Huang LiLi

Cloning and Characterization of a Pathogenesis Related Protein Gene TaPR10 from Wheat Induced by Stripe Rust Pathogen

Pathogenesis-related proteins (PRs) play many important roles in plant defense response against pathogen attack. To better understand the molecular mechanism of PR genes involved in wheat adult plant resistance (APR) to stripe rust, based on a differentially expressed transcribed derived fragment (TDF), a novel PR gene from wheat cv. Xingzi 9104 infected by the Puccinia striiformis Westend f. sp. tritici Erikss. pathotype CY32, which was highly similar to the maize ZmPR10 gene and designated as TaPR10, was identified using in silico cloning and RT-PCR method. This novel TaPR10 gene was predicted to encode a 160-amino acid protein with a deduced molecular weight of 17.06 kDa and an isoelectronic point (pI) of 5.19. An amino acid sequence analysis of TaPR10 demonstrated the presence of a typical conserved domain of pathogenesis related protein Bet_v_I family. Multiple alignment analysis based on the amino acids encoded by 10 different PR10 genes from maize (Zea mays), rice (Oryza sativa), broomcorn (Sorghum bicolor), and wheat (Triticum aestivum) indicated that PR proteins of class 10 was conserved among the 4 plant species with about 80% similarity. DNA sequence of TaPR10 suggested the presence of one 84-bp intron with the splicing sites of GT-AT bi-nucleotide sequence between 188 and 271 bp. Using a real-time quantitative RT-PCR (qRT-PCR), expression profiles of TaPR10 revealed that at the adult-plant stage, TaPR10 transcript was up-regulated as early as 12 h post-inoculation (hpi), with the occurrence of maximum induction at 24 hpi. At the seedling stage, TaPR10 was also slightly induced 18 hpi. However, the transcript amount was relatively lower than that of the adult-plant stage. Taken together, these results suggest that TaPR10 may participate in wheat defense response of APR to stripe rust.

Cloning and Expression Analysis of a CBS Domain Containing Protein Gene TaCDCP1 from Wheat: Cloning and Expression Analysis of a CBS Domain Containing Protein Gene TaCDCP1 from Wheat

利用电子克隆和RT-PCR技术，在本实验室前期构建的非亲和互作的SSH文库基础上，从条锈菌侵染的小麦水原11叶片中首次分离出一个含CBS结构域蛋白的基因，暂命名为 Ta CDCP1 ( T riticum aestivum CBS domain containing protein 1)。 Ta CDCP 1 包含一个完整的654 bp的开放阅读框，编码217个氨基酸。推测编码的蛋白具有两个CBS保守结构域，不含跨膜区且无信号肽，定位在叶绿体基质内；经过同源比对，小麦TaCDCP1氨基酸序列与大麦、水稻和玉米等的同源序列的相似性较高；该基因表达量在小麦叶中显著高于在根和茎中；在小麦与条锈菌的非亲和、亲和组合中， Ta CDCP 1 基因均受到条锈菌诱导，分别在接种后18 h和96 h达到表达高峰，非亲和组合表达量在侵染前期(接种后18~48 h)高于亲和组合，而在侵染后期(接种后96~120 h)低于亲和组合；外源植物激素脱落酸诱导该基因上调表达，苄基腺嘌呤，乙烯，赤霉素，茉莉酸甲酯和水杨酸处理后其表达量在不同程度上受到抑制； Ta CDCP 1 在低温和干旱条件下表达量上升，在机械伤害和高盐处理下表达量无明显差异。表明 Ta CDCP 1 可能通过脱落酸等信号途径参与小麦对条锈菌的防御反应，同时参与低温和干旱环境下的信号转导途径。这些结果对于明确CBS结构域的功能以及CBS结构域蛋白尤其是 Ta CDCP 1 在小麦与条锈菌互作中的作用奠定了基础。

Cloning and expression analysis of a novel calmodulin isoform TaCaM5 from wheat.

Calmodulin is a ubiquitous transducer of calcium signals in eukaryotes.It mediates a lot of cellular processes,such as transcription,cytoskeletal organization and motility,and amino acid metabolism.In diploid plant species,several isoforms of calmodulin have been described.A novel CaM gene was isolated from cDNA library of wheat leaves infected by Puccinia striiformis f.sp.tritici through RT-PCR approach.The gene was tentatively designated as TaCaM5 and encoded a novel CaM isoform based on protein sequence analysis.This open reading frame of TaCaM5 was 450 bp in length and 149 amino acids were encoded with four conserved EF-hand domains.TaCaM5,in which transmembrane domain or signal peptide sequence was absent,was predicted existing in cytoplasm.The amino acid sequence of TaCaM5 shares 97% identify with ZmCaM from Zea mays.TaCaM5 expressed differently in wheat leaf,stem,and root.Challenged by stripe rust fungus(Puccinia striiformis f.sp.tritici),TaCaM5 was induced by this fungus in both incompatible and compatible interactions,with the maximal expression at 6 h and 24 h post inoculation respectively.TaCaM5 was up-regualted by exogenous abscisic acid,ethylene,and jasmonic acid but down-regulated by salicylic acid.TaCaM5 was obviously up-regulated by various abiotic stresses,such as low temperature,mechanical wound,and drought.However,high salinity stress could not induce the expression of TaCaM5.These results suggest that TaCaM5 is probably involved in regulating the host defence responses through ethylene and jasmonic acid pathways,and also participate in Ca2+-CaM signal transmission pathways under mechanical wound,low temperature,and drought conditions.