Fast,simple,efficient Agrobacterium rhizogenes-mediated transformation system to non-heading Chinese cabbage with transgenic roots

Non-heading Chinese cabbage, a variety of Brassica campestris, is an important vegetable crop in the Yangtze River Basin of China. However,the immaturity of its stable transformation system and its low transformation efficiency limit gene function research on non-heading Chinese cabbage. Agrobacterium rhizogenes-mediated(ARM) transgenic technology is a rapid and effective transformation method that has not yet been established for non-heading Chinese cabbage plants. Here, we optimized conventional ARM approaches(one-step and two-step transformation methods) suitable for living non-heading Chinese cabbage plants in nonsterile environments. Transgenic roots in composite non-heading Chinese cabbage plants were identified using phenotypic detection, fluorescence observation, and PCR analysis. The transformation efficiency of a two-step method on four five-day-old non-heading Chinese cabbage seedlings(Suzhouqing, Huangmeigui, Wuyueman, and Sijiu Caixin) was 43.33%-51.09%, whereas using the stout hypocotyl resulted in a transformation efficiency of 54.88% for the 30-day-old Sijiu Caixin.The one-step method outperformed the two-step method;the transformation efficiency of different varieties was above 60%, and both methods can be used to obtain transgenic roots for functional studies within one month. Finally, optimized ARM transformation methods can easily,quickly, and effectively produce composite non-heading Chinese cabbage plants with transgenic roots, providing a reliable foundation for gene function research and non-heading Chinese cabbage genetic improvement breeding.

Effect of Partial Replacement of Nitrate by Amino Acid and Urea on Nitrate Content of Non-heading Chinese Cabbage and Lettuce in Hydroponic Condition

In this paper, the authors studied the effect of different mixtures of glycine (Gly), isoleucine (Iso), proline (Pro), and urea solutions used as a partial (20%) replacment of nitrate in the nitrate content and quality of non-heading Chinese cabbage and lettuce in hydroponice. Five treatments were done 12 d before harvest. Compared to the control group, Gly had the best effect in reducing the nitrate content of both vegetable leaves and petioles; the mixture of Gly, Iso and Pro ranked second and urea the least. Treatments with amino acid could also increase soluble sugar and protein contents and enhance total-N in leaves significantly. In contrast, amino acid enhanced NRA in non-heading Chinese cabbage, while they decreased it slightly in lettuce. The results showed that amino acids and urea could reduce the nitrate content of both vegetables, but they had almost the same effect on non-heading Chinese cabbage. Moreover, amino acids were more effective than urea in lettuce. As a result, it was concluded that partial replacement of nitrate with amino acids not only reduced the nitrate content but also improved the quality of vegetables.

Molecular Cloning,Expression Analysis and Localization of Exo70A1 Related to Self Incompatibility in Non-Heading Chinese Cabbage(Brassica campestris ssp.chinensis)

The exocyst is a conserved protein complex,and required for vesicles tethering,fusion and polarized exocytosis.Exo70A1,the exocyst subunit,is essential for assembly of the exocyst complex.To better understand potential roles of Exo70A1 in non-heading Chinese cabbage（Brassica campestris ssp.chinensis）,we obtained the full-length cDNA of Exo70A1 gene,which consisted of 1 917 bp and encoded a protein of 638 amino acids.BlastX showed BcExo70A1 shared 94.9% identity with Brassica oleracea var.acephala（AEI26267.1）,and clustered into a same group with other homologues in B.oleracea var.acephala and Brassica napus.Subcellular localization analysis showed BcExo70A1 was localized to punctate structures in cytosol of onion epithelial cells.Results showed that BcExo70A1 was widely presented in stamens,young stems,petals,unpollinated pistils,roots and leaves of self compatible and incompatible plants.The transcripts of BcExo70A1 in non- heading Chinese cabbage declined during initial 1.5 h after incompatible pollination,while an opposite trend was presented after compatible pollination.Our study reveals that BcExo70A1 could play essential roles in plant growth and development,and is related to the rejection of self pollen in non-heading Chinese cabbage.

Using a Suppression Subtractive Library-Based Approach to Identify Non-Heading Chinese Cabbage Genes Up-Regulated in Early Response to Elicitor PB90

Monitoring expression at the transcriptional level is the first essential step for the functional analysis of plant genes. Genes-encoding proteins directly involved in early response to elicitor constitute only a small fraction of all the genes affected by elicitor. Transcriptional responses to various elicitors have been extensively studied in different plants including Nicotiana and Arabidopsis thaliana; however, corresponding data aren＇t available for non-heading Chinese cabbage. To address this problem, we describe a suppression subtractive library-based approach to isolate the plant＇s ESTs up-regulated in the early induction/execution of the HR induced by elicitor PB90 from Phytophthora boehmeriae. According to their putative identification in BLAST searches against the three genome databases, 70 up-regulated genes were classified into 9 parts： some aspect of primary ＇metabolism＇ or ＇energy＇ production; ＇protein synthesis＇ or ＇protein fate＇; cellular communication/signal transduction mechanism; cell fates including Beclin, SPT1, and SPT2; HLA-B and AGO1 which participate in transcription; cellular transport and hypothetical proteins or proteins for which a function has yet to be determined. Seven selected genes such as Beclin, thioredoxin, HLA-B, MAP3K, SPT1, SPT2, and AGO1 were up-regulated induced by PB90, suggesting that the genes may play an important role in PB90-triggered HR.

The Variety Screening of Green-stem Non-heading Chinese Cabbage and Dark-green-leaf Non-heading Chinese Cabbage Tolerant to Heat

[Objectives] Introducing and screening leaf vegetable varieties tolerant to heat and humidity has great significance to ensure balanced supply of vegetables in summer and autumn in Hainan Province. [Methods] On the basis of the introduction of green-stem non-heading Chinese cabbage and dark-green-leaf non-heading Chinese cabbage varieties, the varieties tolerant to heat and humidity were identified by the investigation of agronomic traits and evaluation of heat injury index. [Results] The data analysis results showed that four green-stem non-heading Chinese cabbage varieties including Huaguan Qingcaigeng F_1, Zhongguan Qinggeng, Hanshen No. 1 Qinggengcai F_1 and Huaxiawang Piaocai have the characteristics of strong tolerance to heat and high yield, and four dark-green-leaf non-heading Chinese cabbage varieties including Baimeigui, Heijingang, Xiaxue Naibaicai and Siji Baicai have the characteristics of strong tolerance to heat and high yield. [Conclusions] These eight leaf vegetable varieties are suitable for popularization in summer and autumn in Hainan Province.

Zinc chemical forms and organic acid exudation in non-heading Chinese cabbages under zinc stress

As an essential element, zinc also is a heavy metal. Non-heading Chinese cabbage showed obvious tolerance to Zn stress in former research. To further understand the mechanisms involved in Zn adaptability and detoxification, two genotypes Suzhouqing and Aijiaohuang were selected to investigate the chemical forms of Zn and root exudation. Zinc stress obvious strained the plant growth, and Aijiaohuang was more injured than Suhouqing under Zn stress. Under normal Zn levels, the chemical forms of Zn were diverse in three organs between genotypes. Results showed extractions of 2% HAc, 80% ethanol and 1 M NaCl were separately dominant in roots, petioles and leaves. However, under Zn stress (13 mg·L–1 and 52 mg·L–1) most of the Zn was extracted by 1M NaCl, and the subdominant amount of Zn was extracted by 80% ethanol. In the control only four types of organic acid could be detected. While under Zn stress, oxalic acid, tartaric acid, malic acid, lactic acid, acetic acid, citric acid and amber acid were all detected, so it could be speculated Zn detoxification with organic ligands or integrated with pectates and proteins in cells might be responsible for the adaptation of Zn stress in Chinese cabbage.

Isolation and characterization of an ERF-B3 gene associated with flower abnormalities in non-heading Chinese cabbage

BrcERF-B3 gene, a member of ethylene-responsive factor family, was screened from a mutant plant in non-heading Chinese cabbage（Brassica rapa ssp. chinensis） by cDNA-AFLP technology. We got full length cDNA of two BrcERF-B3 genes by homology-based cloning from two materials and found that their nucleotide sequences were the same by sequencing. The BrcERF-B3 protein, belonging to the B3 subgroup of the ERF subfamily, shared a close relationship with B. rapa. RT-PCR result showed that BrcERF-B3 expressed only in mutant stamen rather than maintainer stamen. qRT-PCR results indicated that BrcERF-B3 expressed highly during reproductive growth development and in the early of mutant buds, suggesting BrcERF-B3 might be involved in the formation of abnormal flower in mutant. What＇s more, the expression of BrcERF-B3 was more significant to ABA, Me JA and cold stresses in mutant than in maintainer and was down-regulated in NaCl treatment in two lines, implying BrcERF-B3 might be different roles in biotic and abiotic stresses.

The construction of a genetic linkage map of non-heading Chinese cabbage (Brassica campestris ssp.chinensis Makino)

Non-heading Chinese cabbage （Brassica carnpestris ssp. chinensis Makino） is one of the most important vegetables in eastern China. A genetic linkage map was constructed using 127 doubled haploid （DH） lines, and the DH population was derived from a commercial hybrid ＂Hanxiao＂ （lines SW-13 x L-118）. Out of the 614 polyrnorphic markers, 43.49% were not assigned to any of the linkage groups （LGs）. Chi-square tests showed that 42.67% markers were distorted from expected Mendelian segregation ratios, and the direction of distorted segregation was mainly toward the paternal parent L-118. After sequentially removing the markers that had an interval distance smaller than 1 cM from the upper marker, the overall quality of the linkage map was increased. Two hundred and sixty-eight molecular markers were mapped into 10 LGs, which were anchored to the corresponding chromosome of the B. rapa reference map based on com- mon simple sequence repeat （SSR） markers. The map covers 973.38 cM of the genome and the average interval distance between markers was 3.63 cM. The number of markers on each LG ranged from 18 （R08） to 64 （R07）, with an average interval distance within a single LG from 1.70 cM （R07） to 6.71 cM （R06）. Among these mapped markers, 169 were sequence-related amplified polymorphism （SRAP） molecular markers, 50 were SSR markers and 49 were random amplification polymorphic DNA （RAPD） markers. With further saturation to the LG9 the current map offers a genetic tool for loci analysis for important agronomic traits.

Isolation and Characterization of a Novel Chitosan-Binding Protein from Non-Heading Chinese Cabbage Leaves

Abstract: To know the mechanism of ammonia assimilation in non-heading Chinese cabbage (Brassica campestrish L. ssp. chinensis (L.) Makino) leaves regulated by chitosan (CTS), a CTS-binding protein was isolated from non-heading Chinese cabbage leaves using the chitosan affinity chromatography approach and this CTS-binding protein was partially characterized. The profile of the 53.1 kDa purified protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was compared with the native molecular weight of 106.5 kDa, which indicated that the purified protein was a dimer with identical subunits. After isoelectric focusing, a band was obtained at pH 8.25. The agglutination test and periodic acid-Schiff staining further revealed that the protein was a glycoprotein with lectin activity. Moreover, the purified protein contained 17.4% (w/w) neutral carbohydrate and 82.56% (w/w) protein. The comparison of this protein and the 67 kDa CTS-binding protein isolated previously from Rubus culture tissue exhibited some differences in characterization. According to results of peptide mass fingerprinting analysis, the protein purified in the present study does not show any similarity with any protein in the protein data bank. Thus, it was deduced that the protein purified in the present study is a novel CTS-binding protein.

Fine mapping and cloning of the sterility gene Bra2Ms in nonheading Chinese cabbage(Brassica rapa ssp.chinensis)

The application of a male-sterile line is an ideal approach for hybrid seed production in non-heading Chinese cabbage(Brassica rapa ssp.chinensis).However,the molecular mechanisms underlying male sterility in B.rapa are still largely unclear.We previously obtained the natural male sterile line WS24-3 of non-heading Chinese cabbage and located the male sterile locus,Bra2Ms,on the A2 chromosome.Cytological observations revealed that the male sterility of WS24-3 resulted from disruption of the meiosis process during pollen formation.Fine mapping of Bra2Ms delimited the locus within a physical distance of about 129 kb on the A2 chromosome of B.rapa.The Bra039753 gene encodes a plant homeodomain(PHD)-finger protein and is considered a potential candidate gene for Bra2Ms.Bra039753 was significantly downregulated in sterile line WS24-3 compared to the fertile line at the meiotic anther stage.Sequence analysis of Bra039753 identified a 369 bp fragment insertion in the first exon in male sterile plants,which led to an amino acid insertion in the Bra039753 protein.In addition,the 369 bp fragment insertion was found to cosegregate with the male sterility trait.This study identified a novel locus related to male sterility in non-heading Chinese cabbage,and the molecular marker obtained in this study will be beneficial for the marker-assisted selection of excellent sterile lines in non-heading Chinese cabbage and other Brassica crops.