A Genetic Linkage Map of Brassica campestris L.ssp. pekinensis (syn. B. rapa L. ssp. pekinensis)

A molecular genetic map of Chinese cabbage was constructed with a 102 recombinant inbred (RI) population from a cross of two cultivated Chinese cabbage lines 177 and 276, using AFLP and RAPD markers. 352 markers including 265 AFLP markers and 87 RAPD markers were integrated into 17 linkage groups. It covered a total of 2 665. 7 cM with an average interval of 7. 6 cM. AFLP marker is efficient for map construction while it easily forms clusters to cause big gaps in map. A total of 13.92 % abnormal segregation markers distributed in the map. The molecular genetic map is fundamental for gene localization, comparative genomics, and QTL mapping of important agronomic traits.

镉胁迫对小白菜(Brassica campestris L.)抗氧化机理的影响

采用水培的方法,研究了不同Cd2+水平(0、1、2.5、5、10、15mg·L-1)对小白菜(Brassica campestris L.)抗氧化酶(SOD、POD、CAT)活性、非酶物质(SH、GSH、PCs)含量以及生长的影响。结果表明,小白菜叶片和根系SOD活性随Cd处理质量浓度的增加呈降低的变化趋势,POD、CAT活性以及MDA含量随Cd处理质量浓度的增加而增加。小白菜的生物量、根长、株高、叶绿素质量分数随Cd处理质量浓度的增加显著降低(P<0.05),表明Cd抑制了小白菜的生长,破坏了叶绿素的合成。小白菜地上、地下部镉质量分数均随Cd处理质量浓度的增加而显著增加(P<0.05)。当Cd处理质量浓度为15mg·L-1,小白菜地上、地下部镉质量分数分别达到637.5、1663.0mg·kg-1,表明小白菜对Cd有良好的富集效果。小白菜根系与叶片中SH、GSH和PCs含量均随Cd处理质量浓度增加而增加的变化趋势,表明SH、GSH和PCs在解毒小白菜Cd毒害中起着重要作用。

利用InDel标记鉴定典湖1号杂交种纯度

典湖1号是宁波微萌种业有限公司育成的青梗菜新品种,但种子纯度鉴定速度限制了该品种的推广。为实现对该品种杂交种纯度的高通量快速鉴定,在典湖1号父母本(父本EPG-194,母本EPG-168)基因组重测序的基础上开发用于纯度检测的InDel标记。试验以典湖1号及其父母本DNA样品为材料筛选到亲本带型互补、扩增条带清晰和分离速度快的引物D1-7,并用该引物检测田间典湖1号F_(1)群体(EpG-194×EpG-168群体400株,EpG-168×EpG-194群体400株)。试验中,引物D1-7的分子检测结果显示,EpG-194×EpG-168群体和EpG-168×EpG-194群体分别存在非F_(1)杂株146和32株,田间表型鉴定发现EpG-194×EpG-168群体和EpG-168×EpG-194群体分别存在非F_(1)杂株146和32株,对比后二者鉴定的800株单株结果完全一致。结果表明,InDel分子标记引物D1-7准确鉴定了典湖1号群体中混杂的母本自交种,为之后典湖1号杂交种纯度的快速鉴定奠定了基础。

Role of nitrification inhibitor DMPP(3,4-dimethylpyrazole phosphate) in NO^-_3-N accumulation in greengrocery( Brassica campestris L. ssp. chinensis ) and vegetable soil

The influence of nitrification inhibitor(NI) 3,4 dimethylpyrazole phosphate(DMPP) on nitrate accumulation in greengrocery( Brassica campestris L. ssp. chinensis ) and vegetable soil at surface layer were investigated in field experiments in 2002 and 2003 Results showed that NI DMPP took no significant effect on yields of edible parts of greengrocery, but it could significantly decrease NO - 3 N concentration in greengrocery and in vegetable soil at surface layer. In addition, NI DMPP could reduce the NO - 3 N concentration during the prophase stage of storage.

Effect of Nitrogen and Sulfur Supply on Glucosinolates in Brassica campestris ssp.chinensis

Glucosinolates （GSs） are a group of plant secondary metabolites containing abundant nitrogen （N） and sulfur （S） mainly in Brassica and have the beneficial effects on human health including anti-carcinogenic, cholesterol-reducing and other pharmacological effects. The objective of this study was to investigate the effect of different concentrations of N （5, 10, and 20 mmol L-a, denoted by N5, N10 and N20） and S （0,5, 1, and 2 mmol L^-1, denoted by S0.5, S1 and S2） on the yield and GSs in pakchoi （Brassica campestris L. ssp. chinensis var. communis） in hydroponics. Results showed that N10 and N20 significantly enhanced the yield compared with N5, however, N20 had a negative effect relative to N10. Only with N10 and N20 low S supply （S0.5） reduced the yield. The concentrations of aliphatic GSs, aromatic GS and total GSs were enhanced by N5 and indolyl GSs were enhanced by N20. S2 enhanced the concentration of individual GS and total GSs. The concentrations of indolyl GSs were maximized in N20S2 treatment, whereas the highest concentrations of aliphatic GSs, aromatic GS and total GSs were found in N5S2 treatment. Effects of N and S on aliphatic GSs were higher than on indolyl GSs. The results suggest that the accumulation of aliphatic GSs and aromatic GS could be enhanced by low N and high S and restricted by high N while that of indolyl GSs could be enhanced by high N and high S.

Differential Expression Analysis of Genic Male Sterility A／B Lines in Chinese Cabbage—Pak—Choi（Brassica campestris ssp.chinensis Makino）

To determine differential expression of genie male sterility A/B lines in Chinese cabbage-pak-choi (Brassica campestris ssp. chinensis Makino var. communis Tsen et Lee), we used the RNA fingerprinting technique, cDNA-AFLP analysis, in different developmental stages and different tissues. While no obvious differential expressions were observed in rosette leaves, florescence leaves, and scapes, some differential expressions were found in alabstrums of A/B lines and among leaves, scapes and alabstrums. We analyzed the al-abstrums collected in different developmental stages with 10 primer combinations. We got a unique band between middle size alabstrums and large alabstrums in B line in one of the ten pair primers, and in another one pair, one band reflecting a higher gene-expression level in A line than that in B line was obtained. No unique bands were found with the other primer combinations. The bands reflecting different gene-expression level were confirmed by Northern hybridization. The results indicated that cDNA-AFLP was a suitable tool for studying differential expression of genie male sterility in plants. SDS-polyacrylamide gel electrophoresis patterns of soluble proteins further verified the difference in A/B lines.

Genetic linkage map of Brassica campestris L.using AFLP and RAPD markers

A genetic linkage map comprised of 131 loci was constructed with an F2 population derived from an inter-subspecific cross between Brassica campestris L.ssp.chinensis cv.aijiaohang” and ssp.rapifera cv.,”'isihai”.The genetic map included 93 RAPD loci,36 AFLP loci and 2 morphological loci organized into 10 main linkage groups(LGs) and 2 small groups,covering 1810.9cM with average distance between adjacent markers being approximately 13.8cM.The map is suitable for identification of molecular markers linked to important agronomic traits.QTL analysis,and even for marker-assisted selection in breeding programs of Chinese cabbage and turnip.

Nitrogen Dioxide-Induced Responses in Brassica campestris Seedlings: The Role of Hydrogen Peroxide in the Modulation of Antioxidative Level and Induced Resistance

This article investigates the responses of Brassica campestris seedlings to an acute level of nitrogen dioxide （NO2） exposure in a plant growth chamber, and examines whether pretreating plants with hydrogen peroxide （H2O2） will alleviate NO2-caused injury. Twenty-eight-day-old B. campestris plants sprayed with 10 mmol L^-1 H2O2 aqueous solution （corresponding to approximate 1.0 mg H2O2 per single plant） were exposed to different concentrations of NO2 （0.25, 0.5, 1.0, and 2.0 μL L^-1, respectively） for 24 h under controlled environment. To measure the plant biomass, the plants were fumigated with the same NO2 concentrations as mentioned above for 7 h per day （8.00-15.00） for 7 days. As a control, charcoal filtered air alone was applied. Data were collected on plant biomass, total chlorophyll, photosynthetic rate, stomatal conductance, nitrate and nitrate reductase （NR）, antioxidative enzymes, ascorbate （ASA）, and malondialdehyde （MDA）, immediately after exposure. The results showed that exposure to a moderate dose of NO2 （e.g., 0.25 μL L^-1） had a favorable effect on plants, and the dry weight of the above-ground part increased, whereas the exposure to high NO2 concentrations （e.g., 0.5 μL L^-1 or higher） caused a reduction in the plant biomass and the total chlorophyll, when compared with the control. In addition, at 0.5 μL L^-1 or higher NO2 concentrations, prominent increases in the MDA level and superoxide dismutase （SOD） and NR activities were observed. Exposure to 1 μL L^-1 and higher NO2 resulted in necroses appearing on older leaves, and an increase in catalase （CAT） activity, decrease in ASA content, increased accumulation of NO3^-, and reduction in photosynthesis, when compared with the controls. No changes were detected in stomatal conductance under NO2 fumigation. The pretreatment with 10 mmol L^-1 H2O2 alleviated significantly NO2- caused biomass decrease and photosynthetic inhibition when compared with H2O2-untreated plants. Under NO2 fumigation, further induction in SOD and CAT activities occurred in H2O2 treated plants when compared with H2O2- untreated plants. The effect of NO2 on the ASA and MDA contents was also absent in H2O2-treated plants. However, the H2O2 treatment did not alter the nitrate content and NR activity in plants under NO2 fumigation. The H2O2 treatment caused a lower rate of stomatal conductance. Taken together, these data suggest that fumigation with an acute level of NO2 causes oxidative damage to B. campestris seedlings. The H2O2 pretreatment markedly protects plants against NO2 stress and this may be associated with inducible antioxidative level. NO2 fumigation contributes, at least in part, to the enhanced levels of nitrate in B. campestris leaves.

Effect of the Antisense BcMF12 Driven by the BcA9 Promoter on Gene Silencing in Brassica campestris L.ssp.chinensis

The study analyzed the silencing of BcMF12 gene regulated by BcA9 promoter in the transgenic pakchoi and confirmed the effect of antisense BcMF12 gene on the pollen development. A conserved BcMF12 gene fragment was amplified from the cDNA of flower buds in pakchoi （Brassica campestris L. ssp. chinensis, syn. B. rapa L. ssp. chinensis） and was fused to the anther specific BcA9 promoter. The plant antisense expression vector was constructed and then introduced into pakchoi via Agrobacterium-mediated transformation. The transgenic plants were screened by antibiotics and molecular analysis. PCR and Southern blot revealed that the antisense BcMF12-GUS fusion gene regulated by BcA9 promoter was integrated into transgenic plants. Northern blot suggested that the expression of BcMF12 gene was down-regulated significantly. The pollen germination rate of transgenic plants with antisense BcMF12 gene decreased as compared with that of the control plants. The expression of the gene BcMF12 related to the pollen development was inhibited by the antisense BcMF12 driven by BcA9 promoter, which consequently affected the pollen development in pakchoi.

Effects of Fermented Nori (<i>Pyropia yezoensis</i>) Liquid Fertilizer on Plant Growth Characteristics and Nutrient Content of Komatsuna (<i>Brassica rapa</i>L. var. Wakana Komatsuna) Cultivated in Vermiculite

We conducted plant growth experiments in microbe-free vermiculite to study the effects of four types of fermented seaweed liquid fertilizer (SLF) made from nori (Pyropia yezoensis) seaweed on the germination, plant growth characteristics, SPAD value, and nutrient content and uptake of komatsuna (Brassica rapa L. var. wakana komatsuna). The four types of fermented nori SLF used in this study were prepared by anaerobic fermentation of unwashed nori (SLF1), aerobic fermentation of unwashed nori (SLF2), anaerobic fermentation of washed nori (SLF3), and aerobic fermentation of washed nori (SLF4). Komatsuna seeds treated with 200-, 300-, and 400-fold dilutions of SLFs exhibited improved relative germination ratios (RGRs) at 3 and 4 days after sowing (DAS). At 4 DAS, the RGRs of seeds treated with 10-, 100-, 200-, 300-, and 400-fold SLF dilutions showed no differential effect. Seeds treated with undiluted SLFs did not germinate by 4 DAS. SLF1 may promote komatsuna seed germination. The nitrogen (N), calcium, magnesium, sodium (Na), and iodine (I) contents of plants treated with SLF1 were significantly increased relative to plants treated with the other SLFs. Moreover, the I and Na contents of plants were significantly increased by foliar spray application of different dilutions of SLF1. However, SLF treatment markedly reduced the shoot dry weight compared with 1/2-strength modified Hoagland nutrient (MHN) solution, although the same amounts of N and K were applied. SPAD values of the plants treated with SLFs were significantly higher than those of plants treated with MHN. Foliar treatment with SLFs had no significant effect on plant growth, SPAD value, or uptake of nutrients (except Na) relative to the control, but the I content was increased. Plants treated with SLF1 and SLF2 exhibited the highest Na uptake. Foliar spray treatments with SLF1 resulted in the highest I contents in plants. Based on our results, SLF1 is suitable for use as a liquid fertilizer to promote germination and increase nutrient content in komatsuna. These results need to be followed up in soil experiments in the presence of microbes in the rhizosphere.

Identifying Cytoplasmic Male Sterile Types of Chinese Cabbage(Brassica campestris L.)by Molecular Markers

Different sterile cytoplasm types of nine cabbage cytoplasmic male sterile materials were identified by molecular marker in the study, in order to better use molecular marker to conduct the assisted breeding in the future. Genomic DNA was isolated from Chinese cabbage by CTAB method. The design of two pairs of specific primers was performed on conserved flanking region of orf138 gene in the GenBank. PCR was performed with genomic DNA of the nine Chinese cabbage materials. The bands were sequenced. The homologous comparison was conducted in NCBI, and finally, the type of sterile cytoplasm was determined. The results showed that the bands were amplified only in four Chinese cabbage male sterile materials with two pairs of specific primers PUPIl and PIII/PIV, while the other five materials did not obtain the relative bands. The result was consistent with the field sterility identification. And then four molecular markers of Chinese cabbage Ogura cytoplasmic male sterility （CMS） were obtained. After conducting a homologous comparative analysis with BLAST in GenBank, it was found that the homologous degree was 100% in specific segments of tbe tbree sterility materials （L1-CI, L3-CI and L3- F1 ） and Ogu orf138 gene （GenBank accession No. ： HQ149728） of the reported broccoli Ogu CMS. The homologous degree of L1-F1 was 99% with a variation point. The type of cytoplasmic male sterility of the other five materials needed further research. Four materials of the nine were identified as the radish cytoplasmic male sterility materials and four molecular markers were obtained.

Genetic Variation and Correlation Analysis on Seed Oil Content and Protein Content of Brassica campestris L.Germplasm Resources

The content of oil and protein in 84 germplasm materials of Brassica campestris L. from all over the country was tested. In Guiyang environment,the oil content and protein content of these materials showed abundant variation,and were divided into three groups. Correlation analysis showed that there was a significant negative correlation between oil content and protein content of B. campestris L. seeds,and the application and formation mechanism of the negative correlation were also discussed.

Effects of Different Cadmium Levels on Active Oxygen Metabolism and H_2O_2-Scavenging System in Brassica campestris L.ssp.chinensis

The effects of different Cd (Cadmium) levels on generation of active oxygen speceies(AOS) and H2O2-scavenging system in the leaves of Brassica campestris L. ssp. chinensiswere studied. The results showed that generation rate, and H2O2 content were enhancedand malondialdehyde (MDA) content increased with the increase of Cd concentrations inthe growth medium. The activities of ascorbate peroxidase (APX), dehydroascorbatereductase (DR) and glutathione reductase (GR) were promoted by the addition of Cd.Exposed to Cd also increased the contents of ascorbate (AsA) and glutathione (GSH) in theleaves.

乌菜BcVIL2基因克隆及春化响应表达分析

【目的】从乌菜(Brassica campestris L.)中克隆VIN3⁃LIKE 2(VIL2)基因并进行生物信息学与表达分析,为进一步研究BcVIL2基因功能奠定基础。【方法】以‘徽乌19’为试验材料,利用同源克隆等技术获得BcVIL2基因,对其序列进行蛋白分子量(MW)、糖基化位点、二级结构等生物信息学分析;利用荧光定量PCR等技术分析其应答春化表达模式。【结果】BcVIL2基因ORF(Open reading fragment)全长为1296 bp,共编码431个氨基酸,属于亲水性蛋白,预测含有1个潜在的糖基化位点和59个潜在的磷酸化位点,蛋白分子量为47.91 kDa,理论等电点(pI)为8.27;其二级结构主要由α⁃螺旋与无规则卷曲组成;亚细胞定位预测其可能在细胞核中发挥作用;同源序列比对分析显示该基因与芸薹属植物VIL2基因同源性较高;亲缘进化树分析也显示该基因与白菜(B.rapa,syn.B.campestris)和芥菜(B.juncea)VIL2基因亲缘关系很近;荧光定量PCR结果表明,盛花期时BcVIL2基因在根中表达量最低,在荚中表达量最高,而叶中表达量显著低于茎和花中表达量;一月苗龄植株春化后BcVIL2基因的表达量显著低于未春化时的表达量。【结论】乌菜VIL2基因响应春化负表达,与春化反应相关基因BraA01g032910.3C、BraA05g026410.3C与BraA10g016160.3C密切相关,可能存在互作关系,本研究结果为深入分析BcVIL2基因的功能提供了良好基础。

国五国六柴油车尾气颗粒物对小白菜(Brassica campestris L.)的生态毒性

为研究柴油车尾气颗粒物对植物的毒性效应,本研究以执行国五、国六两种排放标准的柴油车为研究对象,测定其尾气颗粒物中重金属含量,并以小白菜(Brassica campestris L.)为受试植物,分析了两种颗粒物对小白菜的毒性效应.结果表明:(1)国五、国六柴油车尾气颗粒物中13种重金属总含量分别为1.04 g·kg^(-1)和0.47 g·kg^(-1),国五柴油车尾气颗粒物重金属含量高于国六柴油车;(2)在两种颗粒物的高浓度胁迫下,小白菜体内叶绿素以及蛋白质的含量均出现显著降低趋势;(3)随着两种颗粒物浓度的升高,植物体内部分氧化应激的酶指标超氧化物歧化酶(SOD)、过氧化氢酶(CAT)呈先上升后下降的趋势,而过氧化物酶(POD)以及丙二醛(MDA)、谷胱甘肽(GSH)呈上升趋势,颗粒物的污染能对小白菜产生较为显著的氧化胁迫效应.(4)21 d的毒性实验后,国五、国六柴油车尾气颗粒物对小白菜鲜重的半数有效浓度(EC50)值分别为76.27 mg·L^(-1)和104.48 mg·L^(-1).综合来看,国六标准的实施有效控制了颗粒物中重金属的含量,并一定程度地降低了其对小白菜的毒性.

Selenium-oxidizing Agrobacterium sp.T3F4 decreases arsenic uptake by Brassica rapa L.under a native polluted soil

Toxic arsenic(As)and trace element selenium(Se)are transformed by microorganisms but their complex interactions in soil-plant systems have not been fully understood.An Asand Se-oxidizing bacterium,Agrobacterium sp.T3F4,was applied to a native seleniferous As-polluted soil to investigate As/Se uptake by the vegetable Brassica rapa L.and As-Se interaction as mediated by strain T3F4.The Se content in the aboveground plants was significantly enhanced by 34.1%,but the As content was significantly decreased by 20.5% in the T3F4-inoculated pot culture compared to the control(P<0.05).Similar result was shown in treatment with additional 5 mg/kg of Se(IV)in soil.In addition,the As contents in roots were significantly decreased by more than 35% under T3F4 or Se(IV)treatments(P<0.05).Analysis of As-Se-bacterium interaction in a soil simulation experiment showed that the bioavailability of Se significantly increased and As was immobilized with the addition of the T3F4strain(P<0.05).Furthermore,an As/Se co-exposure hydroponic experiment demonstrated that As uptake and accumulation in plants was reduced by increasing Se(IV)concentrations.The 50% growth inhibition concentration(IC50)values for As in plants were increased about one-fold and two-fold under co-exposure with 5 and 10μmol/L Se(IV),respectively.In conclusion,strain T3F4 improves Se uptake but decreases As uptake by plants via oxidation of As and Se,resulting in decrease of soil As bioavailability and As/Se competitive absorption by plants.This provides a potential bioremediation strategy for Se biofortification and As immobilization in As-polluted soil.

白菜抽薹性状相关基因的cDNA-AFLP分析

先期抽薹严重制约着我国春大白菜及高原或高海拔地区的春夏白菜栽培,给生产造成重大损失,因而研究白菜作物低温春化途径抽薹和开花的相关基因对白菜育种具有重要意义。文章选取极早抽薹株系DH-54和极晚抽薹株系DH-43为材料,利用256对引物组合进行cDNA-AFLP分析,筛选到与抽薹开花相关的差异表达的转录衍生片段(Transcript-derived fragment,TDF)191条,对其中82个TDF进行克隆测序。结果表明:52个TDF与NCBI或TIGR已有序列同源,功能涉及代谢、胁迫及逆境应答、信号转导、转录调控等等;22个TDF在NCBI或TIGR找到同源序列,但功能未知;有8个TDF在NCBI或TIGR找不到同源序列,可能是一些新基因。

利用cDNA-AFLP技术分析白菜核雄性不育两用系的表达差异

利用cDNA AFLP技术 ,对白菜核雄性不育两用系可育株群和不育株群分析显示 :花蕾之间基因表达存在差异 ;莲座期叶片之间、开花期叶片之间和花茎之间基因表达无差异 ;叶片、花茎和花蕾之间基因表达存在差异。对开花期的小蕾、中蕾和大蕾分析显示 :10对引物中 ,1对在可育株中蕾和大蕾中扩增出 1条特异带 ,另 1对扩增出 1条在可育株中蕾中表达丰度高于不育株中蕾的条带 ,其余 8对未发现差异条带。对上述表达丰度有差异的基因进行Northern验证 ,结果与之一致 ,表明cDNA AFLP技术可以用于植物雄性不育基因表达差异的研究。

白菜核雄性不育两用系AFLP扩增特异片段的克隆及序列分析

利用AFLP技术从白菜核不育两用系"矮脚黄"可育系DNA中得到一个特异扩增片段MF-14.Southern杂交结果证实不育系也存在该片段的同源序列;回收该特异扩增片段并将其克隆到pGEM-TEasy载体进行序列测定.测序结果表明,该片段全长289bp,其碱基组成为A+T=46.02%.与白菜RAPD特异片段S107及其他育性相关基因核苷酸序列比较,同源性均低于50%.表明该片段为一新的序列.

NaCl和Na2SO4胁迫对白菜型冬油菜种子萌发的影响及其耐盐性分析

以6个耐盐性不同的白菜型冬油菜种子为材料,将NaCl和Na_2SO_4按摩尔比1:1混合,设浓度为0(CK)、45、90、135、180、240 mmol·L^(-1)6个处理,测定其对种子萌发和生长参数的影响,研究不同冬油菜种子萌发时的耐盐能力,以期为耐盐冬油菜的筛选提供依据。结果表明:(1)45 mmol·L^(-1)和90 mmol·L^(-1)盐浓度对种子萌发的影响不明显;135 mmol·L^(-1)和180 mmol·L^(-1)盐浓度明显抑制了种子萌发,表现为种子发芽势、发芽率、发芽指数、活力指数、胚根长、胚芽长、鲜重和干重均随盐浓度的增大而明显降低。因此,135~180 mmol·L^(-1)盐浓度可认为是耐盐性鉴定的适宜范围。(2)不同指标对盐胁迫的敏感程度差异较大,发芽指数、活力指数、胚芽长和鲜重与盐浓度均为极显著负相关,对盐胁迫最为敏感。(3)3个耐盐冬油菜ky-1、ky-10-191和ky-NDJ的种子发芽指数、活力指数、胚芽长和鲜重的耐盐临界值显著高于原有品系平油1号、10-191和ky-NDJ。ky-1、ky-10-191、ky-NDJ的耐盐临界值分别为133.895、123.264、107.399 mmol·L^(-1),而平油1号、10-191、NDJ的耐盐临界值分别为117.394、103.947、93.834 mmol·L^(-1)。用隶属函数法综合评价,耐盐性强弱顺序依次为ky-1>ky-10-191>平油1号>kyNDJ>10-191>NDJ。可见,ky-1、ky-10-191和ky-NDJ的耐盐性较平油1号、10-191和NDJ均有所提高。