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。

Study on Agrobacterium tumefaciens-mediated Transformation of Brassica campestris L. with Fusion Gene Ycoil-bFGF

[ Objective] The study is to generate pharmaceutical protein via plant transgenic technique. [Methed] Using the cotyledons with petiole as transformation receptor, the fusion gene of rapeseed oil-body gene and bFGF was introduced into the rapeseed （ Brassica campestris L. ） by Agrobacterium tumefaciens-mediated transformation; meanwhile regeneration conditions of rapeseed were also optimized, and the regenerated resistant plantlets were detected by PCR and Southern blot. [ Result] This fusion gene had been integrated into rapeseed genome successfully, and the optimized conditions of transformation and regeneration were as follows： explants pre-culture for 2 d, co-culture for 3 d, bacteria solution OD600 for 0.3 and infection time for 5 min. [ Conclusion] The results laid a solid foundation for extraction, isolation and purification of protein in transgenic plant seeds.

Effect of Combined Use of Boron and Manganese Fertilizers on the Nutritional Quality and Physiological Indices of Brassica campestris L.ssp.chinensis L.var.utilis

[Objective] The purpose of this study is to determine the effects of com-bined use of boron and manganese fertilizers on the nutritional quality and physio-logical indices of Brassica campestris. [Method] In the nutrient solutions for growing B. campestris by hydroponics, boric acid and manganese sulfate were added at 0.5, 2.5, and 7.5 mg/L respectively. Another treatment without boron and manganese was prepared as the control. Quality and physiological indices of B. campestris in the 10 treatments were measured. [Result] Boron and manganese shared obvious in-teraction in improving the quality and physiological indices of B. campestris. To cul-tivate B. campestris with high quality and strong resistance, the optimum concentra-tions of boron and manganese in the nutrient solution should be 2.5 mg/L boric acid and 2.5-7.5 mg/L manganese sulfate. [Conclusion] The findings wil provide refer-ence for studying effects of trace elements on nutrient composition of vegetables.

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.

Analysis on Cloning and Expression of WRKY Gene Segment in Brassica campestris L.

[Objective]The aim was to explore the relative expression trends of WRKY transcription factor gene under ABA treatment in Brassica campestris.[Method]Actin gene and WRKY gene were cloned by using the homology cloning method.The sequences of nucleic acid and amino acid were analyzed using BLAST and DNAMAN software.Relative expression trends of WRKY gene were detected by applying real-time relative quantification PCR（RT-qPCR）under ABA（100 μmol/L）treatment.[Result]A 680 bp WRKY gene segment and a 933 bp β-actin gene segment were acquired in Brassica campestris.The result of RT-qPCR analysis revealed that BcWRKY expression could be induced by ABA and that the relative expression of WRKY gene reached the peak at 1 h with ABA treatment.[Conclusion]Actin and WRKY gene in B.campestris were cloned successfully,which was proved to play an important role in ABA signal pathway.

Differential Expression Analysis of Genie 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 F 2 population derived from an inter subspecific cross between Brassica campestris L. ssp. chinensis cv. 'aijiaohuang' and ssp. rapifera cv. 'qisihai'. 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.

Effects of Different Simple Cultivation Facilities on Yield and Quality of Over-summer Brassica campestris L. in South China

The effects of different simple cultivation facilities on yield and quality of Brassica campestris L. were compared to explore the optimal facility type for pro- duction of B. campestris, thereby providing a technical reference for efficient cultiva- tion of B. campestris during hot season. A total of four treatments were designed, open field cultivation （CK）, ordinary anti-insect net shed cultivation, ordinary plastic film greenhouse cultivation and Japanese translucent anti-insect net shed cultivation. The results showed that compared with those in open field cultivation, the plant height, leaf area, yield, Vc content, soluble sugar content and soluble protein con- tent of B. campestris in Japanese translucent anti-insect net shed cultivation were increased by 22%, 63%, 49%, 14%, 13% and 10%, respectively, and Japanese translucent anti-insect net shed was the optimal facility type for cultivation of B. campestris in hot season.

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.

Preliminary Study on Tissue Culture Technique of Brassica campestris L. ssp. chinensis (L.) Makino var. utilis

In order to establish a rapid propagation system for Brassica campestris L. ssp. chinensis var. utilis, the anther and cotyledon-cotyledonary petioles were used as explants to conduct tissue culture research. The results showed that not bloomed flower buds with higher styles than petals were appropriate for anther cul- ture. Moreover, most microspores were at the mid-late uninucleate stage at that time. The pollen germination rates of Brassica campestris L. ssp. chinensis var. u- tills were low and the germination rates in autumn and winter were higher than that in summer. The induction medium of anther callus of Brassica campestris L. ssp. chinensis var. utilis was MS＋1.0 mg/L KT＋1.0 mg/L 2, 4-D＋3% sugar＋6 g/L agar＋ 8% coconut milk and the induction media of adventitious bud were MS＋2.0 mg/L 6-BA＋ 0.5 mg/L NAA＋I.0 g/L activated carbon＋2% sugar＋6 g/L agar or MS＋2.0 mg/L ZT＋ 0.5 mg/L IAA＋0.5 g/L AgNO3＋1.0 g/L activated carbon＋2% sugar＋6 g/L agar. The induction percentage of adventitious bud by anther culture was 36.7%. Browning phenomenon appeared during the culture of adventitious bud and regeneration plant could not be formed. The plant regeneration rate reached 80% when cotyledon- cotyledonary petioles were used as explant.

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.

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。

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。