Phylogenetic relationship of a new class of LMW-GS genes in the M genome of Aegilops comosa

A new class of low molecular weight glutenin subunit (LMW-GS) genes was isolated and characterized from Aegilops comosa (2n?=?2x?=?14, MM). Although their DNA structure displayed high similarity to LMW-i type genes, there are some key differences. The deduced amino acid sequences of their mature proteins showed that the first amino acid residue of each gene was leucine and therefore they were designated as LMW-l type subunits. An extra cysteine residue was present in the signal peptide and the first cysteine residue of mature proteins located at the end of repetitive domain. Additionally, a long insertion of 10?C22 residues (LGQQPQ_5?C17) occurred in the end of the C-terminal II. Comparative analysis demonstrated that LMW-l type glutenin genes possessed a great number of single-nucleotide polymorphisms and insertions/deletions. A new classification system was proposed according to the gene structure and phylogenetic analysis. In this new system, LMW-GS is classified into two major classes, LMW-M and LMW-I, with each including two subclasses. The former included LMW-m and LMW-s types while the latter contained LMW-l and LMW-i types. Analysis of their evolutionary origin showed that the LMW-l genes diverged from the group 2 of LMW-m type genes at about 12?C14?million years ago (MYA) while LMW-i type evolved from LMW-l type at approximately 8?C12 MYA. The LMW-s type was a variant form of group 1 of LMW-m type and their divergence occurred about 4?C6 MYA. In addition to homologous recombination, non-homologous illegitimate recombination could be an important molecular mechanism for the origin and evolution of LMW-GS gene family. The secondary structure prediction suggested that the novel LMW-l type subunits, such as AcLMW-L1 and AcLMW-L2, may have positive effects on dough properties.     

Characterization of low-molecular-weight glutenin subunit genes and their protein products in common wheats

To characterize the low-molecular-weight glutenin subunit (LMW-GS), we developed specific PCR primer sets to distinguish 12 groups of LMW-GS genes of Norin 61 and to decide their loci with nullisomic–tetrasomic lines of Chinese Spring. Three, two, and ten groups were assigned to Glu-A3, Glu-B3, and Glu-D3 loci, respectively. To identify the proteins containing the corresponding amino acid sequences, we determined the N-terminal amino acid sequence of 12 spots of LMW-GSs of Norin 61 separated by two-dimensional gel electrophoresis (2DE). The N-terminal sequences of the LMW-GS spots showed that 10 of 12 groups of LMW-GSs were expressed as protein products, which included LMW-i, LMW-m, and LMW-s types. Four spots were encoded by Glu-A3 (LMW-i). Three spots were encoded by Glu-B3 (LMW-m and LMW-s). Five spots were encoded by Glu-D3 (LMW-m and LMW-s). A minor spot of LMW-m seemed to be encoded by the same Glu-B3 gene as a major spot of LMW-s, but processed at a different site. Comparing among various cultivars, there were polymorphic and non-polymorphic LMW-GSs. Glu-A3 was highly polymorphic, i.e., the a, b, and c alleles showed one spot, the d allele showed four spots, and the e allele had no spot. Insignia used as one of the Glu-A3 null standard cultivars had a LMW-GS encoded by Glu-A3. We also found that Cheyenne had a new Glu-D3 allele. Classification of LMW-GS by a combination of PCR and 2DE will be useful to identify individual LMW-GSs and to study their contribution to flour quality.     

Phylogenetic analysis of C, M, N, and U genomes and their relationships with Triticum and other related genomes as revealed by LMW-GS genes at Glu-3 loci

Phylogenetic relationships between the C, U, N, and M genomes of Aegilops species and the genomes of common wheat and other related species were investigated by using three types of low-molecular-weight glutenin subunit (LMW-GS) genes at Glu-3 loci. A total of 20 LMW-GS genes from Aegilops and Triticum species were isolated, including 11 LMW-m type and 9 LMW-i type genes. Particularly, four LMW-m type and three LMW-i type subunits encoded by the genes on the C, N, and U genomes possessed an extra cysteine residue at conserved positions, which could provide useful information for understanding phylogenetic relationships among Aegilops and Triticum genomes. Phylogenetic trees constructed by using either LMW-i or the combination of LMW-m and LMW-s, as well as analysis of all the three types of LMW-GS genes together, demonstrated that the C and U genomes were closely related to the A genome, whereas the N and M genomes were closely related to the D genome. Our results support previous findings that the A genome was derived from Triticum uratu, the B genome was from Aegilops speltoides, and the D genome was from Aegilops tauschii. In addition, phylogenetic relationships among different genomes analysed in this study support the concept that Aegilops is not monophyletic.     

LMW-GS genes in Agropyron elongatum and their potential value in wheat breeding

To study the usefulness of low-molecular-weight glutenin subunits (LMW-GS) of Agropyron elongatum (Host) Nevski to wheat (Triticum aestivum L.) quality improvement, we characterized LMW-GS genes of A. elongatum. Nine LMW-GS genes of A. elongatum, which were named AeL1 to AeL9, were cloned by genomic PCR. After sequencing, we obtained complete open reading frames from AeL2 to AeL8 and partial genes of AeL1 and AeL9. All nine sequences are homoeologous to those of wheat and related grasses. Comparison of the deduced amino acid sequences with those of published LMW-GS suggests that the basic structures of all the subunits are very similar. However, except for AeL4 and AeL5, which contain the identical N-terminal sequence with LMW-m, other LMW-GS sequences separated from A. elongatum cannot be classified according to previous criteria for the three types: LMW-m (methionine), LMW-s (serine), and LMW-i (isoleucine), and then 12 groups. In addition, there are some characters in the LMW-GS sequences of A. elongatum: AeL2, AeL3, and AeL6 involve a Cys residue in the signal peptide respectively, which is absent in most of LMW-GS; AeL3, AeL6, AeL8, and AeL9 start their first Cys residues in the N-terminal repetitive domains, respectively; both AeL2 and AeL5 have nine Cys residues, with an extra Cys residue in the N-terminal repetitive domain and the repetitive and glutamine-rich domain; AeL2, AeL3, AeL6, and AeL9 comprise long repetitive domains. Phylogenetic analysis indicates that there is a relatively weak sequence identity between the LMW-GS genes from A. elongatum cloned in this study and those reported from other plants. Three LMW-GS sequences, AeL2, AeL3, and AeL6, are clustered to Glu-A3 from wheat than to those from other plants. The possible use of these genes in relation to the high quality of hybrid wheat is discussed.     

Characterization of low-molecular-weight glutenin subunit genes from Hordeum brevisubulatum ssp. turkestanicum

Three novel low-molecular-weight glutenin subunit (LMW-GS) genes (designated as Ht1, Ht2, and Ht3) were isolated from the genomic DNA of Hordeum brevisubulatum ssp. turkestanicum by PCR amplification (accession no. Y0695). The coding regions of Ht1, Ht2, and Ht3 were 924, 924, and 903 bp, respectively. The deduced amino acid sequences were 306, 306, and 299 amino acid residues each with a signal peptide, a central repetitive region rich in proline and glutamine, and N-and C-terminal non-repetitive domains. A comparison was carried out of these genes with other known B hordein genes from cultivated barley and LMW glutenin genes from wheat. The results indicated that Ht1, Ht2, and Ht3 had a more similar structure and a higher level of homology with the LMW-GS genes than the B hordein genes. In order to investigate the evolutionary relationship of the novel genes with the prolamin genes from barley and wheat, the phylogenetic tree was constructed and the subfamilies of these prolamin genes were identified. The results suggested that the three novel genes were glutenin-like proteins designated as LMW-m type genes. The text was submitted by the authors in English.     

A novel chimeric low-molecular-weight glutenin subunit gene from the wild relatives of wheat Aegilops kotschyi and Ae. juvenalis: evolution at the Glu-3 loci

Four LMW-m and one novel chimeric (between LMW-i and LMW-m types) low-molecular-weight glutenin subunit (LMW-GS) genes from Aegilops neglecta (UUMM), Ae. kotschyi (UUSS), and Ae. juvenalis (DDMMUU) were isolated and characterized. Sequence structures showed that the 4 LMW-m-type genes, assigned to the M genome of Ae. neglecta, displayed a high homology with those from hexaploid common wheat. The novel chimeric gene, designed as AjkLMW-i, was isolated from both Ae. kotschyi and Ae. juvenalis and shown to be located on the U genome. Phylogentic analysis demonstrated that it had higher identity to the LMW-m-type than the LMW-i-type genes. A total of 20 single nucleotide polymorphisms (SNPs) were detected among the 4 LMW-m genes, with 13 of these being nonsynonymous SNPs that resulted in amino acid substitutions in the deduced mature proteins. Phylogenetic analysis demonstrated that it had higher identity to the LMW-m-type than the LMW-i-type genes. The divergence time estimation showed that the M and D genomes were closely related and diverged at 5.42 million years ago (MYA) while the differentiation between the U and A genomes was 6.82 MYA. We propose that, in addition to homologous recombination, an illegitimate recombination event on the U genome may have occurred 6.38 MYA and resulted in the generation of the chimeric gene AjkLMW-i, which may be an important genetic mechanism for the origin and evolution of LMW-GS Glu-3 alleles as well as other prolamin genes.     

Cloning and molecular characterization of three novel LMW-i glutenin subunit genes from cultivated einkorn (Triticum monococcum L.)

Three novel low molecular weight (LMW) glutenin subunits from cultivated einkorn (Triticum monococcum L., A^mA^m, 2n = 2x = 14) were characterized by SDS-PAGE and molecular weights determined by MALDI-TOF-MS. Their coding genes were amplified and cloned with designed AS-PCR primers, revealing three complete gene sequences. All comprised upstream, open reading frame (ORF), downstream and no introns were present. The deduced amino acid sequences showed that all three genes, named as LMW-M1, LMW-M3 and LMW-M5, respectively, belonged to the LMW-i type subunits with the predicted molecular weight between 38.5206 and 38.7028 kDa. They showed high similarity with other LMW-i type genes from hexaploid bread wheats, but also displayed unique features. Particularly, LMW-M5 subunit contained an extra cysteine residue in the C-terminus except for eight conserved cysteines, which resulted from a single-nucleotide polymorphism (SNP) of the T–C transition, namely arginine → cysteine substitution at position 242 from the N-terminal end. This is the first report that the LMW-i subunit contained nine cysteines residues that could result in a more highly cross-linked and more elastic glutenin suggesting that LMW-M5 gene may associates with good quality properties. In addition, a total of 25 SNPs and one insertions/deletions (InDels) were detected among three LMW-i genes, which could result in significant functional changes in polymer formation of gluten. It is anticipated that these SNPs could be used as reliable genetic markers during wheat quality improvement. The phylogenetic analysis indicated that LMW-i type genes apparently differed from LMW-m and LMW-s type genes and diverged early from the primitive LMW-GS gene family, at about 12.92 million years ago (MYA) while the differentiation of A^m and A genomes was estimated at 3.98 MYA.Q. Zhang had the same contribution to this work as the first author.     

糜子抗旱节水相关基因PmMYB的克隆及表达分析

根据在糜子抗旱节水分子基础研究中获得的一个糜子MYB基因的EST序列, 以其序列及水稻MYB18基因的序列为基础设计引物, 扩增得到1 739 bp的全长基因组序列。序列分析表明, 其包含121 bp(347~467 bp)和93 bp(599~691 bp)的两个内含子, 3个外显子; 全长cDNA序列为1 525 bp, 其中3′非翻译区为212 bp, 5′非翻译区为41 bp, 编码区为1 272 bp, 共编码424个氨基酸, C-端存在一个丝氨酸(Ser, S)丰富区。该基因具有两个典型的MYB类转录因子基因的DNA结合区(DNA-binding domain), 分别为13~63、66~114位氨基酸, 属于典型的R2R3-MYB转录因子。对其与水稻、玉米、火炬松、拟南芥、辣椒、陆地棉、大麦及茄子等9种植物的MYB基因的R2、R3重复区的氨基酸序列多重比较, 表明R2R3重复序列在植物中具有较高的保守性; 基于氨基酸序列的编码区系统进化树分析表明, 不同植物的MYB基因遗传分化很大, 序列相似性为32%~84%, 其中糜子MYB基因与水稻的MYB18相似程度最高(84%), 与大麦和玉米的相似性分别为46%和41%。通过半定量RT-PCR对其表达模式分析表明, 该基因在水分胁迫和干旱后复水条件下上调表达, 与糜子抗旱节水紧密相关。该基因的克隆为进一步探讨利用该基因改良其他植物的抗旱节水性奠定了良好的基础。     

小麦PAL基因的克隆及赤霉菌诱导下的表达分析

利用苯丙氨酸解氨酶(PAL,phenylalanine ammonia-lyase)基因保守区域从小麦抗赤霉病材料苏麦3号中克隆获得4个PAL基因,分别命名为Ta PAL1、Ta PAL2、Ta PAL3、Ta PAL4。4个基因的开放阅读框(ORF,open reading frame)长度分别为2142 bp、2016 bp、2118 bp和2139 bp,分别编码714个、672个、706个和713个氨基酸。基因序列比对发现其相似性达到88.35%,所编码的氨基酸相似性为91.92%,氨基酸序列分析表明4个基因都包含HAL-PAL结构域及PAL结构域。通过接种禾谷镰刀菌,利用荧光定量PCR对PAL基因进行表达分析发现,4个PAL基因全部为上调表达,其中Ta PAL2、Ta PAL3和Ta PAL4最为明显。PAL基因的上调表达,说明PAL基因在小麦抵抗赤霉病菌侵染的机制中可能起着重要作用。     

Identification and molecular characterization of a novel y-type Glu-Dt 1 glutenin gene of Aegilops tauschii

A novel y-type high-molecular-weight glutenin subunit possessing a slightly faster mobility than that of subunit 1Dy12 in SDS-PAGE, designated 1Dy12.1^t in Aegilops tauschi, was identified by one- and two-dimensional gel and capillary electrophoresis. Its coding gene at the Glu-D ^t 1 locus was amplified with allele-specific-PCR primers, and the amplified products were cloned and sequenced. The complete nucleotide sequence of 2,807 bp containing an open reading frame of 1,950 bp and 857 bp of upstream sequence was obtained. A perfectly conserved enhancer sequence and the –300 element were present at positions of 209–246 bp and 424–447 bp upstream of the ATG start codon, respectively. The deduced mature protein of 1 Dy12.1^t subunit comprised 648 amino acid residues and had a Mr of 67,518 Da, which is slightly smaller than the 1Dy12 (68,695 Da) but larger than the 1Dy10 (67,495 Da) subunits of bread wheat, respectively, and corresponds well with their relative mobilities when separated by acid-PAGE. The deduced amino acid sequence indicated that the 1Dy12.1^t subunit displayed a greater similarity to the 1Dy10 subunit, with only seven amino acid substitutions, suggesting that this novel gene could have positive effect on bread-making quality. A phenetic tree produced by nucleotide sequences showed that the x- and y-type subunit genes were respectively clustered together and that the Glu-D ^t 1y12.1 gene of Ae. tauschii is closely related to other y-type subunit genes from the B and D genomes of hexaploid bread wheat.Communicated by H.F. Linskens     

The exon organization of the triple-helical coding regions of the human alpha 1(VI) and alpha 2(VI) collagen genes is highly similar.

The alpha 1(VI) and alpha 2(VI) chains, two of the three constituent chains of type VI collagen, are highly similar in size and domain structure. They are encoded by single-copy genes residing in close proximity on human chromosome 21. To study the evolution of the type VI collagen genes, we have isolated and characterized genomic clones coding for the triple-helical domains of the human alpha 1(VI) and alpha 2(VI) chains, which consist of 336 and 335 amino acid residues, respectively. Nucleotide sequencing indicates that, in both genes, the exons are multiples of 9 bp in length (including 27, 36, 45, 54, 63, and 90 bp) except for those encoding for regions with triple-helical interruptions. In addition, the introns are positioned between complete codons. The most predominant exon size is 63 bp, instead of 54 bp as seen in the fibrillar collagen genes. Of particular interest is the finding that the exon structures of the alpha 1(VI) and alpha 2(VI) genes are almost identical. A significant deviation is that a segment of 30 amino acid residues is encoded by two exons of 54 and 36 bp in the alpha 1(VI) gene, but by a single exon of 90 bp in the alpha 2(VI) gene. The exon arrangement therefore provides further evidence that the two genes have evolved from tandem gene duplication. Furthermore, comparison with the previously reported gene structure of the chick alpha 2(VI) chain indicates that the exon structure for the triple-helical domain of the alpha 2(VI) collagen is strictly conserved between human and chicken.     

Cloning and characterization of a non-TIR-NBS-LRR type disease resistance gene analogue from peach.

Cloning of plant disease resistant genes is greatly helpful for disease resistant breeding in plants and the insight of resistance mechanism. However, there are less relevant researches in peach [prunus persica (L.) Batch]. In this study, four NBS-LRR type resistance gene analogs (RGAs) were cloned from genomic DNA of peach. The PNBS2 fragment was also amplified from peach cDNA and the full-length cDNA of PNBS2 (PRPM1, GenBank accession no. AY599223) has been cloned. Sequence analysis indicated that the cDNA of PRPM1 is 3007 bp in length and that the contained ORF encodes for a polypeptide of 917 amino acids. Compared with known NBS-LRR genes, it presented relatively high amino acid sequence identity. The polypeptide has typical structure of non-TIR-NBS-LRR genes, with NB-ARC, LZ, LRR and transmembrane domains. Southern analysis indicated that the PRPM1 gene might be a single copy in peach genome. Northern blot and RT-PCR analysis showed that the expression of PRPM1 was not induced by salicylic acid (SA) in peach young leaves. The isolation of putative resistance genes from peach provided useful bases for studying the structure and function of peach disease-resistance relating genes and disease resistant genetic breeding in peach.     

Identification of LMW glutenin-like genes from Secale sylvestre host

Three low-molecular-weight (LMW) glutenin-like genes (designated as Ssy1, Ssy2 and Ssy3) from Secale sylvestre Host were isolated and characterized. The three genes consist of a predicted highly conservative signal peptide with 20 amino acids, a short N-terminal region with 13 amino acids, a highly variable repetitive domain and a less variable C-terminal domain. The deduced amino acid sequences of the three genes were the LMW-m type due to a methionine residue at the N-terminus. The phylogenic analysis indicated that the prolamin genes could be perfectly clustered into five groups, including HMW-GS, LMW-GS, alpha/beta-, gamma- and omega-prolamin. The LMW glutenin-like genes of S. sylvestre were more orthologous with the LMW-GS genes of wheat and B hordein genes of barley, which also had been confirmed by the homology analysis with the LMW-GS of wheat at Glu-A3, Glu-B3 and Glu-D3 loci. These results indicated that a chromosome locus (designated as Glu-R3) might be located on the R genome of S. sylvestre with the functions similar to the Glu-3 locus in wheat and its related species.     

Cloning, nucleotide sequence, and expression of the HincII restriction-modification system.

Two genes, coding for the HincII from Haemophilus influenzae Rc restriction-modification system, were cloned and expressed in Escherichia coli RR1. Their DNA sequences were determined. The HincII methylase (M.HincII) gene was 1,506 base pairs (bp) long, corresponding to a protein of 502 amino acid residues (Mr = 55,330). The HincII endonuclease (R.HincII) gene was 774 bp long, corresponding to a protein of 258 amino acid residues (Mr = 28,490). The amino acid residues predicted from the R.HincII and the N-terminal amino acid sequence of the enzyme found by analysis were identical. These methylase and endonuclease genes overlapped by 1 bp on the H. influenzae Rc chromosomal DNA. The clone, named E. coli RR1-Hinc, overproduced R.HincII. The R.HincII activity of this clone was 1,000-fold that from H. influenzae Rc. The amino acid sequence of M.HincII was compared with the sequences of four other adenine-specific type II methylases. Important homology was found between tne M.HincII and these other methylases.     

ChAy/Bx, a novel chimeric high-molecular-weight glutenin subunit gene apparently created by homoeologous recombination in Triticum turgidum ssp. dicoccoides

High-molecular-weight glutenin subunits (HMW-GSs) are of considerable interest, because they play a crucial role in determining dough viscoelastic properties and end-use quality of wheat flour. In this paper, ChAy/Bx, a novel chimeric HMW-GS gene from Triticum turgidum ssp. dicoccoides (AABB, 2n = 4x = 28) accession D129, was isolated and characterized. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that the electrophoretic mobility of the glutenin subunit encoded by ChAy/Bx was slightly faster than that of 1Dy12. The complete ORF of ChAy/Bx contained 1671 bp encoding a deduced polypeptide of 555 amino acid residues (or 534 amino acid residues for the mature protein), making it the smallest HMW-GS gene known from Triticum species. Sequence analysis showed that ChAy/Bx was neither a conventional x-type nor a conventional y-type subunit gene, but a novel chimeric gene. Its first 1305 nt sequence was highly homologous with the corresponding sequence of 1Ay type genes, while its final 366 nt sequence was highly homologous with the corresponding sequence of 1Bx type genes. The mature ChAy/Bx protein consisted of the N-terminus of 1Ay type subunit (the first 414 amino acid residues) and the C-terminus of 1Bx type subunit (the final 120 amino acid residues). Secondary structure prediction showed that ChAy/Bx contained some domains of 1Ay subunit and some domains of 1Bx subunit. The special structure of this HMW glutenin chimera ChAy/Bx subunit might have unique effects on the end-use quality of wheat flour. Here we propose that homoeologous recombination might be a novel pathway for allelic variation or molecular evolution of HMW-GSs.     

Isolation and molecular characterization of high molecular weight glutenin subunit genes 1Bx13 and 1By16 from hexaploid wheat

The high molecular weight glutenin subunit （HMW-GS） pair 1Bx13＋1By16 are recognized to positively correlate with bread-making quality; however, their molecular data remain unknown. In order to reveal the mechanism by which 1By16 and 1Bx13 creates high quality, their open reading frames （ORFs） were amplified from common wheat Atlas66 and Jimai 20 using primers that were designed based on published sequences of HMW glutenin genes. The ORF of 1By16 was 2220bp, deduced into 738 amino acid residues with seven cysteines including 59 hexapeptides and 22 nanopeptides motifs. The ORF of 1Bx13 was 2385bp, deduced into 795 amino acid residues with four cysteines including 68 hexapeptides, 25 nanopeptides and six tripepUdes motifs. We found that 1By16 was the largest y-type HMW glutenin gene described to date in common wheat. The 1By16 had 36 amino acid residues inserted in the central repetitive domain compared with 1By15. Expression in bacteria and western-blot tests confirmed that the sequence cloned was the ORF of HMW-GS 1By16, and that 1Bx13 was one of the largest 1Bx genes that have been described so far in common wheat, exhibiting a hexapeptide （PGQGQQ） insertion in the end of central repetitive domain compared with 1Bx7. A phylogenetic tree based on the deduced full-length amino acid sequence alignment of the published HMW-GS genes showed that the 1By16 was clustered with Glu-1B-2, and that the 1Bx13 was clustered with Glu-1B-1 alleles.     

鲤鱼sGnRH基因克隆及其在成熟个体的表达分析

采用RACE方法,从鲤鱼脑组织克隆了两个差异的sGnRH(salmon GnRH[Trp^7Leu^8]GnRH)cDNAs,即cDNA1和cDNA2,其长度分别为393和478bp。两个cDNAs都包括一个285bp开放阅读框,编码的sGnRH前体为94个氨基酸残基,由一个信号肽、sGnRH十肽和一个由蛋白水解位点(Gly-Lys-Arg)连接的促性腺激素释放激素相关肽共3部分组成。用内含子捕获得到相应的两个差异sGnRH基因,即sGnRH genel和gene2,其基本结构都包括4个外显子和3个内含子,3个内含子的核苷酸相似性分别为71．1％、76．1％和88．0％。鲤鱼sGnRH cDNAs及基因的基本结构和编码特点与已报道的不同形式GnRH cDNAs和GnRH基因相似,由此推测所有类型的GnRH可能来自一个共同的祖分子。Southern杂交进一步证实鲤鱼基因组存在两个不同的sGnRH基因座位。相对定量RT-PCR检测发现,两个sGnRH基因除在精巢的表达存在差异外,在脑区、垂体和成熟卵巢共表达。其中两个sGnRH基因在端脑和下丘脑的表达水平明显高于后脑区。根据sGnRH mRNAs在多个脑区、性腺和垂体的共存推测,sGnRH可能对鲤鱼下丘脑-垂体-性腺轴的调节有至关重要作用,同时可能起神经调节剂或自分泌和旁分泌调节因子的作用。