Molecular tagging of erucic acid trait in oilseed mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>) by QTL mapping and single nucleotide polymorphisms in<Emphasis Type="Italic"> FAE1</Emphasis> gene

Molecular mapping and tagging of the erucic acid trait (C22:1) in Brassica juncea was done by a candidate gene approach. Two QTLs underlying the variation of seed erucic acid content were assigned to two linkage groups of a B. juncea map using a doubled haploid (DH) mapping population derived from high × low erucic acid F₁ hybrid. Two consensus primers corresponding to the full-length Fatty Acid Elongase 1 (FAE1) gene, reported to be involved in the elongation of C18:1 to C22:1, were designed. PCR amplification and subsequent cloning and sequencing identified two FAE1 genes (FAE1.1 and FAE1.2) in both high and low erucic acid mustard lines. Sequence alignment of corresponding FAE1 genes between high and low erucic acid mustard lines identified four substitution type single nucleotide polymorphisms (SNPs) in FAE1.1 and three in FAE1.2. Using the SNuPE method of SNP genotyping, these two genes were mapped to two independent loci that co-segregated with the two QTLs governing the erucic acid trait. Association of wild (E1E2) and mutant (e1e2) haplotypes of two FAE1 genes with erucic acid variation in two segregating populations revealed that the e1e1e2e2 genotype identified low erucic acid individuals (<2%) and E1E1E2E2 identified individuals with highest erucic acid content (>40%). The E1e1E2e2 heterozygote was found to be intermediate in phenotype. The applicability of these SNPs in marker-assisted manipulation of the erucic acid trait was verified by genotyping a set of contrasting germplasm of B. juncea belonging to two distinct gene pools (Indian and east European) and other oil-yielding Brassica species.Communicated by C. Möllers     

Cloning of TTG1 gene and PCR identification of genomes A,B and C in Brassica species

Arabidopsis Transparent Testa Glabra 1 (TTG1) genes were cloned from three diploid Brassica species (B. rapa, B. nigra and B. oleracea) and two amphidiploids species (B. juncea and B. carinata) by homology cloning. TTG1 homologues identified in all the accessions of the investigated species had a coding sequence of 1,014 bp. One copy was obtained from each diploid species and two copies from each amphidiploid species. Combined analysis of the TTG1 sequences cloned in this study with those obtained from public databases demonstrated that three, forty-five and seven nucleotides were specific variations in TTG1 genes from genomes A, B and C, respectively. Primers designed with genome-specific nucleotide variations were able to distinguish among TTG1 genes originating from genomes A, B and C in Brassica. Therefore, the TTG1 gene could serve as a candidate marker gene to detect the pollen flow of Brassica and provide an alternative method for the detection of pollen drift and risk assessment of gene flow in Brassica species.     

Molecular Cloning and Characterization of FATTY ACID ELONGATION1 (BjFAE1) Gene of Brassica juncea

The Brassica juncea homologue of Arabidopsis thaliana FAE1 gene, which is responsible for elongation of fatty acid chain length from C18 to C20 and C22 was amplified via PCR (Polymerase Chain Reaction) using heterologous primers. The PCR product was cloned into pGEM-T vector, subcloned and sequenced. The BjFAE1 has 1536-nucleotides and shares 93.6% homology with the A. thaliana counterpart. Southern analysis, using the PCR product as probe, indicated that FAE1 gene is of the same size in all the cultivated Brassica species, i.e. B. juncea, B. nigra, B. campestris, B. oleracea, B. napus and B. carinata. It expresses strongly only in the developing seed and podwall.     

Restoring enzyme activity in nonfunctional low erucic acid Brassica napus fatty acid elongase 1 by a single amino acid substitution.

Genomic fatty acid elongation 1 (FAE1) clones from high erucic acid (HEA) Brassica napus, Brassica rapa and Brassica oleracea, and low erucic acid (LEA) B. napus cv. Westar, were amplified by PCR and expressed in yeast cells under the control of the strong galactose-inducible promoter. As expected, yeast cells expressing the FAE1 genes from HEA Brassica spp. synthesized very long chain monounsaturated fatty acids that are not normally found in yeast, while fatty acid profiles of yeast cells expressing the FAE1 gene from LEA B. napus were identical to control yeast samples. In agreement with published findings regarding different HEA and LEA B. napus cultivars, comparison of FAE1 protein sequences from HEA and LEA Brassicaceae revealed one crucial amino acid difference: the serine residue at position 282 of the HEA FAE1 sequences is substituted by phenylalanine in LEA B. napus cv. Westar. Using site directed mutagenesis, the phenylalanine 282 residue was substituted with a serine residue in the FAE1 polypeptide from B. napus cv. Westar, the mutated gene was expressed in yeast and GC analysis revealed the presence of very long chain monounsaturated fatty acids (VLCMFAs), indicating that the elongase activity was restored in the LEA FAE1 enzyme by the single amino acid substitution. Thus, for the first time, the low erucic acid trait in canola B. napus can be attributed to a single amino acid substitution which prevents the biosynthesis of the eicosenoic and erucic acids.     

芥菜型油菜脂肪酸含量的变异、相关性分析及芥酸调控基因FAE1特异引物设计

芥菜型油菜是我国芸苔属的三大油料作物之一,具有耐旱、抗病虫等优良特性;而我国是芥菜型油菜的重要起源中心,具有丰富的种质资源.该研究从全国各地搜集了34份芥菜型油菜,在贵阳环境条件下种植,其脂肪酸含量(芥酸、油酸、硬脂酸、亚麻酸和亚油酸)表现出丰富的变异,并呈正态分布.结果表明:这些芥菜型油菜种质资源的不同脂肪酸含量间的相关性发现,芥酸和油酸间呈极显著负相关,亚麻酸和硬脂酸呈极显著的正相关,亚麻酸和亚油酸呈现负相关.利用这些材料的脂肪酸含量进行主成分分析,发现绝大部分材料(30份,占88.2%)集中在二维图的特定区域,只有少数其它材料散落在图中其它区域,他们分别是SL63、棱角油菜、T6342和长阳黄芥,这些变异较大的材料在芥菜型油菜的育种中可以发挥特殊作用.此外,运用来自甘蓝型油菜和甘蓝的芥酸调控基因FAE1的已知序列,并设计了FAE1特异引物,而引物在全部34份芥菜型油菜种质资源中均表现出了较好的扩增效果.因此证实芥菜型油菜中至少含有一个FAE1拷贝.该研究结果对于芥菜型油菜育种在我国的开展及其未来的分子育种具有重要的指导意义.     

Cloning of fatty acid elongase1 gene and molecular identification of A and C genome in <Emphasis Type="Italic">Brassica</Emphasis> species

The fatty acid elongase 1 (FAE1) genes of Brassic napus were cloned from two cultivars, i.e. Zhongshuan No. 9 with low erucic acid content, and Zhongyou 821 with high erucic acid content, using the degenerate PCR primers. The sequence analysis showed that there was no intron within the FAE1 genes. The FAE1 genes from Zhongyou 821 contained a coding sequence of 1521 nucleotides, and those cloned from Zhongshuan No. 9 contained a 1517 bp coding sequence. Alignment of the FAE1 sequences from Brassica rapa, B. oleracea and B. napus detected 31 single nucleotide polymorphic sites (2.03%), which resulted in 7 amino-acid substitutions. Further analysis indicated that 19 SNPs were genome-specific, of which, 95% were synonymous mutations. The nucleotide substitution at position 1217 in the FAE1 genes led to a specific site of restricted cleavage. An AvrII cleavage site was present only in the C genome genes and absent in the A genome FAE1 genes. Digestion profile of the FAE1 sequences from B. rapa, B. oleracea and B. napus produced with AvrII confirmed that the FAE1 genes of B. oleracea origin was recognized and digested, while that of B. rapa origin could not. The results indicated that by AvrII cleavage it was possible to distinguish B. rapa from B. oleracea and between the A and C genome of B. napus. In addition, the FAE1 genes could be used as marker genes to detect the pollen flow of B. napus, thus providing an alternative method for risk assessment of gene flow.     

Cloning of fatty acid elongase1 gene and molecular identification of A and C genome in Brassica species

The fatty acid elongase 1 (FAE1) genes of Brassic napus were cloned from two cultivars, i.e. Zhongshuan No. 9 with low erucic acid content, and Zhongyou 821 with high erucic acid content, using the degenerate PCR primers. The sequence analysis showed that there was no intron within the FAE1 genes. The FAE1 genes from Zhongyou 821 contained a coding sequence of 1521 nucleotides, and those cloned from Zhongshuan No. 9 contained a 1517 bp coding sequence. Alignment of the FAE1 sequences from Brassica rapa, B. oleracea and B. napus detected 31 single nucleotide polymorphic sites (2.03%), which resulted in 7 amino-acid substitutions. Further analysis indicated that 19 SNPs were genome-specific, of which, 95% were synonymous mutations. The nucleotide substitution at position 1217 in the FAE1 genes led to a specific site of restricted cleavage. An AvrII cleavage site was present only in the C genome genes and absent in the A genome FAE1 genes. Digestion profile of the FAE1 sequences from B. rapa, B. oleracea and B. napus produced with AvrII confirmed that the FAE1 genes of B. oleracea origin was recognized and digested, while that of B. rapa origin could not. The results indicated that by AvrII cleavage it was possible to distinguish B. rapa from B. oleracea and between the A and C genome of B. napus. In addition, the FAE1 genes could be used as marker genes to detect the pollen flow of B. napus, thus providing an alternative method for risk assessment of gene flow. Supported by the National Natural Science Foundation of China (Grant No. 30471099), Development Plan of the State Key Fundamental Research of China (Grant No. 2006CB101600), and the National High Technology and Development Program of China (Grant No. 2006AA10A113)     

The two genes homologous to Arabidopsis FAE1 co-segregate with the two loci governing erucic acid content in Brassica napus

 KCS (β-keto-acyl-CoA synthase) has been proposed as a candidate gene for explaining the erucic acid level in rapeseed. Degenerate PCR primers corresponding to the FAE1 gene have been designed. Two B. napus genes BN-FAE1.1 and BN-FAE1.2, corresponding to the parental species B. rapa and B. oleracea FAE1 genes, were amplified. Polymorphism was revealed for these two genes by acrylamide electrophoresis of the amplification products. These two genes could then be mapped and a co-segregation of these genes with the E1 and E2 loci controlling erucic acid content was found. Furthermore, mutations observed for one of these genes could explain part of the low erucic trait of the three LEAR types used in this study. Received: 3 November 1997 / Accepted: 25 November 1997     

Selection of genetically transformed Brassica juncea L. cv. Varuna (Indian mustard) based on Positech system

The use of antibiotic and herbicide resistance based negative selection in plant transformation experiments remains a major impediment in the acceptance of transgenic crops. To overcome this, Positech selection system involving the use of phosphomannose isomerase (pmi) gene from Escherichia coli and mannose as selection agent was exploited for the selection of transgenic Brassica juncea L. cv. Varuna. The transgenic plants were generated by transformation with Agrobacterium tumefaciens harbouring the pmi gene driven by a constitutive Cestrum leaf curling viral promoter. Supplementing the selection medium with 0.09 gl^-1 mannose and 5 gl^-1 glucose provided the optimal condition for the selection of transformed explants. Stable integration and expression of pmi gene was confirmed by Southern and northern blot analysis, respectively. Our results show that the pmi gene driven by the constitutive Cestrum leaf curling viral promoter can be successfully used for positive selection in transformation of B.juncea, an important agronomic oil-seed crop, and that a combination of mannose and glucose rather than mannose alone is more suitable for the selection. To the best of our knowledge, Positech system has not been used so far in transformation of Brassica juncea.     

Genome-wide identification and characterization of Chitinase gene family in Brassica juncea and Camelina sativa in response to Alternaria brassicae

Chitinases belong to the group of Pathogenesis-related (PR) proteins that provides protection against fungal pathogens. This study presents the, genome-wide identification and characterization of chitinase gene family in two important oilseed crops B. juncea and C. sativa belonging to family Brassicaceae. We have identified 47 and 79 chitinase genes in the genomes of B. juncea and C. sativa, respectively. Phylogenetic analysis of chitinases in both the species revealed four distinct sub-groups, representing different classes of chitinases (I-V). Microscopic and biochemical study reveals the role of reactive oxygen species (ROS) scavenging enzymes in disease resistance of B. juncea and C. sativa. Furthermore, qRT-PCR analysis showed that expression of chitinases in both B. juncea and C. sativa was significantly induced after Alternaria brassicae infection. However, the fold change in chitinase gene expression was considerably higher in C. sativa compared to B. juncea, which further proves their role in C. sativa disease resistance to A. brassicae. This study provides comprehensive analysis on chitinase gene family in B. juncea and C. sativa and in future may serve as a potential candidate for improving disease resistance in B. juncea through transgenic approach.     

Modification of erucic acid content in Indian mustard (Brassica juncea) by up-regulation and down-regulation of the Brassica juncea FAT TY ACID ELONGATION1 (BjFAE1) gene

In Brassicas, the Fatty Acid Elongation1 (FAE1) gene product, a 3-ketoacyl-CoA synthase, is the first in a 4-enzyme complex involved in the synthesis of erucic acid from oleic acid. The FAE1 homologue from Brassica juncea cv. Pusa Bold was cloned in a binary vector both in sense and antisense orientations under the control of the CaMV35S promoter. The recombinant binary vectors were used to transform B. juncea cv. RLM 198 via Agrobacterium tumefaciens. The presence of the transgene was confirmed by polymerase chain reaction and Southern hybridization. Northern and western analyses showed the expression of the gene and protein, respectively, in the transgenic plants. Analyses of the fatty acid profile of the seed oil from homozygous T4 generation seeds revealed that over-expression of the FAE1 gene caused a 36% increase in the percent of erucic acid (37–49% compared to 36% in untransformed control). The down-regulation of FAE1 caused an 86% decrease in the percent of erucic acid to as low as 5% in the seed oil of transgenic plants. Thus, it is clearly possible to alter erucic acid content of mustard by altering the expression level of the FAE 1 gene. S. Kanrar and J. Venkateswari equally contributed to this work.     

Evolutionary Pattern of the FAE1 Gene in Brassicaceae and Its Correlation with the Erucic Acid Trait

The fatty acid elongase 1 (FAE1) gene catalyzes the initial condensation step in the elongation pathway of VLCFA (very long chain fatty acid) biosynthesis and is thus a key gene in erucic acid biosynthesis. Based on a worldwide collection of 62 accessions representing 14 tribes, 31 genera, 51 species, 4 subspecies and 7 varieties, we conducted a phylogenetic reconstruction and correlation analysis between genetic variations in the FAE1 gene and the erucic acid trait, attempting to gain insight into the evolutionary patterns and the correlations between genetic variations in FAE1 and trait variations. The five clear, deeply diverged clades detected in the phylogenetic reconstruction are largely congruent with a previous multiple gene-derived phylogeny. The Ka/Ks ratio (<1) and overall low level of nucleotide diversity in the FAE1 gene suggest that purifying selection is the major evolutionary force acting on this gene. Sequence variations in FAE1 show a strong correlation with the content of erucic acid in seeds, suggesting a causal link between the two. Furthermore, we detected 16 mutations that were fixed between the low and high phenotypes of the FAE1 gene, which constitute candidate active sites in this gene for altering the content of erucic acid in seeds. Our findings begin to shed light on the evolutionary pattern of this important gene and represent the first step in elucidating how the sequence variations impact the production of erucic acid in plants.     

Identification and characterization of an acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2) gene from the microalga O. tauri

In order to identify novel genes encoding enzymes involved in the terminal step of triacylglycerol (TAG) formation, a database search was carried out in the genome of the unicellular photoautotrophic green alga Ostreococcus tauri. The search led to the identification of three putative type 2 acyl-CoA:diacylglycerol acyltransferase-like sequences (DGAT; EC 2.3.1.20), and revealed the absence of any homolog to type 1 or type 3 DGAT sequence in the genome of O. tauri. For two of the cDNA sequences (OtDGAT2A and B) enzyme activity was detected by heterologous expression in Saccharomyces cerevisiae mutant strains with impaired TAG metabolism. However, activity of OtDGAT2A was too low for further analysis. Analysis of their amino acid sequences showed that they share limited identity with other DGAT2 from different plant species, such as Ricinus communis and Vernicia fordii with ～25 to 30% identity. Lipid analysis of the mutant yeast cells revealed that OtDGAT2B showed broad substrate specificity accepting saturated as well as mono- and poly-unsaturated acyl-CoAs as substrates.     

A rapeseed FAE1 gene is linked to the E1 locus associated with variation in the content of erucic acid

 The synthesis of very long chain fatty acids occurs in the cytoplasm via an elongase complex. A key component of this complex is the β-ketoacyl-CoA synthase, a condensing enzyme which in Arabidopsis is encoded by the FAE1 gene. Two sequences homologous to the FAE1 gene were isolated from a Brassica napus immature embryo cDNA library. The two clones, CE7 and CE8, contain inserts of 1647 bp and 1654 bp, respectively. The CE7 gene encodes a protein of 506 amino acids and the CE8 clone, a protein of 505 amino acids, each having an approximate molecular mass of 56 kDa. The sequences of the two cDNA clones are highly homologous yet distinct, sharing 97% nucleotide identity and 98% identity at the amino acid level. Southern hybridisation showed the rapeseed β-ketoacyl-CoA synthase to be encoded by a small multigene family. Northern hybridisation showed the expression of the rapeseed FAE1 gene(s) to be restricted to the immature embryo. One of the FAE1 genes is tightly linked to the E1 locus, one of two loci controlling erucic acid content in rapeseed. The identity of the second locus, E2, is discussed. Received: 4 April 1997 / Accepted: 30 July 1997     

Exogenous jasmonic acid modulates the physiology,antioxidant defense and glyoxalase systems in imparting drought stress tolerance in different Brassica species

This study examined the ability of jasmonic acid (JA) to enhance drought tolerance in different Brassica species in terms of physiological parameters, antioxidants defense, and glyoxalase system. Ten-day-old seedlings were exposed to drought (15 % polyethylene glycol, PEG-6000) either alone or in combination with 0.5 mM JA. Drought significantly increased lipoxygenase activity and oxidative stress, levels of malondialdehyde and H₂O₂. Drought reduced seedling biomass, chlorophyll (chl) content, and leaf relative water content (RWC). Drought increased proline, oxidized ascorbate (DHA) and glutathione disulfide (GSSG) levels. Drought affected different species differently: in B. napus, catalase (CAT) and glyoxalase II (Gly II) activities were decreased, while glutathione-S-transferase (GST) and glutathione peroxidase (GPX) activities were increased in drought-stressed compared to unstressed plants; in B. campestris, activities of glutathione reductase (GR), glyoxalase I (Gly I), GST, and GPX were increased, monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), CAT and other enzymes were decreased; in B. juncea, activities of ascorbate peroxidase, GR, GPX, Gly I were increased; Gly II activity was decreased and other enzymes did not change. Spraying drought-stressed seedlings with JA increased GR and Gly I activities in B. napus; increased MDHAR activity in B. campestris; and increased DHAR, GR, GPX, Gly I and Gly II activities in B. juncea. JA improved fresh weight, chl, RWC in all species, dry weight increased only in B. juncea. Brassica juncea had the lowest oxidative stress under drought, indicating its natural drought tolerance capacity. The JA improved drought tolerance of B. juncea to the highest level among studied species.     

Active-site residues of a plant membrane-bound fatty acid elongase β-ketoacyl-CoA synthase,FAE1 KCS

The fatty acid elongase-1 β-ketoacyl-CoA synthase, FAE1 KCS, a seed-specific elongase condensing enzyme from Arabidopsis, is involved in the production of eicosenoic (C20:1) and erucic (C22:1) acids. Alignment of the amino acid sequences of FAE1 KCS, KCS1, and five other putative elongase condensing enzymes (KCSs) revealed the presence of six conserved cysteine and four conserved histidine residues. Each of the conserved cysteine and histidine residues was individually converted by site-directed mutagenesis to both alanine and serine, and alanine and lysine respectively. After expression in yeast cells, the mutant enzymes were analyzed for their fatty acid elongase activity. Our results indicated that only cysteine 223 is an essential residue for enzyme activity, presumably for acyl chain transfer. All histidine substitutions resulted in complete loss of elongase activity. The loss of activity of these mutants was not due to their lower expression level since immunoblot analysis confirmed each was expressed to the same extent as the wild type FAE1 KCS.     

A Cointegrate Ti Plasmid Vector for Agrobacterium tumefaciens - mediated Transformation of indica Rice cv Pusa Basmati 1

An intermediate vector pSSJ1 was constructed by cloning a hph gene and a gus gene with catalase intron in pGV1500. pSSJ1 was cointegrated into a disarmed receptor Ti plasmid pGV2260 harboured in Agrobacterium tumefaciens strain C58C1Rif^R. The resulting A. tumefaciens strain C58C1Rif^R (pGV2260::pSSJ1) stably transformed Oryza sativa L. cv Pusa Basmati 1 scutellum-derived calli at 26% frequency. Introduction of the plasmid pSSJ3 (3′virB, virG and virC of pTiB0542) into A. tumefaciens C58C1Rif^R (pGV2260::pSSJ1) resulted in the elevation of acetosyringone-induced T -strand accumulation. Rice transformation efficiency of the cointegrate plasmid pGV2260::pSSJ1 increased from 26% to 33% in the presence of pSSJ3 and from 26% to 35% in the presence of pToK47 (complete virB, virG and virC). T-DNA integration in To plants was confirmed by Southern hybridization analysis. Inheritance analysis of the T₀ plants with single-copy T-DNA insertions revealed segregation of hygromycin resistance in 3:1 ratio. The feasibility of rice transformation with a cointegrate Ti plasmid vector is clearly established.