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.

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。

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.

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.

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.

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.

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.

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 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.

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.

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.

The influence of Lactobacillus plantarum fermentation in selenium-enriched Brassica napus L.:changes in the nutritional constituents,bioactivities and bioaccessibility

Selenium(Se)-enriched Brassica napus L.is a valuable organic Se supplement.In this study,the fermentation broth enriched with organic Se(FFS)was prepared using Lactobacillus plantarum to ferment the substrate of Se-enriched Brassica napus L.Significant increases were observed after fermentation in total sugars,reducing sugars,soluble proteins,total phenolic content(TPC),and total flavonoid content(TFC).The organic Se was retained at a concentration of 54.75 mg/g in the freeze-dried sample.Principal component analysis and cluster analysis showed good separation between the FFS and unfermented(FS)groups.Fragrant 2-ethyloxetane had the highest content among all volatiles,while sinapine had the highest content among all phenolic compounds.The fermentation process showed remarkable improvement in the abundance and concentration of volatile compounds and phenolic contents,making FFS exhibit strong antioxidant activity and inhibitory capacity againstα-glucosidase activity.The bioaccessibility of phenolic compounds was significantly greater in FFS compared to FS.ADMET analysis revealed that the majority of phenolic compounds contained in FFS did not exhibit mutagenicity toxicity,hepatotoxicity,skin sensitization,or blood-brain barrier penetration,indicating a favorable level of biosafety.Overall,our study provides a new insight into the further utilization of Se-enriched Brassica napus L.in foods.

Effect of Foliar Fertilizer Spraying at Seedling Stage on the Growth Physiological Characteristics and Yield of Brassica napus L.

In order to select a suitable foliar fertilizer for Brassica napus L.at the seedling stage,using‘Fengyou 958’as the material,different foliar fertilizers including BR,Se,Si,BR+Si,BR+Se,Se+Si and BR+Se+Si were sprayed at the seedling stage to study their effect on the physiological characteristics,growth and yield of Brassica napus L..The results showed that the growth,chlorophyll content,soluble sugar content,soluble protein content,and yield of different treatments at the budding stage improved compared with the control.The effect of the Si+Se treatment was the best,followed by the Se and Si treatments.The chlorophyll content of the flower decreased continuously during the whole flowering period,and the chlorophyll content of the mature silique peel was higher than that of the seed.The content of soluble sugar in flowers was the highest in the early flowering stage,and the content of soluble sugar in leaves at the flowering stage was higher than that at the budding stage.The soluble sugar content in the harvested silique peel decreased gradually with the increase of time,and that in the silique peel and seed was similar at 35 d after pollination.The soluble protein content in the silique peel and seed decreased gradually at the mature stage,and the soluble protein content in the silique peel was higher than that in the seed at the same stage.Si+Se foliar fertilizer spraying at the seedling stage can promote the growth and yield of Brassica napus L.and can be applied in Brassica napus L.production.

Development of a KASP Marker on Chromosome A05 for Seed Oil Content of Brassica napus

In order to identify the molecular markers that can be widely used in the breeding of Brassica napus L.varieties with high seed oil content under different genetic backgrounds,we developed a Kompetitive Allele Specific PCR(KASP)marker for seed oil content on the basis of the results from available studies.The verification in the F2 population showed that the marker was closely linked to the quantitative trait locus(QTL)for oil content on chromosome A05.The findings helped to breed the‘Fengyou’varieties with high seed oil content in the middle reaches of the Yangtze River.

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.

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.

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.