Cloning, characterization and expression analysis of the receptor-like kinase gene (RLK) in common wheat.

To investigate whether homologs of wheat (Triticum aestivum L.) LRK10 gene was expressed in powdery mildew-resistant lines after inoculation with Blumeria graminis f. sp. tritici, one degenerate primer for 5'-RACE was designed according to the 6th kinase subdomain of LRK10 and other plant kinases. 5'-RACE was performed with the template cDNA synthesized with RNA extracted from seedling leaves of a powdery mildew-resistant wheat line "99-2439" after inoculation with B. graminis. One 1551 bp cDNA fragment representing a protein kinase gene was obtained (S1125, GenBank accession number: AY584533). Subsequently, a 2255-bp full-length cDNA clone with a complete encoding region (open reading frame, ORF) was obtained by RACE. The clone encodes a polypeptide consisting of 637 amino acid residues. The result of homology search showed that it belongs to a receptor-like kinase gene family in wheat, which was named as wlrk (wheat leaf rust kinase) previously. Similar to LRK10, this protein kinase has five distinct domains: a hydrophobic signal sequence at the amino-terminus, a putative extracellular domain, a transmembrane domain, a highly charged sequence and a serine/threonine kinase domain at the carboxy-terminus, and thus it was named as TaLRK (Triticum aestivum LRK). The expression pattern of TaLRK at transcription level in leaves after B. graminis infection was investigated by semi-quantitative RT-PCR (semi-QRT-PCR), using wheat actin gene as a control. The result showed that the transcription of TaLRK was significantly enhanced by B. graminis infection. The expression pattern of TaLRK in different tissues showed that this new wheat RLK gene was expressed only in green parts of wheat. This study suggests that TaLRK may function in wheat powdery mildew resistance responses.

Niu JS ，Zhang LN ，Wang YH ，Hong DF ... -  《-》

Cloning, characterization and expression of wheat EDR1 (enhanced disease resistance) gene.

To investigate if there is an EDR1 pathway in wheat (Triticum aestivum L.), a pair of degenerate primers was designed according to the cDNAs of Arabidopsis thaliana EDR1 gene and its homologs were used to isolate EDR1 gene homologs from wheat. RT-PCR was conducted on the cDNA template synthesized with RNA of wheat leaves. A 627-bp cDNA fragment representing an EDR1 gene (named as TaEDR1) was isolated (GenBank accession number: AY743662). Subsequently, the 3050-bp full-length cDNA sequence of TaEDR1, which encodes a polypeptide consisting of 959 amino acid residues, was obtained by RACE technique. The amino acid sequence of TaEDR1 and that of barley (Hordeum vulgare) EDR1 (signed as HvEDR1) show 92% identity. There is a highly conserved catalytic domain of serine/threonine protein kinases in the C-terminus of TaEDR1. Because this protein has a putative nuclear localization motif, it probably functions in the nucleus. This study provides the first molecular biological evidence of the presence of an EDR1 homolog in common wheat. The transcription pattern of TaEDR1 was investigated in leaves after inoculation with Blumeria graminis (DC.) E.O. Speer f. sp. tritici Em. Marchal (Bgt) through semi-quantitative RT-PCR (semi-QRT-PCR). The result showed that the transcribing of TaEDR1 was enhanced by Bgt. The expression pattern of the TaEDR1 gene in different tissues showed that it expressed in leaves, stems, spikes and roots. This study suggests that the TaEDR1, a MAP kinase kinase kinase, may function in wheat defense responses.

Niu JS ，Zhang LN ，Hong DF ，Wang YH ... -  《-》

A novel homeobox-like gene associated with reaction to stripe rust and powdery mildew in common wheat.

Stripe rust and powdery mildew, caused by Puccinia striiformis f. sp. tritici and Blumeria graminis f. sp. tritici, respectively, are severe diseases in wheat (Triticum aestivum) worldwide. In our study, differential amplification of a 201-bp cDNA fragment was obtained in a cDNA-amplified fragment length polymorphism (AFLP) analysis between near-isogenic lines Yr10NIL and Avocet S, inoculated with P. striiformis f. sp. tritici race CYR29. A full-length cDNA (1,357 bp) of a homeobox-like gene, TaHLRG (GenBank accession no. EU385606), was obtained in common wheat based on the sequence of GenBank accession AW448633 with high similarity to the above fragment. The genomic DNA sequence (2,396 bp) of TaHLRG contains three exons and two introns. TaHLRG appeared to be a novel homeobox-like gene, encoding a protein with a predicted 66-amino-acid homeobox domain. It was involved in race-specific responses to stripe rust in real-time quantitative polymerase chain reaction (PCR) analyses with Yr9NIL, Yr10NIL, and Avocet S. It was also associated with adult-plant resistance to stripe rust and powdery mildew based on the field trials of doubled haploid lines derived from the cross Bainong 64/Jingshuang 16 and two F(2:3) populations from the crosses Lumai 21/Jingshuang 16 and Strampelli/Huixianhong. A functional marker, THR1 was developed based on the sequence of TaHLRG and located on chromosome 6A using a set of Chinese Spring nulli-tetrasomic lines.

Liu D ，Xia X- ，He Z- ，Xu S- ... -  《PHYTOPATHOLOGY》

Molecular cloning, characterization and mapping of a rhodanese like gene in wheat.

To isolate genes related to resistance to Erysiphe graminis (Blumeria graminis) DC. f. sp. tritici in wheat (Triticum aestivum L.), differential display analysis was conducted for mRNA extracted from seedlings of a wheat-Haynaldia villosa 6VS/6AL translocation line 92R137 that contains a powdery mildew resistance gene Pm21. A full-length cDNA sequence named TaTST (Triticum aestivum thiosulfate sulfurtransferase) homologous to the thiosulfate sulfurtransferase (rhodanese) in Datisca glomerata was isolated. Northern blot showed that the expression of TaTST was enhanced after infection with Erysiphe graminis. TaTST was mapped on the short arm of 6B chromosomes of wheat through Southern blot and GSP-PCR using Chinese Spring nullisomic/tetrasomic lines and ditelosomic lines. There is a homologue of TaTST on 6VS too.

Niu JS ，Yu L ，Ma ZQ ，Chen PD ，Liu DJ ... -  《-》

Cloning, sequence and expression analysis of a zinc finger protein gene in wheat.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was performed on total RNA of resistant-line TAM104R and susceptible-line TAM104S using 33 primers designed according to conservative domain of plant R-genes and the sequence of barley powdery mildew resistance genes Mlo, Mla1, Mla6 and wheat leaf rust resistance gene Lrk10. A polymorphic cDNA fragment TaZF was cloned and sequenced. The Open Reading Frame (ORF) of TaZF is comprised of 822 base pairs which encodes a zinc finger-like DNA or RNA-binding protein with 273 amino acids and molecular weight of 31 kD. TaZF is more than one copy gene in TAM104R genome based on southern blot. It is constitutively expressed, but level of expression was enhanced in tissue infected by Erysiphe Graminis. There is no intron in TaZF genome DNA.

Wan P ，Ling LJ ，Zhou WJ ，Zhang WJ ，Ling HQ ，Zhu LH ，Zhang XQ ... -  《-》