Molecular characterization and genetic origin of the Brassica napus acetohydroxyacid synthase multigene family

Summary The Brassica napus rapeseed cultivar Topas contains an acetohydroxyacid synthase (AHAS) multigene family consisting of five members (AHAS 1–5). DNA sequence analysis indicate that AHAS1 and AHAS3 share extensive homology. They probably encode the AHAS enzymes essential for plant growth and development. AHAS2 has diverged significantly from AHAS1 and AHAS3 and has unique features in the coding region of the mature polypeptide, transit peptide and upstream non-coding DNA, which raises the possibility that it has a distinct function. AHAS4 and AHAS5 have interrupted coding regions and may be defective. The complexity of the AHAS multigene family in the allotetraploid species B. napus is much greater than reported for Arabidopsis thaliana and Nicotiana tabacum. Analysis of the presumptive progenitor diploid species B. campestris and B. oleracea indicated that AHAS2, AHAS3 and AHAS4 originate from the A genome, whereas AHAS1 and AHAS5 originate from the C genome. Further variation within each of the AHAS genes in these species was found.     

Isolation and characterization of a Brassica napus cDNA corresponding to a B-class floral development gene

B-class floral homeotic genes are required for the proper formation and identity of petals and stamens in dicot flowers. A partial cDNA clone encoding a B-class gene, BnAP3 (Brassica napus APETALA3), was isolated from a B. napus cDNA library derived from young inflorescence meristems. The 5' region of the cDNA was retrieved by RACE. The deduced amino acid sequence of the full-length clone exhibited high similarity to APETALA3 of Arabidopsis thaliana and functionally homologous proteins from other species. 5' RACE and Southern analysis suggests that BnAP3 has multiple alleles in B. napus. Expression analysis assayed by RT-PCR shows that BnAP3 is expressed in floral tissues, as well as non-floral tissues such as root and bract. Transformation of wild-type A. thaliana and B. napus plants with BnAP3 under the control of a promoter specific to reproductive organs converts carpels to stamens, while the expression of this construct in A. thaliana plants mutant for AP3 restores the development of third-whorl stamens in addition to directing a carpel to stamen conversion in the fourth whorl.     

The protein kinases AtMAP3Kepsilon1 and BnMAP3Kepsilon1 are functional homologues of S. pombe cdc7p and may be involved in cell division

We identified an Arabidopsis thaliana gene, AtMAP3Kepsilon1, and a Brassica napus cDNA, BnMAP3Kepsilon1, encoding functional protein serine/threonine kinases closely related to cdc7p and Cdc15p from Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. This is the first report of cdc7-related genes in non-fungal eukaryotes; no such genes have as yet been identified in Metazoans. The B. napus protein is able to partially complement a cdc7 loss of function mutation in S. pombe. RT-PCR and in situ hybridisation revealed that the A. thaliana and B. napus genes are expressed in both the sporophytic and the gametophytic tissues of the respective plant species and revealed further that expression is highest in dividing cells. Moreover, AtMAP3Kepsilon1 gene expression is cell cycle-regulated, with higher expression in G2-M phases. Our results strongly suggest that the plant cdc7p-related protein kinases are involved in a signal transduction pathway similar to the SIN pathway, which positively regulates cytokinesis in S. pombe.     

Identification of FAD2 and FAD3 genes in Brassica napus genome and development of allele-specific markers for high oleic and low linolenic acid contents

Modification of oleic acid (C18:1) and linolenic acid (C18:3) contents in seeds is one of the major goals for quality breeding after removal of erucic acid in oilseed rape (Brassica napus). The fatty acid desaturase genes FAD2 and FAD3 have been shown as the major genes for the control of C18:1 and C18:3 contents. However, the genome structure and locus distributions of the two gene families in amphidiploid B. napus are still not completely understood to date. In the present study, all copies of FAD2 and FAD3 genes in the A- and C-genome of B. napus and its two diploid progenitor species, Brassica rapa and Brassica oleracea, were identified through bioinformatic analysis and extensive molecular cloning. Two FAD2 genes exist in B. rapa and B. oleracea, and four copies of FAD2 genes exist in B. napus. Three and six copies of FAD3 genes were identified in diploid species and amphidiploid species, respectively. The genetic control of high C18:1 and low C18:3 contents in a double haploid population was investigated through mapping of the quantitative trait loci (QTL) for the traits and the molecular cloning of the underlying genes. One major QTL of BnaA.FAD2.a located on A5 chromosome was responsible for the high C18:1 content. A deleted mutation in the BnaA.FAD2.a locus was uncovered, which represented a previously unidentified allele for the high oleic variation in B. napus species. Two major QTLs on A4 and C4 chromosomes were found to be responsible for the low C18:3 content in the DH population as well as in SW Hickory. Furthermore, several single base pair changes in BnaA.FAD3.b and BnaC.FAD3.b were identified to cause the phenotype of low C18:3 content. Based on the results of genetic mapping and identified sequences, allele-specific markers were developed for FAD2 and FAD3 genes. Particularly, single-nucleotide amplified polymorphisms markers for FAD3 alleles were demonstrated to be a reliable type of SNP markers for unambiguous identification of genotypes with different content of C18:3 in amphidiploid B. napus.     

Comparative mapping reveals similar linkage of functional genes to QTL of yield-related traits between <Emphasis Type="Italic">Brassica napus</Emphasis> and <Emphasis Type="Italic">Oryza sativa</Emphasis>

Oryza sativa and Brassica napus-two important crops for food and oil, respectively-share high seed yield as a common breeding goal. As a model plant, O. sativa genomics have been intensively investigated and its agronomic traits have been advanced. In the present study, we used the available information on O. sativa to conduct comparative mapping between O. sativa and B. napus, with the aim of advancing research on seed-yield and yield-related traits in B. napus. Firstly, functional markers (from 55 differentially expressed genes between a hybrid and its parents) were used to detect B. napus genes that co-localized with yield-related traits in an F(2:3) population. Referring to publicly available sequences of 55 B. napus genes, 53 homologous O. sativa genes were subsequently detected by screening, and their chromosomal locations were determined using silico mapping. Comparative location of yield-related QTL between the two species showed that a total of 37 O. sativa and B. napus homologues were located in similar yield-related QTL between species. Our results indicate that homologous genes between O. sativa and B. napus may have consistent function and control similar traits, which may be helpful for agronomic gene characterization in B. napus based on what is known in O. sativa.     

Genetic analysis of pathway regulation for enhancing branched‐chain amino acid biosynthesis in plants

The branched‐chain amino acids (BCAAs) valine, leucine and isoleucine are essential amino acids that play critical roles in animal growth and development. Animals cannot synthesize these amino acids and must obtain them from their diet. Plants are the ultimate source of these essential nutrients, and they synthesize BCAAs through a conserved pathway that is inhibited by its end products. This feedback inhibition has prevented scientists from engineering plants that accumulate high levels of BCAAs by simply over‐expressing the respective biosynthetic genes. To identify components critical for this feedback regulation, we performed a genetic screen for Arabidopsis mutants that exhibit enhanced resistance to BCAAs. Multiple dominant allelic mutations in the VALINE‐TOLERANT 1 (VAT1) gene were identified that conferred plant resistance to valine inhibition. Map‐based cloning revealed that VAT1 encodes a regulatory subunit of acetohydroxy acid synthase (AHAS), the first committed enzyme in the BCAA biosynthesis pathway. The VAT1 gene is highly expressed in young, rapidly growing tissues. When reconstituted with the catalytic subunit in vitro, the vat1 mutant‐containing AHAS holoenzyme exhibits increased resistance to valine. Importantly, transgenic plants expressing the mutated vat1 gene exhibit valine tolerance and accumulate higher levels of BCAAs. Our studies not only uncovered regulatory characteristics of plant AHAS, but also identified a method to enhance BCAA accumulation in crop plants that will significantly enhance the nutritional value of food and feed.     

Identification and characterization of candidate Rlm4 blackleg resistance genes in Brassica napus using next-generation sequencing

A thorough understanding of the relationships between plants and pathogens is essential if we are to continue to meet the agricultural needs of the world's growing population. The identification of genes underlying important quantitative trait loci is extremely challenging in complex genomes such as Brassica napus (canola, oilseed rape or rapeseed). However, recent advances in next-generation sequencing (NGS) enable much quicker identification of candidate genes for traits of interest. Here, we demonstrate this with the identification of candidate disease resistance genes from B.?napus for its most devastating fungal pathogen, Leptosphaeria maculans (blackleg fungus). These two species are locked in an evolutionary arms race whereby a gene-for-gene interaction confers either resistance or susceptibility in the plant depending on the genotype of the plant and pathogen. Preliminary analysis of the complete genome sequence of Brassica rapa, the diploid progenitor of B.?napus, identified numerous candidate genes with disease resistance characteristics, several of which were clustered around a region syntenic with a major locus (Rlm4) for blackleg resistance on A7 of B.?napus. Molecular analyses of the candidate genes using B.?napus NGS data are presented, and the difficulties associated with identifying functional gene copies within the highly duplicated Brassica genome are discussed.     

Expression levels of meristem identity and homeotic genes are modified by nuclear-mitochondrial interactions in alloplasmic male-sterile lines of Brassica napus

Homeotic conversions of anthers were found in cytoplasmic male sterile (CMS) plants of Brassica napus derived from somatic hybrids of B. napus and Arabidopsis thaliana. CMS line flowers displayed petals reduced in size and width and stamens replaced by carpelloid structures. In order to investigate when these developmental aberrations appeared, flower development was analysed histologically, ultrastructurally and molecularly. Disorganized cell divisions were detected in the floral meristems of the CMS lines at stage 4. As CMS is associated with mitochondrial aberrations, ultrastructural analysis of the mitochondria in the floral meristems was performed. Two mitochondrial populations were found in the CMS lines. One type had disrupted cristae, while the other resembled mitochondria typical of B. napus. Furthermore, expression patterns of genes expressed in particular floral whorls were determined. In spite of the aberrant development of the third whorl organs, BnAP3 was expressed as in B. napus during the first six stages of development. However, the levels of BnPI were reduced. At later developmental stages, the expression of both BnAP3 and BnPI was strongly reduced. Interestingly the expression levels of genes responsible for AP3 and PI activation such as LFY, UFO and ASK1 were higher in the CMS lines, which indicates that activation of B-genes in the CMS lines does not occur as in B. napus. Disrupted and dysfunctional mitochondria seem to be one of the first aberrations manifested in CMS which result in a retrograde influence of the expression levels of genes responsible for the second and third whorl organ differentiation.     

甘蓝型油菜的BR响应及BnBZL2基因的功能分析

对甘蓝型油菜(Brassica napus L.)与拟南芥(Arabidopsis thaliana L.)中保守的油菜素甾醇(Brassinosteroids,BR)信号相关基因进行对比分析,并以甘蓝型油菜品种‘沪油15’为材料,对BR信号通路相关同源基因进行了组织表达分析。结果显示,BR合成基因与信号组分在花和幼嫩种子中表达量更高;低浓度BR处理可以促进幼苗根的生长,高浓度BR处理则起抑制作用; BR合成抑制剂(Brassinozole,BRZ)处理可抑制黑暗条件下幼苗下胚轴的伸长; BR处理可以降低BR合成基因的表达水平,而BRZ处理则相反,表明甘蓝型油菜中BR信号增加能反馈抑制BR的合成。烟草(Nicotiana tabacum L.)瞬时表达实验结果发现,与拟南芥BZR1基因同源的甘蓝型油菜BnBZL2编码蛋白定位在细胞质和细胞核中,BR处理可增加BnBZL2的核定位。蛋白质免疫印迹检测结果显示,BR处理可增加去磷酸化BnBZL2的比例。本研究进一步模拟了拟南芥bzr1-1D功能获得性突变体对BnBZL2蛋白进行点突变(BnBZL2*),并构建载体转化拟南芥,黑暗条件下转基因植株幼苗对BRZ处理不敏感,提示BnBZL2*可提高转基因植株的BR信号水平。本研究结果表明甘蓝型油菜中存在与拟南芥相似且保守的BR信号通路和调控机制。     

Expression, mapping, and genetic variability of Brassica napus disease resistance gene analogues.

Numerous sequences analogous to resistance (R) genes exist in plant genomes and could be involved in resistance traits. The aim of this study was to identify a large number of Brassica napus sequences related to R genes and also to test the adequacy of specific PCR-based tools for studying them. Different consensus primers were compared for their efficiency in amplifying resistance-gene analogues (RGAs) related to the nucleotide-binding-site subgroup of R genes. Specific primers were subsequently designed to fine-study the different RGAs and we tested their efficiency in three species related to B. napus: Brassica oleracea, Brassica rapa, and Arabidopsis thaliana. Forty-four B. napus RGAs were identified. Among 29 examined, at least one-third were expressed. Eighteen RGAs were mapped on 10 of the 19 B. napus linkage groups. The high variability within these sequences permitted discrimination of each genotype within a B. napus collection. The RGA-specific primers amplified RGAs in the B. oleracea and B. rapa genomes, but the sequences appear to be poorly conserved in A. thaliana. Specific RGA primers are a precise tool for studying known-sequence RGAs. These sequences represent interesting markers that could be correlated with resistance traits in B. napus or related Brassica genomes.     

油菜沪油15中AP2/ERF-B3亚族转录因子的克隆和生物信息学分析

AP2/ERF转录因子家族广泛存在于植物中,参与植物细胞周期、生长发育以及生物和非晌镄财认喙鼗虮泶锏骺?本文利用油菜UniGene数据库.以拟南芥AP2/ERF-B3亚族转录因子保守序列为信息探针,分离得到2个油菜AP2/ERF-B3亚族的转录因子BnaERFB3-1和BnaERFB3-2.通过PCR和RT-PCR方法分别从双低甘蓝型油菜沪油15的DNA和cDNA中克隆了上述基因.序列分析显示.克隆的BnaERFB3-1-Hv15和BnaERFB3-2-Hv15转录因子与电子克隆的基因序列差异很小,均只有1个氨基酸位点不同.且都没有内舍子.从氨基酸序列的相似性、组成成分、理化性质、疏水性/亲水性、序列比对、进化树、功能域、二级结构、三级结构、无序化特性等方面进行了预测和较为全面的分析.结果显示BnaERFB3-1-Hv15和BnaERFB3-2-Hv15是亲水性蛋白,在蛋白质的三级结构上与AtERF5相似,BnaERFB3-1-Hv15和BnaERFB3-2-Hv15蛋白无序化程度大于拟南芥AtERF5.通过分析EST丰度显示,BnaERFB3-1的表达集中在种子中,而BnaERFB3-2的表达则集中在根中.另外.将上述基因分别构建入酵母表达载体和植物双元表达载体,为深入研究该基因在油菜抗逆调控中的作用奠定了基础.     

AGL15, a MADS domain protein expressed in developing embryos.

To extend our knowledge of genes expressed during early embryogenesis, the differential display technique was used to identify and isolate mRNA sequences that accumulate preferentially in young Brassica napus embryos. One of these genes encodes a new member of the MADS domain family of regulatory proteins; it has been designated AGL15 (for AGAMOUS-like). AGL15 shows a novel pattern of expression that is distinct from those of previously characterized family members. RNA gel blot analyses and in situ hybridization techniques were used to demonstrate that AGL15 mRNA accumulated primarily in the embryo and was present in all embryonic tissues, beginning at least as early as late globular stage in B. napus. Genomic and cDNA clones corresponding to two AGL15 genes from B. napus and the homologous single-copy gene from Arabidopsis, which is located on chromosome 5, were isolated and analyzed. Antibodies prepared against overexpressed Brassica AGL15 lacking the conserved MADS domain were used to probe immunoblots, and AGL15-related proteins were found in embryos of a variety of angiosperms, including plants as distantly related as maize. Based on these data, we suggest that AGL15 is likely to be an important component of the regulatory circuitry directing seed-specific processes in the developing embryo.     

Agrobacterium tumefaciens-mediated transformation of Brassica napus winter cultivars

An efficient protocol for Agrobacterium tumefaciens-mediated transformation of six commercial Brassica napus winter cultivars is described. Two B. napus spring cultivars were analysed for comparison. Five strains of A. tumefaciens with different combinations of nopaline and octopine chromosomal backgrounds and virulence plasmids were used for cocultivation. Selection of putative regenerated transgenic plants was performed on kanamycin- or hygromycin-containing media. The scores of transgenic plants were calculated on the basis of GUS (-glucuronidase) activity, detected by the histochemical X-Gluc test. Target tissue derived from the cut surface of cotyledon petioles resulted in successful transformation with all the winter cultivars tested. Target tissue from hypocotyl segments resulted in a successful transformation with only one winter cultivar. The transformation rates for B. napus winter cultivars in this study were higher than in previous reports. Southern blot analysis revealed that integration of marker genes occurred in single and in multiple copies and at multiple loci in the genome. The transgenic plants all grew normally and developed fertile flowers after a vernalization period. After self-pollination, Southern blot analysis of selected GUS active F1 plants revealed that introduced marker genes were stably inherited to the next generation. These data demonstrate that morphologically normal, fertile transgenic plants of B. napus winter cultivars can be achieved with both nopaline- and octopine-derived A. tumefaciens strains. This protocol should have a broad application in improvement of Brassica napus winter cultivars by introduction of foreign genes     

Characterization of interploid hybrids from crosses between Brassica juncea and B. oleracea and the production of yellow-seeded B. napus

Yellow-seeded Brassica napus was for the first time developed from interspecific crosses using yellow-seeded B. juncea (AABB), yellow-seeded B. oleracea (CC), and black-seeded artificial B. napus (AACC). Three different mating approaches were undertaken to eliminate B-genome chromosomes after trigenomic hexaploids (AABBCC) were generated. Hybrids (AABCC, ABCC) from crosses AABBCC?×?AACC, AABBCC?×?CC and ABCC?×?AACC were advanced by continuous selfing in approach 1, 2 and 3, respectively. To provide more insight into Brassica genome evolution and the cytological basis for B. napus resynthesis in each approach, B-genome chromosome pairing and segregation were intensively analyzed in AABCC and ABCC plants using genomic in situ hybridization methods. The frequencies at which B-genome chromosomes underwent autosyndesis and allosyndesis were generally higher in ABCC than in AABCC plants. The difference was statistically significant for allosyndesis but not autosyndesis. Abnormal distributions of B-genome chromosomes were encountered at anaphase I, including chromosome lagging and precocious sister centromere separation of univalents. These abnormalities were observed at a significantly higher frequency in AABCC than in ABCC plants, which resulted in more rapid B-genome chromosome elimination in the AABCC derivatives. Yellow or yellow-brown seeds were obtained in all approaches, although true-breeding yellow-seeded B. napus was developed only in approaches 2 and 3. The efficiency of the B. napus construction approaches was in the order 1?>?3?>?2 whereas this order was 3?>?2?>?1 with respect to the construction of yellow-seeded B. napus. The results are discussed in relation to Brassica genome evolution and the development and utilization of the yellow-seeded B. napus obtained here.     

Characterization and immunolocalization of a cytosolic calcium-binding protein from Brassica napus and Arabidopsis pollen.

Two low-molecular-weight proteins have been purified from Brassica napus pollen and a gene corresponding to one of them has been isolated. The gene encodes an 8.6-kD protein with two EF-hand calcium-binding motifs and is a member of a small gene family in B. napus. The protein is part of a family of pollen allergens recently identified in several evolutionarily distant dicot and monocot plants. Homologs have been detected in Arabidopsis, from which one gene has been cloned in this study, and in snapdragon (Antirrhinum majus), but not in tobacco (Nicotiana tabacum). Expression of the gene in B. napus was limited to male tissues and occurred during the pollen-maturation phase of anther development. Both the B. napus and Arabidopsis proteins interact with calcium, and the potential for a calcium-dependent conformational change was demonstrated. Given this affinity for calcium, the cloned genes were termed BPC1 and APC1 (B. napus and Arabidopsis pollen calcium-binding protein 1, respectively). Immunolocalization studies demonstrated that BPC1 is found in the cytosol of mature pollen. However, upon pollen hydration and germination, there is some apparent leakage of the protein to the pollen wall. BPC1 is also concentrated on or near the surface of the elongating pollen tube. The essential nature of calcium in pollen physiology, combined with the properties of BPC1 and its high evolutionary conservation suggests that this protein plays an important role in pollination by functioning as a calcium-sensitive signal molecule.     

Cellular responses of two rapid-cycling Brassica species, B. napus and B. carinata, to seawater salinity

Cellular responses of two rapid-cycling Brassica species. B. napus and B. carinata , to seawater salinity were characterized to determine whether callus showed a tolerance similar to that of whole plants. Callus was initiated from the leaves of 7-day-old seedlings of B. napus and B . carinata and then subcultured with two different levels of seawater salinity (2.3 and 5.2 g l⁻¹ Instant Ocean. Aquarium Systems, Inc. Mentor. OH, USA) for 14 days. Callus growth of both species was reduced by seawater salinity. Based on the percentage of the reduction in the relative fresh weight gain. B. napus was more salt-tolerant than B. carinata . consistent with the response of whole plants of the same species to seawater salinity. Seawater salinity caused changes in the concentrations of Na, K. Ca, Mg and Cl in both B. napus and B. carinata . The growth expressed as the percentage of control was significantly (P = 0.05) positively correlated with the concentration of Ca. and K/Na and Ca/Na ratios. It was also negatively correlated ( P = 0.01) with the concentrations of Na. Cl and Mg. In comparison with B. carinala . the salt-tolerant species, B . napus , showed a small reduction in the concentrations of Ca and K in the salt-stressed plants relative to the control.