M2(ac) Allele in Maize

We have shown before that the Ac element from the maize bz-m2(Ac) allele, located in the short arm of chromosome 9 (9S), transposes preferentially to sites that are linked to the bz donor locus. Yet, about half of the Ac transpositions recovered from bz-m2(Ac) are in receptor sites not linked to the donor locus. In this study, we have analyzed the distribution of those unlinked receptor sites. Thirty-seven transposed Ac (trAc) elements that recombined independently of the bz locus were mapped using a set of wx reciprocal translocations. We found that the distribution of unlinked receptor sites for trAs was not random. Ten trAcs mapped to 9L, i.e., Ac had transposed to sites physically, if not genetically, linked to the donor site. Among chromosomes other than 9, the Ac element of bz-m2(Ac) appeared to have transposed preferentially to certain chromosomes, such as 5 and 7, but infrequently to others, such as 1, the longest chromosome in the maize genome. The seven trAc elements in chromosome 5 were mapped relative to markers in 5S and 5L and localized to both arms of 5. We also investigated the transposition of Ac to the homolog of the donor chromosome. We found that Ac rarely transposes from bz-m2(Ac) to the homologous chromosome 9. The clustering of Ac receptor sites around the donor locus has been taken to mean that a physical association between the donor site and nearby receptor sites occurs during transposition. The preferential occurrence of 9L among chromosomes harboring unlinked receptor sites would be expected according to this model, since sites in 9L would tend to be physically closer to 9S than sites in other chromosomes. The nonrandom pattern seen among the remaining chromosomes could reflect an underlying nuclear architecture, i.e., an ordering of the chromosomes in the interphase nucleus, as suggested from previous cytological observations.     

Molecular marker diversity among current and historical maize inbreds

Advanced-cycle pedigree breeding has caused maize (Zea mays L.) inbreds to become more-elite but more-narrow genetically. Our objectives were to evaluate the genetic distance among current and historical maize inbreds, and to estimate how much genetic diversity has been lost among current inbreds. We selected eight maize inbreds (B14, B37, B73, B84, Mo17, C103, Oh43 and H99) that largely represented the genetic background of current elite inbreds in the U.S. seed industry. A total of 32 other inbreds represented historical inbreds that were once important in maize breeding. Cluster analysis of the inbreds, using data for 83 SSR marker loci, agreed well with pedigree information. Inbreds from Iowa Stiff Stalk Synthetic (BSSS), Reid Yellow Dent, and Lancaster clustered into separate groups with only few exceptions. The average number of alleles per locus was 4.9 among all 40 inbreds and 3.2 among the eight current inbreds. The reduction in the number of alleles per locus was not solely due to sample size. The average genetic distance (D _ij ) was 0.65 among the eight current inbreds, 0.67 among the 32 historical inbreds, and 0.67 among all 40 inbreds. These differences were statistically insignificant. We conclude that genetic diversity among current inbreds has been reduced at the gene level but not at the population level. Hybrid breeding in maize maintained, rather than decreased, genetic diversity, at least during the initial subdivision of inbreds into BSSS and non-BSSS heterotic groups. We speculate, however, that exploiting other germplasm sources is necessary for sustaining long-term breeding progress in maize. Received: 21 August 2000 / Accepted: 5 January 2001     

DNA methylation in wheat. Purification and properties of DNA methyltransferase

The origin and function of the large amount of 5-methylcytosine in plant DNA is not well understood. As a tool for in vitro studies of methylcytosine formation in plants we have isolated and characterized the DNA methyltransferase present in germinating wheat embryo. An enzyme fraction enriched 300-fold over the tissue homogenate was obtained by salt extraction of nuclei, chromatography on DEAE-cellulose, Sephadex G-75, blue Sepharose and on DNA immobilized on cellulose. It catalyzes the methylation of cytosine residues in double-stranded DNAs isolated from wheat, maize, calf thymus or bacteria using S-adenosylmethionine as methyl donor. The efficient methylation of both an unmethylated plasmid DNA and its hemimethylated derivative indicate that the wheat DNA methylase can function de novo and in maintenance methylation. A relative molecular mass of 50,000-55,000 was estimated by gel permeation chromatography and sucrose density gradient centrifugation. Polyacrylamide gel electrophoresis showed the presence of a protein of Mr = 50,000 and one other component (Mr = 35,000). The preference for endogenous, double-stranded DNA as substrate and the lower molecular mass distinguish wheat DNA methyltransferase from the DNA methylases obtained from mammalian sources. The properties of the wheat enzyme resemble, however, those of the DNA methylase isolated from the alga Chlamydomonas reinhardii, suggesting that plant cells possess their own type of DNA methyltransferase for the biosynthesis of their high methylcytosine content in DNA.     

Variation of DNA fingerprints among accessions within maize inbred lines and implications for identification of essentially derived varieties: II. Genetic and technical sources of variation in AFLP data and comparison with SSR data

Accuracy and reproducibility of genetic distances (GDs) based on molecular markers are crucial issues for identification of essentially derived varieties (EDVs). Our objectives were to investigate (1) the amount of variation for amplified fragment length polymorphism (AFLP) markers found among different accessions within maize inbreds and doubled haploid (DH) lines, (2) the proportion attributable to genetic and technical components and marker system specific sources, (3) its effect on GDs between maize lines and implications for identification of EDVs, and (4) the comparison to published SSR data from the same plant materials. Two to five accessions from nine inbred lines and five DH lines were taken from different sources of maintenance breeding or drawn as independent samples from the same seed lot. Each of the 41 accessions was genotyped with 20 AFLP primer combinations revealing 988 AFLP markers. Map positions were available for 605 AFLPs covering all maize chromosomes. On average, six (0.6%) AFLP bands were polymorphic between different accessions of the same line. GDs between two accessions of the same line averaged 0.013 for inbreds and 0.006 for DH lines. The correlation of GDs based on AFLPs and SSRs was tight (r = 0.97**) across all 946 pairs of accessions but decreased (r = 0.55**) for 43 pairs of accessions originating from the same line. On the basis of our results, we recommend specific EDV thresholds for marker systems with different degree of polymorphism. In addition, precautions should be taken to warrant a high level of homogeneity for DNA markers within maize lines before applying for plant variety protection.     

In Situ Nitrogen Mineralization,Nitrification, and Ammonia Volatilization in Maize Field Fertilized with Urea in Huanghuaihai Region of Northern China

Nitrogen (N) fertilization potentially affects soil N mineralization and leaching, and can enhance NH₃ volatilization, thus impacting crop production. A fertilizer experiment with five levels of N addition (0, 79, 147, 215 and 375 kg N ha^-1) was performed in 2009 and 2010 in a maize field in Huanghuaihai region, China, where > 300 kg N ha^-1 has been routinely applied to soil during maize growth period of 120 days. Responses of net N mineralization, inorganic N flux (0–10cm), NH₃ volatilization, and maize yield to N fertilization were measured. During the growth period, net N mineralization and nitrification varied seasonally, with higher rates occurring in August and coinciding with the R1 stage of maize growth. Soil NO₃ ⁻-N contributed to more than 60% of inorganic N flux during maize growth. Cumulative NH₃ volatilization increased with N additions, with total NH₃ volatilization during maize growth accounting for about 4% of added N. Relative to the control, mean maize yield in the fertilizer treatments increased by 17% and 20% in 2009 and 2010, respectively. However, grain yield, aboveground biomass, and plant N accumulation did not increase with added N at levels > 215 kg N ha^-1. These results suggest that the current N rate of 300 kg N ha^-1 is not only excessive, but also reduces fertilizer efficacy and may contribute to environmental problems such as global warming and eutrophication of ground water and streams.