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.

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.

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.

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.

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 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.

不结球白菜(Brassi cacampestris ssp.chinensis Makino)种质资源SRAP遗传分化分析

利用SRAP分子标记分析了国内外64份不结球白菜种质资源的DNA遗传多样性和遗传分化。21对引物组合共检测出215个位点,其中112个为多态性位点,多态性比率达52.09%,平均每对引物组合产生10.24个位点和5.33个多态性位点。不结球白菜各类型中普通白菜的Nei’s基因多样性指数(0.1410)和遗传丰富度[(190)88.37%]最高;各生态区域中江淮流域不结球白菜的Nei’s基因多样性指数(0.1451)和遗传丰富度[(185)86.05%]最高;国内的Nei’s基因多样性指数(0.1293)和遗传丰富度[(188)87.44%]分别高于国外。遗传变异估算表明,不结球白菜遗传分化系数58.22%,大部分变异存在于种群间;基因流为0.4031,说明群体间基因流动较少,遗传分化程度较高。以遗传相似系数0.872为截值,可把不结球球白菜分为Ⅵ个类群。

Research Progress of Heat-tolerance of Brassica campestris ssp. Chinensis

Brassica campestris ssp. chinensis, also known as non-heading Chinese cabbage, is an important vegetable widely distributed in southern China. High temperature is the most common adversity factor in vegetable production, because Brassica campestris ssp. chinensis is a thermophilic vegetable, which can＇t well grow at high temperature. In summer and autumn, high temperature stress would prevent Brassica campestris ssp. chinensis from growing and result in poor quality of its seedlings, seriously influencing yield and quality of the vegetable in later period. In this paper, present situation and latest advances in heat resistance research of Brassica campestris ssp. chinensis in recent years were reviewed at home and abroad. The research tendency in Bassica campestris ssp. chinensis was also described, providing reference for breeding of heat-resistant Brassica campestris ssp. chinensis.

Genetic Inheritance and Molecular Marker of Clubroot Resistance Genes in Brassica campestris ssp. chinensis

Objective] This study was conducted to investigate the genetic inheritance of clubroot resistance in Chinese non-heading cabbage （Brassica campestris ssp. chinensis）. [Method] The clubroot resistance gene was introduced from a Brassica campestris ssp. pekinensis cultivar to non-heading Chinese cabbage, and the inheri-tance and molecular markers of clubroot resistance gene in parental lines, F1, F2 and BC1 of non-heading Chinese cabbage were studied through pathogen inoculation at seedling stage and ISSR-PCR. [Result] Clubroot resistance in non-heading Chi-nese cabbage was control ed by a single dominant gene. ISSR molecular markers with Bulk segregant analysis （BSA） found that primer-873 was linked to resistance gene, named CR-873, and the genetic distance between the marker and the resis-tance gene was 9.72 cM. [Conclusion] The results provide references for the molecular marker assisted breeding of non-heading Chinese cabbage.

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.

Measurement of Some Physiological and Biochemical Indexes of the Antisense Fragment of CYP86MF Gene Transgenic Male Sterile Plantlets in Chinese Cabbage-Pak-Choi

In order to study the mechanism of the transgenic male sterility induced by the antisense fragment of gene CYP86MF, some physiological and biochemical indexes were compared between the transgenic plantlets of Chinese cabbage-pak-choi(Brassica campestris L. ssp. chinensis Makino)and their controls. Results showed that there was significant difference between content of the endogenous hormones in leaves and floral buds. GA3 contents in leaves of the transgenic plantlets of B. campestris L. ssp. chinensis Makino cv. Shanghai-qing and cv. Youqing were reduced by 31. 72 and 46. 88% respectively as compared with those of their controls, and GA3 contents in floral buds of them were reduced by 62. 92 and 80. 57 % respectively. In addition, ZT contents in leaves of cv. Shanghai-qing and cv. Youqing were reduced by 11. 81 and 181. 20% respectively as compared with those of their controls and those in floral buds were reduced by 105. 94 and 128.75 % respectively. But, ABA content in transgenic plantlets was significantly higher than that in non-transgenic plantlets. However, differences among respiratory rate and cyanide-resistant respiratory rate, the photosynthesic rate and the content of total flavones in floral buds were not significant. In this study, the results indicated that the gene CYP86MF might be one of the key functional genes to control fertility of Chinese cabbage.

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.

Factors Affecting Transformation Efficiency by Micro-Injecting Agrobacterium into Flower Bud of Chinese Cabbage

Three main parameters were selected to study their importance in transformation by budmicroinjection in non-head Chinese cabbage [Brassica campestris ssp. chinensis (L.) Makinovar. communis Tsen et Lee]. The results showed that the developmental stage of floral bud, theconcentrations of sucrose and surfactant Silwet L-77 were critical for the successfultransformation by this new method. The suitable bud size is 2-3 mm in length, the favorableconcentration of sucrose and surfactant Silwet L-77 are 8 and 0.02% respectively. When thesucrose concentration was greater than 10% or that of Silwet L-77 was above 0.1%, the treatedbuds became yellow and finally blighted. 4/6 T1 seedlings resistant to kanamycin were positiveby PCR analysis, and T2 progeny of all these positive T1 plants have one or more hybridizingbands by Southern blot. Under 5% sucrose, 0.02% Silwet L-77 and grade 2 bud (2-3 mm in itslength) parameters, the most favorable transformation efficiency is about 0.56%, and meanefficiency reaches 0.16% in all experiments indicating that bud microinjection is potentialtransformation way in non-head Chinese cabbage.

Chinese Cabbage-pak-choi Transcriptome Map Construction with cDNA-AFLP Techniques

Chinese cabbage-pak-choi （Brassica campestris L. ssp. chinensis Makino） transcriptome map with cDNA-AFLP techniques was constructed. The inbred line Aijiaohuang 97-3-2, the inbred line Baimanjing 001-24 of turnip [B. campestris ssp. rapifera （Matzg.） Sinsk] and 183 F6 （recombinant inbred population） plants were used as experimental materials, cDNAs were synthesized from total RNA extracted from young leaves at rosette stage. 256 pairs of cDNA-AFLP primers were used to detect the polymorphisms between parents Aijiaohuang 97-3-2 and Baimanjing 001-24. 56 pairs of cDNA-AFLP primers with high polymorphisms were screened from 256 pairs of primer by DNA-AFLP techniques. The genetic diversity of parents and 183 F6 progenies was detected by 56 pairs of cDNA-AFLP primers. The segregation and distribution of cDNA-AFLPs molecular marker were analyzed to construct transcriptome map amongst parents and F6 plants. A total of 164 cDNA-AFLPs marker loci were mapped into 13 linkage groups which covered 1401.2 cM with an average distance of 9.7 cM. It was the first transcriptome map of Chinese cabbage using cDNA-AFLP technique.

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.

不结球白菜BcCHC1蛋白参与调控TuMV侵染的初步研究

【目的】为增强不结球白菜对芜菁花叶病素养(TuMV)的抗性,研究探讨了不结球白菜中BcCHC1蛋白与TuMV之间的相互作用机制。【方法】首先从不结球白菜中鉴定出重链网格蛋白(CHC)基因家族的成员BcCHCs,随后克隆了1个代表性的CHC1基因,并对其进行了亚细胞定位分析。通过双分子荧光互补实验(BiFC)筛选出与BcCHC1蛋白相互作用的TuMV蛋白,并利用病毒诱导的基因沉默技术(VIGS)来沉默BcCHC1基因,以评估其功能。【结果】(1)成功克隆了不结球白菜的BcCHC1基因,该基因的编码序列长5124碱基对,编码1708个氨基酸。(2)在TuMV感染不结球白菜30 d后,实时荧光定量PCR分析显示,感染TuMV的不结球白菜中BcCHC1基因的表达量显著降低。(3)亚细胞定位研究表明,BcCHC1蛋白主要定位于烟草表皮细胞的细胞膜和细胞核。(4)BiFC实验进一步证实,BcCHC1蛋白能够与TuMV的CI和6K2蛋白发生相互作用,其中与CI的互作主要发生在细胞核内,而与6K2的互作则主要位于细胞膜。(5)通过对BcCHC1基因沉默株系的观察发现,沉默BcCHC1基因会导致植株死亡。【结论】BcCHC1蛋白通过与TuMV的CI和6K2蛋白相互作用,影响网格蛋白依赖的内吞作用途径及病毒复制,从而调控TuMV对不结球白菜的侵染。然而,BcCHC1基因调控TuMV侵染不结球白菜的具体机制仍需进一步研究。

不结球白菜细胞质雄性不育材料的筛选及其保持系选育

用多种不同基因型的可育不结球白菜(Brassica campestris ssp. chinensis Makino)材料对2个不结球白菜雄性不育株进行授粉,根据F1代群体的育性表现,筛选出细胞质雄性不育株;对16个自交品系的纯度进行调查,各品系筛选出5个或10个典型植株对其株高、开展度、叶长、叶宽、叶柄长、色泽等性状进行测试和比较分析。结果表明,16个品系的纯度均不低于50%;1-5、3-31、2-16、2-61、1-79、华冠4的开展度、株高均较大;聚类结果显示,3-31、1-9、2-61、2-16和华冠4聚在第三类,品系间性状差异较小。1-5、1-79、3-31、2-61、1-26、2-11、2-25和1-98各性状的变异系数均低于30%,一致性好;16个自交品系叶色空间分布较叶柄色集中,说明品系间叶色差异较小;3-31、1-79、1-34-17、2-11、2-16、1-5和华冠4的叶色亮丽。综合分析,1-5、1-79、3-31和2-11色泽好,变异系数小,且纯度高,是选育保持系的优良品系;而华冠4和墩子黄既可以作保持系又可以作父本。3-31和华冠4的植株高大,分别适宜作为保持系和父本,用于选配高产的不结球白菜杂交组合。

Cloning and Characterization of the Microspore Development-Related Gene BcMF2 in Chinese Cabbage Pak-Choi （Brassica campestris L. ssp. chinensis Makino）

: For the sake of providing some important information relevant to the study of the molecular mechanism of genic male sterility in plants, gene differential expression in flower buds at different developmental stages, as well as in rosette leaves, florescence leaves, and scapes was analyzed using cDNA amplified fragment length polymorphism (cDNA-AFLP) in the genic male sterile A and fertile B line of Chinese cabbage pak-choi. Following amplification of 125 pairs of primer combinations, 11 differential fragments were obtained, of which eight were from the B line and the other three were from the A line. Of 11 differential fragments, four were verified by Northern hybridization that were expressed preferentially in fertile flower buds. Results of GenBank BLAST showed that one fragment was with unknown function, whereas the other fragments have strong nucleotide sequence similarities with the polygalacturonase (PG) gene, the pectinesterase (PE) gene, and the polygalacturonase inhibitory protein (PGIP4) gene. Only full-length cDNA from the differential fragment BcMF-A18T16-1 was amplified by rapid amplification of cDNA ends (RACE) and Northern analysis showed that this fragment was expressed only in medium and large-sized flower buds of the B line. The full-length cDNA, designated as BcMF2 (Brassica campestris Male Fertile 2), was 1 485 bp long and was composed of a 1 263-bp open reading frame, which had 83% nucleotide similarity to a PG gene from Arabidopsis encoding polygalacturonase. Analysis of the basic structure of the protein revealed that it had one polygalacturonase active site (RVTCGPGHGLSVGS) at 256th site of amino acids and was classified as being a member of family 28 of the glycosyl hydrolases. The role of the BcMF2 gene on microspore development is discussed in the present paper.

镉对小白菜光合作用特性影响的研究

研究表明,微量镉(Cd)(0.01、0.1 mg/L)促进小白菜(Brassica campestris ssp.chinensis)生长,对苏州青的促进作用更明显;高浓度Cd显著抑制地上部生长,对根系生长影响相对较小.Cd降低小白菜叶绿素含量,特别是苏州青降低幅度较大.低浓度Cd对小白菜净光合速率Pn影响不大,高浓度Cd可能是由于非气孔限制而引起Pn降低.Cd降低气孔导度,但高浓度Cd增加胞间CO2浓度.叶绿素荧光测定表明,Cd对小白菜叶片光化学效率Fv/Fm影响不大,而高浓度Cd明显降低光合电子传递量子效率PSⅡ、光化学淬灭系数qP和电子传递速率ETR.Cd增加沪青一号非光化学淬灭系数qNP,而苏州青在高Cd浓度下qNP下降.