Microtubules in epidermal and cortical root cells of Brassica rapa during clinorotation

Using confocal microscopy the organization of tubulin cytoskeleton including endoplasmic and cortical microtubules (CMTs) has been studied in epidermal and cortical cells of the different growth zones of main root of Brassica rapa L. 6-days-old seedlings in control conditions and under clinorotation. It was shown that changes in CMTs orientation occured only in the distal elongation zone (DEZ). In the control, CMT arrays oriented transversely to the root long axis. Under clinorotation appearance of the shorter randomly organized CMTs was observed. Simultaneously, a significant decrease in the cell length in the central elongation zone (CEZ) under clinorotation was detected. It is suggested that the decline of anisotropic growth typical for CEZ cells is connected with CMTs disorientation under clinorotation.     

白菜型油菜自交亲和性变异分析

对不同来源的85份白菜型油菜品种的自交亲和指数研究表明,白菜型油菜的自交亲和性存在较大幅度的变异,自交亲和指数在0．00～9．28之间,这种变异不仅存在于品种间,而且存在于品种内不同个体间。85份参试材料中,自交亲和指数小于1的有50个品种;35个品种自交亲和指数大于1,自交亲和指数在1．00～9．28之间,其中亲和指数大于4的材料有28、44、45、55x、75、119、123、139号品种等。依据自交亲和指数,可将参试材料分为4种类型:高自交亲和类型(自交亲和指数>4．00);自交亲和类型(自交亲和指数1．00～3．99);自交不亲和类型(0．00< 自交亲和指数<1．00);高自交不亲和类型(自交亲和指数=0．00)。自交亲和性因地区而异,一般西部地区的品种自交亲和性高于其它地区的品种。     

Biomass, reproductive output, and physiological responses of rapid-cycling Brassica (Brassica rapa) to ozone and modified root temperature

Brassica rapa L. (rapid-cycling Brassica), was grown in environmentally controlled chambers to determine the interactive effects of ozone (O₃) and increased root temperature (RT) on biomass, reproductive output, and photosynthesis. Plants were grown with or without an average treatment of 63 ppb O₃. RT treatments were 13°C (LRT) and 18°C (HRT). Air temperatures were 25°C/15°C day/night for all RT treatments.
Ozone affected plant biomass more than did root temperature. Plants in O₃ had significantly smaller total plant d. wt, shoot weight, leaf weight, leaf area and leaf number than plants grown without O₃. LRT plants tended to have slightly smaller total plant d. wt, shoot weight, root weight, leaf weight, leaf area, and leaf number than HRT plants. For all variables, LRT plants grown in O₃ had the smallest biomass, and plants grown in HRT without O₃ had the largest biomass.
Ozone reduced both fruit weight and fruit number; LRT also reduced fruit weight but had no effect on fruit number. Ozone reduced photosynthesis but RT had no effect. Conductance and internal CO₂ were unaffected by O₃ or RT.
These studies indicate that plant growth with LRT might be more reduced in the presence of O₃ than growth in plants with HRT, which might be able to compensate for O₃-caused reductions in photosynthesis to avoid decreased biomass and reproductive output.     

扫帚菜-白菜轮作对白菜镉吸收的影响

通过盆栽和大田试验研究扫帚菜Kochia scoparia对土壤中镉(Cd)的富集效率,并利用盆栽试验将其与4个白菜品种进行轮作试验,验证扫帚菜对土壤Cd污染的修复效果。结果表明,扫帚菜各部位富集能力表现为叶>根>茎,富集系数分别为15.07、5.44和2.96;种植扫帚菜后土壤总Cd降低6.02%-13.60%;土壤脲酶和酸性磷酸酶活性也有所提高。盆栽轮作结果表明,扫帚菜-白菜轮作系统中白菜地上部Cd含量与未轮作的对照组相比平均降低17.21%,生物量有略微增加,地上部对Cd的转运系数无明显变化。结果显示,通过扫帚菜与白菜轮作不仅可以增加白菜产量,而且可以有效降低白菜可食部Cd含量,实现边生产边治理的绿色农业理念。     

白菜型油菜黄子资源的初步遗传研究

对随机选取的国内外22份黄子白菜型油菜和22份褐子白菜型油菜进行了种皮色泽的显隐性关系鉴定、黄子性状的等位性测验以及遗传多样性分析。结果表明,黄子白菜型油菜与褐子白菜型油菜配组的杂交组合中,部分组合的F1种皮色泽呈现父本的种皮色泽,表现出花粉直感现象;自然界中存在多种白菜型油菜黄子类型,鉴定出的3种黄子白菜型油菜与褐色白菜型油菜的F2种皮色泽均为褐色,表明黄子性状对于褐子性状为隐性;分子标记方差分析结果显示,白菜型油菜的生长习性所解释的遗传变异大于种皮颜色所解释的遗传变异,表明国外不同生长习性的黄子白菜型油菜资源可用于国内黄子白菜型油菜遗传基础的拓宽。     

芜菁SSR引物的开发及多态性分析

目的:用于芜菁的简单序列重复(SSR)标记数量有限,初步探索多态性较好SSR的结构特性,开发更多的SSR引物,有助于芜菁的研究。方法:开发680对新的SSR引物,以2个芜菁品种为模板进行多态性扩增,筛选出多态性较好的引物。结果:565(83.1%)对引物在2种芜菁之间能够有效扩增,有多态性的引物有141对(20.7%);SSR的长度与其多态性水平之间没有直接的线性关系,但SSR基序碱基数与重复次数和多态性水平之间存在一定的联系,即3碱基为基序、7次重复的SSR多态性较好。结论:SSR的类型与引物的多态性之间有一定的联系。     

芜菁的诱导同源四倍体减数分裂分析

细胞学上芜菁 (Brassicarapa)为正常的二倍体 ,具有 1 1的联会和 1 - 1的染色体分离。它的花粉母细胞 (PMC’s )在小孢子发生过程中的减数分裂前期 (M1 )无“B染色体” ,也无任何次级缔合。用 0 0 2 %的秋水仙碱水溶液处理 6h对多倍体的诱导形成明显。诱导出的同源四倍体构型中无联会基因 ,而成 1或 2次交叉的二价体、三价体和四价体。从多倍体逆转的有趣现象中发现 ,4 6 7%的诱导多倍体后来恢复成二倍体。不平衡的染色体分离普遍存在于诱导出的同源四倍体中 ,这就是导致生存能力不同的异常配子体出现的主要原因。异常配子体的结合 ,在芸苔属 (Brassica)现存种的演化中起到了重要作用     

Fitness of hybrids between rapeseed (Brassica napus) and wild Brassica rapa in natural habitats

Fitness of hybrids between genetically modified (GM) crops and wild relatives influences the likelihood of ecological harm. We measured fitness components in spontaneous (non-GM) rapeseed x Brassica rapa hybrids in natural populations. The F1 hybrids yielded 46.9% seed output of B. rapa, were 16.9% as effective as males on B. rapa and exhibited increased self-pollination. Assuming 100% GM rapeseed cultivation, we conservatively predict < 7000 second-generation transgenic hybrids annually in the United Kingdom (i.e. approximately 20% of F1 hybrids). Conversely, whilst reduced hybrid fitness improves feasibility of bio-containment, stage projection matrices suggests broad scope for some transgenes to offset this effect by enhancing fitness.     

Agrobacterium tumefaciens-mediated transformation of greenhouse-grown Brassica rapa ssp. oleifera

Greenhouse-grown plants of turnip rape Brassica rapa ssp. oleifera (syn. B. campestris) cv. Valtti and Sisu were transformed by Agrobacterium tumefaciens infection. Of the three A. tumefaciens strains tested (C58C1, EHA105 and LBA4404), LBA4404 gave the best results. Segments excised from one to two upper internodes of an inflorescence-carrying stem served as explants for the Agrobacterium infection. Cultivation of the explants horizontally during the first 3 days of co-cultivation with A. tumefaciens following immediate selection of transformed tissue of the stem segments placed vertically basal side down were critical. Use of silver nitrate (5–10 mg/l) in the culture medium and Micropore (3 M) paper tape for sealing plates was also beneficial. Transgenic shoots were recovered using either hygromycin or kanamycin (20–25 mg/l) selection. Hygromycin was preferable, as the proportion of `escapes' was 90% under kanamycin and 10% under hygromycin selection. Regeneration was achieved by culturing the explants for 3–6 days on 0.5 mg/l of 2,4-di-chlorophenoxyacetic acid and 1–2 weeks on 2–3 mg/l of 6-benzyl aminopurine with/without 0.05 mg/l α-naphthaleneacetic acid. Recovered shoots were then cultured on hormone-free MS medium. This culture program gave 60–80% shoot regeneration. Regenerants were tested by histological β-glucuronidase staining and Southern blotting. The recovery rate of transgenic shoots was 4–9% of the number of explants used in the experiments. Received: 28 November 1997 / Revision received: 25 March 1998 / Accepted: 22 November 1998     

Within-population variation in hybridisation and transgene transfer between wild Brassica rapa and Brassica napus in the UK

Several studies in Europe and North America have shown that cultivated Brassica napus will readily hybridise with wild Brassica rapa but at widely different frequencies. To understand the implications of this phenomenon with regard to transgene flow, we examined the rate at which cultivated B. napus cv. Westar containing a capsid (coat protein, CP)‐coding sequence from Turnip mosaic virus (Potyvirus) hybridised under glasshouse conditions with wild B. rapa from Culham, in Oxfordshire, UK. We found that the hybridisation rate, as judged using simple sequence repeat (SSR)‐PCR and primer oligonucleotides specific for either the C or the A genomes in progeny from individual crosses varied from 5% to 100%. In hybrids (F₁ progeny), transgene transfer was always observed (inferred by SSR‐PCR) when hybrids were detected. Our observations revealed a hitherto unrecorded source of variability in transgene flow to wild UK B. rapa.     

我国部分白菜型油菜 RAPD 的研究

本研究用RAPD技术和统计学方法,对以湖南和湖北省油菜为主的34个白菜型油菜品种的遗传多样性进行了分析。根据离差平方和,用Ward's聚类方法进行聚类。结果表明：白菜型油菜的遗传变异与生态地理分布有密切的关系;湖南和湖北两省的白菜型油菜品种存在广泛的遗传变异;在DNA水平上可将所分析的34个品种分成8个类群。作者对研究结果在遗传育种中的应用进行了讨论。     

Rapid,nonparallel genomic evolution of Brassica rapa (field mustard) under experimental drought

While we know that climate change can potentially cause rapid phenotypic evolution, our understanding of the genetic basis and degree of genetic parallelism of rapid evolutionary responses to climate change is limited. In this study, we combined the resurrection approach with an evolve-and-resequence design to examine genome-wide evolutionary changes following drought. We exposed genetically similar replicate populations of the annual plant Brassica rapa derived from a field population in southern California to four generations of experimental drought or watered conditions in a greenhouse. Genome-wide sequencing of ancestral and descendant population pools identified hundreds of SNPs that showed evidence of rapidly evolving in response to drought. Several of these were in stress response genes, and two were identified in a prior study of drought response in this species. However, almost all genetic changes were unique among experimental populations, indicating that the evolutionary changes were largely nonparallel, despite the fact that genetically similar replicates of the same founder population had experienced controlled and consistent selection regimes. This nonparallelism of evolution at the genetic level is potentially because of polygenetic adaptation allowing for multiple different genetic routes to similar phenotypic outcomes. Our findings help to elucidate the relationship between rapid phenotypic and genomic evolution and shed light on the degree of parallelism and predictability of genomic evolution to environmental change.     

Phylogenetic Analysis of Brassica rapa MATH-Domain Proteins

The MATH (meprin and TRAF-C homology) domain is a fold of seven anti-parallel β-helices involved in protein-protein interaction. Here, we report the identification and characterization of 90 MATH-domain proteins from the Brassica rapa genome. By sequence analysis together with MATH-domain proteins from other species, the B. rapa MATH-domain proteins can be grouped into 6 classes. Class-I protein has one or several MATH domains without any other recognizable domain; Class-II protein contains a MATH domain together with a conserved BTB (Broad Complex, Tramtrack, and Bric-a-Brac ) domain; Class-III protein belongs to the MATH/Filament domain family; Class-IV protein contains a MATH domain frequently combined with some other domains; Class-V protein has a relative long sequence but contains only one MATH domain; Class-VI protein is characterized by the presence of Peptidase and UBQ (Ubiquitinylation) domains together with one MATH domain. As part of our study regarding seed development of B. rapa, six genes are screened by SSH (Suppression Subtractive Hybridization) and their expression levels are analyzed in combination with seed developmental stages, and expression patterns suggested that Bra001786, Bra03578 and Bra036572 may be seed development specific genes, while Bra001787, Bra020541 and Bra040904 may be involved in seed and flower organ development. This study provides the first characterization of the MATH domain proteins in B. rapa     

Wall-associated kinase BrWAK1 confers resistance to downy mildew in Brassica rapa

The plant cell wall is the first line of defence against physical damage and pathogen attack. Wall-associated kinase (WAK) has the ability to perceive the changes in the cell wall matrix and transform signals into the cytoplasm, being involved in plant development and the defence response. Downy mildew, caused by Hyaloperonospora brassicae, can result in a massive loss in Chinese cabbage (Brassica rapa L. ssp. pekinensis) production. Herein, we identified a candidate resistant WAK gene, BrWAK1, in a major resistant quantitative trait locus, using a double haploid population derived from resistant inbred line T12–19 and the susceptible line 91–112. The expression of BrWAK1 could be induced by salicylic acid and pathogen inoculation. Expression of BrWAK1 in 91–112 could significantly enhance resistance to the pathogen, while truncating BrWAK1 in T12–19 increased disease susceptibility. Variation in the extracellular galacturonan binding (GUB) domain of BrWAK1 was found to mainly confer resistance to downy mildew in T12–19. Moreover, BrWAK1 was proved to interact with BrBAK1 (brassinosteroid insensitive 1 associated kinase), resulting in the activation of the downstream mitogen-activated protein kinase (MAPK) cascade to trigger the defence response. BrWAK1 is the first identified and thoroughly characterized WAK gene conferring disease resistance in Chinese cabbage, and the plant biomass is not significantly influenced by BrWAK1, which will greatly accelerate Chinese cabbage breeding for downy mildew resistance.     

Expression of GFP and Bt transgenes in Brassica napus and hybridization with Brassica rapa

It is possible to monitor the movement of transgenes by tagging them with green fluorescent protein (GFP). In order to develop a model to study transgene flow, canola (Brassica napus cv Westar) was transformed with two GFP constructs, mGFP5er (GFP only) and pSAM 12 [GFP linked to a synthetic Bacillus thuringiensis (Bt) cry1Ac endotoxin gene]. Transformed callus sectors that fluoresced green were preferentially selected in the tissue culture process. Four independent GFP canola events and 12 events of GFP/Bt canola were regenerated through tissue culture. GFP fluorescence was macroscopically detectable throughout the entire life cycle of canola. The GFP/Bt events were insecticidal to neonate corn earworm (Helicoverpa zea) larvae and prevented herbivory damage. Fluorescence intensity at 508 nm varied between the independent transformation events, and ranged from 7.6×10⁵ to 13.8×10⁵ (counts per second) in contrast with the wild-type at 5.3×10⁵ cps. Nine GFP/Bt and three GFP events were hybridized with three wild accessions of B. rapa. The resultant hybrids fluoresced green and were insecticidal to neonate corn earworm larvae to the same degree as the transgenic canola parents. However, fluorescence intensities of the hemizygous F₁ hybrid lines were lower than the respective original homozygous canola parents. Each F₁ hybrid line was backcrossed by hand onto the B. rapa parent, and transgenic backcrosses were produced at rates ranging from 15% to 34%. These data suggest that GFP can be used as a tool to monitor transgene flow from crop species to wild relatives. Received: 11 September 2000 / Accepted: 1 February 2001     

白菜型油菜品种萌发期的抗旱性鉴定与筛选

秋旱影响我国长江流域油菜的播种和生长。从遗传基础广泛的白菜型油菜资源中筛选抗旱材料,对于培育抗旱油菜品种具有重要意义。以不同浓度的PEG-6000溶液于萌发期对5份不同遗传背景的白菜型油菜进行模拟干旱胁迫处理,并测定种子萌发抗旱指数、相对发芽率、相对发芽势、相对根长、相对芽长。与对照相比,干旱胁迫下各指标均有显著差异,对各指标进行主成分分析,确定了抗旱性鉴定参数,并确立了白菜型油菜资源抗旱性筛选的工作液为200 g/L的PEG-6000。选用该工作液于萌芽期对203份白菜型油菜资源进行了抗旱筛选,结果表明,模拟干旱胁迫下,大部分材料的抗旱性与对照有显著差异,用隶属函数法和聚类分析对抗旱性鉴定指标进行分析,并对所有供试材料的抗旱性进行了排序,鉴定出了抗旱性最强的PI226505白菜型油菜,其来源为Iran,为油菜下一步抗旱性遗传改良奠定了基础。     

津田芜菁MYB77蛋白的原核表达及纯化

目的:建立津田芜菁转录因子MYB77的原核表达系统,并在大肠杆菌中获得表达。方法:RT-PCR获得MYB77的编码序列,将其克隆至pGEM-T载体中,在上下游引物中分别引入HindⅢ和EcoRⅠ酶切位点,PCR获得带酶切位点的目的片段并将其连接到重组表达载体pGEX-KG中,转化大肠杆菌DE3工程菌株,IPTG诱导重组质粒pGEX-KG-MYB77在大肠杆菌DE3中表达带有GST标签的融合蛋白,超声裂解大肠杆菌,用MagneGST ProteinPurification System纯化目的蛋白,通过SDS-PAGE和Western印迹验证GST-MYB77融合蛋白的表达。结果:重组菌株可以表达GST-MYB77融合蛋白,用Western印迹鉴定纯化的融合蛋白,在相对分子质量为55.56×103处检测到目的条带。结论:利用大肠杆菌表达系统获得了较高纯度的GST-MYB77融合蛋白,为进一步研究津田芜菁MYB77蛋白的功能奠定了基础。     

Quantitative trait loci for glucosinolate accumulation in Brassica rapa leaves

Glucosinolates and their breakdown products have been recognized for their effects on plant defense, human health, flavor and taste of cruciferous vegetables. Despite this importance, little is known about the regulation of the biosynthesis and degradation in Brassica rapa. Here, the identification of quantitative trait loci (QTL) for glucosinolate accumulation in B. rapa leaves in two novel segregating double haploid (DH) populations is reported: DH38, derived from a cross between yellow sarson R500 and pak choi variety HK Naibaicai; and DH30, from a cross between yellow sarson R500 and Kairyou Hakata, a Japanese vegetable turnip variety. An integrated map of 1068 cM with 10 linkage groups, assigned to the international agreed nomenclature, is developed based on the two individual DH maps with the common parent using amplified fragment length polymorphism (AFLP) and single sequence repeat (SSR) markers. Eight different glucosinolate compounds were detected in parents and F(1)s of the DH populations and found to segregate quantitatively in the DH populations. QTL analysis identified 16 loci controlling aliphatic glucosinolate accumulation, three loci controlling total indolic glucosinolate concentration and three loci regulating aromatic glucosinolate concentrations. Both comparative genomic analyses based on Arabidopsis-Brassica rapa synteny and mapping of candidate orthologous genes in B. rapa allowed the selection of genes involved in the glucosinolate biosynthesis pathway that may account for the identified QTL.     

芜菁原生质体的分离及绿色荧光蛋白的瞬时表达

目的：分离芜菁叶片原生质体,建立蛋白质在芜菁原生质体的瞬时表达系统。方法：以津田芜菁成叶为试材,酶解分离原生质体;通过PEG介导的转化,将编码绿色荧光蛋白（GFP）的瞬时表达载体转入原生质体中,用激光扫描共聚焦显微镜检测原生质体中GFP的表达情况。结果：分离出大量的津田芜菁原生质体,并获得了较高的转化效率,GFP在整个原生质体中都有表达。结论：建立了津田芜菁原生质体瞬时表达系统。     

白菜和白菜型油菜角果相关性状遗传分析

以种荚差异显著的白菜自交不亲和系（P1）和白菜型油菜自交系（P2）为亲本及杂交获得的4个基本世代（P1、P2、F1、F2）为材料,应用植物数量性状主基因+多基因混合遗传模型对其种荚相关性状进行遗传分析。结果表明,芸薹种作物的种荚长度(SPL)性状及喙长（SBL）性状均受加性-显性-上位性多基因控制（C-0模型）,多基因遗传率分别为83.16%和68.67%;种荚宽度(SPW)性状受2对加性-显性-上位性主基因+加性-显性-上位性多基因控制（E-0模型）,其主基因遗传率为20.40%,多基因遗传率为78.34%。种荚相关各性状均以多基因遗传为主,种荚宽度性状受环境因素影响较小,为1.26%;种荚长度、喙长受环境因素影响较大,分别达16.89%和25.36%。针对芸薹种作物种荚性状的改良要以多基因为主,并注意环境条件影响。以种荚差异显著的白菜自交不亲和系（P1）和白菜型油菜自交系（P2）为亲本及杂交获得的4个基本世代（P1、P2、F1、F2）为材料,应用植物数量性状主基因+多基因混合遗传模型对其种荚相关性状进行遗传分析。结果表明,芸薹种作物的种荚长度(SPL)性状及喙长（SBL）性状均受加性-显性-上位性多基因控制（C-0模型）,多基因遗传率分别为83.16%和68.67%;种荚宽度(SPW)性状受2对加性-显性-上位性主基因+加性-显性-上位性多基因控制（E-0模型）,其主基因遗传率为20.40%,多基因遗传率为78.34%。种荚相关各性状均以多基因遗传为主,种荚宽度性状受环境因素影响较小,为1.26%;种荚长度、喙长受环境因素影响较大,分别达16.89%和25.36%。针对芸薹种作物种荚性状的改良要以多基因为主,并注意环境条件影响。