籼稻品种地谷抗稻瘟病基因的遗传

籼稻地谷是我国杂效裟育种上重要的稻瘟病抗源之一。利用我国稻区的稻瘟病菌系ＺＢ１３和ＺＢ１５对地谷与感病品种江南香糯的杂效Ｆ１、Ｆ２和Ｂ１Ｆ１群体,以及地谷与感病品种丽江新团黑谷、冈４６Ｂ和８９８７的Ｆ２群体进行接种鉴定,根据抗病性的分离确认地谷对ＺＢ１３和ＺＢ１５的抗性受显性基因控制。利用菌系ＺＢ１３接种地谷和１０个具有已知抗病基因的鉴别品种及其杂交Ｆ１和Ｆ２群体,进一步证明了地谷的抗温性受１对显     

XA21和PI-D2激酶蛋白质在酿酒酵母中的表达及其自我磷酸化研究

白叶枯病和稻瘟病是最主要的水稻病害。Xa21是水稻白叶枯病抗性基因,Pi-d2是稻瘟病抗性基因,二者都编码类受体激酶蛋白质。在前期研究中,曾系统地研究了细菌中表达XA21激酶蛋白质的生化活性。在此实验中利用真核表达系统酿酒酵母对Xa21和Pi-d2编码的蛋白激酶进行了表达、纯化及自我磷酸化活性分析,为进一步的生化分析、蛋白质-蛋白质相互作用研究、底物筛选等奠定了基础。     

水稻白叶枯病抗性基因Xα21和稻瘟病抗性基因Pi-d2激酶蛋白质在毕赤酵母中的表达及条件优化

[目的]白叶枯病和稻瘟病是最主要的水稻病害,Xα21是水稻白叶枯病抗性基因,Pi-d2是稻瘟病抗性基因,二者都编码类受体激酶蛋白质.本研究旨在毕赤酵母系统中表达XA21和PI-D2激酶蛋白质.[方法]用Xα21和Pi-d2的激酶区PCR产物,构建了pPICZαA-Xα21K、pPICZαA-Pi-d2K重组质粒,酶切及测序验证后,将重组质粒线性化,转化到毕赤酵母菌株中,系统地比较了不同酵母菌株(KM71、GS115、X33),不同甲醇浓度(1%、2%、3%),不同pH(pH5、pH6、pH7、pH8)值,不同诱导时间(24 h、48 h、72 h)条件下激酶蛋白质的表达情况.[结果]XA21和PI-D2激酶蛋白质可以在毕赤酵母中表达,但表达的蛋白质不能分泌到培养基上清中,而只能在菌体中检测到,对表达条件的系统比较发现,毕赤酵母菌株KM71和X33、2%的甲醇诱导浓度、pH5和48 h以上的诱导时间有利于激酶蛋白质的表达,最后我们在酵母裂解物上清中获得了纯化的考染可见的激酶蛋白质.[结论]在毕赤酵母中表达了XA21和PI-D2激酶蛋白质,为下一步生化特性研究奠定了基础.     

Identification of Two Blast Resistance Genes in a Rice Variety, Digu

Blast, caused by Magnaporthe grisea is one of most serious diseases of rice worldwide. A Chinese local rice variety, Digu, with durable blast resistance, is one of the important resources for rice breeding for resistance to blast (M. grisea) in China. The objectives of the current study were to assess the identity of the resistance genes in Digu and to determine the chromosomal location by molecular marker tagging. Two susceptible varieties to blast, Lijiangxintuanheigu (LTH) and Jiangnanxiangnuo (JNXN), a number of different varieties, each containing one blast resistance gene, Pik^s, Pia, Pik, Pi‐b, Pi‐k^p, Pi‐ta², Pi‐ta, Pi‐z, Pi‐i, Pi‐k^m, Pi‐z^t, Pi‐t and Pi‐11, and the progeny populations from the crosses between Digu and each of these varieties were analysed with Chinese blast isolates. We found that the resistance of Digu to each of the two Chinese blast isolates, ZB13 and ZB15, were controlled by two single dominant genes, separately. The two genes are different from the known blast resistance genes and, therefore, designated as Pi‐d(t)1 and Pi‐d(t)2. By using bulked segregation method and molecular marker analysis in corresponding F₂ populations, Pi‐d(t)1 was located on chromosome 2 with a distance of 1.2 and 10.6 cM to restriction fragment length polymorphism (RFLP) markers G1314A and G45, respectively. And Pi‐d(t)2 was located on chromosome 6 with a distance of 3.2 and 3.4 cM to simple sequence repeat markers RM527 and RM3, respectively. We also developed a novel strategy of resistance gene analogue (RGA) assay with uneven polymerase chain reaction (PCR) to further tag the two genes and successfully identified two RGA markers, SPO01 and SPO03, which were co‐segregated toPi‐d(t)1 and Pi‐d(t)2, respectively, in their corresponding F₂ populations. These results provide essential information for further utilization of the Digu's blast resistance genes in rice disease resistance breeding and positional cloning of these genes.     

Expression of the chimeric receptor between the chitin elicitor receptor CEBiP and the receptor-like protein kinase Pi-d2 leads to enhanced responses to the chitin elicitor and disease resistance against Magnaporthe oryzae in rice

We previously reported that rice plants expressing the chimeric receptor consisting of rice chitin oligosaccharides binding protein (CEBiP) and the intracellular protein kinase region of Xa21, which confers resistance to rice bacterial blight, showed enhanced cellular responses to a chitin elicitor N-acetylchitoheptaose and increased resistance to the rice blast fungus Magnaporthe oryzae. Here, we investigated whether CEBiP fused with another type of receptor-like protein kinase (RLK) also functions as a chimeric receptor. Fusion proteins CRPis consist of CEBiP and the intracellular protein kinase region of a true resistance gene Pi-d2. Transgenic rice expressing a CRPi showed enhanced cellular responses specifically to N-acetylchitoheptaose in cultured cells and increased levels of disease resistance against M. oryzae in plants. These responses depended on the amino acid sequences predicted to be essential for the protein kinase activity of CRPi. The structure of the transmembrane domain in CRPi affected the protein accumulation, cellular responses, and disease resistance in transgenic rice. These results suggest that the chimeric receptor consisting of CEBiP and Pi-d2 functions as a receptor for chitin oligosaccharides and CEBiP-based chimeric receptors fused with other RLKs may also act as functional receptors.     

水稻抗稻瘟病基因Pi-d（t）^1、Pi-b、Pi-tα^2的聚合及分子标记选择

冈46B(G46B)是水稻生产应用中的一个农艺性状十分优良的保持系,其主要的缺陷是稻瘟病抗性较弱,通过对地谷,BL-1,Pi-4号等三个分别含抗病基因Pi-d(t)^1、Pi-b、Pi-tα^2的稻瘟病抗性材料与G46B聚合杂交,并利用抗病基因连锁的分子标记对杂交后代进行辅助选择,在聚合杂交的F2代及B1C1代群体中共获得了15株含Pi-d(t)^1、Pi-b、Pi-tα^2等三个抗稻瘟病基因的材料,其可能的基因型分别为：三基因杂合体Pi-d(t)^1pi-d(t)^1,Pi-bpi-b／Pi-tα^2 pi-tα^2 4株,双基因杂合体10株,其中Pi-d(t)^1Pi-d(t)^1／Pi-bpi-b／Pi-tα^2pi-tα^2 6株,Pi-d(t)^1pi-d(t)^1／Pi-bpi-b／Pi-tα^2Pi-tα^2 3株,Pi-d(t)^1pi-d(t)^1,Pi-bPi-6,Pi-tα^2 pi-tα^2 1株,双基因纯合体Pi-d(t)^1Pi-d(t)^1/Pi-bpi-b/Pi-tα^2Pi-tα^2仅1株,这一研究结果为进一步改良G46B的稻瘟病抗性奠定了基础,同时这一研究结果表明利用分子标记可快速、有效地实现多个抗病基因的聚合,大大提高水稻抗病育种的效率。     

水稻广谱抗稻瘟病基因研究进展

稻瘟病是水稻生产中的最严重病害之一,由于稻瘟菌小种的高度变异性,垂直抗性基因难以持续控制稻瘟病的危害,因此,克隆和利用广谱持久抗瘟基因被认为是解决稻瘟病问题最经济有效的策略。本文从广谱抗源的筛选与利用,广谱抗瘟基因的定位、克隆与应用等方面对水稻广谱抗稻瘟病基因研究取得的进展进行了概述,并介绍了广谱抗性分子机理的最新研究进展。基于国内外稻瘟病抗性基因研究的现状及趋势,以及我国丰富的抗瘟水稻种质资源,克隆越来越多的广谱抗瘟基因具有重要的理论与应用价值。     

Transgenic Rice Plants Expressing the Antifungal AFP Protein from Aspergillus Giganteus Show Enhanced Resistance to the Rice Blast Fungus Magnaporthe Grisea

The Aspergillus giganteus antifungal protein (AFP), encoded by the afp gene, has been reported to possess in vitro antifungal activity against various economically important fungal pathogens, including the rice blast fungus Magnaporthe grisea. In this study, transgenic rice ( Oryza sativa ) constitutively expressing the afp gene was generated by Agrobacterium -mediated transformation. Two different DNA constructs containing either the afp cDNA sequence from Aspergillus or a chemically synthesized codon-optimized afp gene were introduced into rice plants. In both cases, the DNA region encoding the signal sequence from the tobacco AP24 gene was N-terminally fused to the coding sequence of the mature AFP protein. Transgenic rice plants showed stable integration and inheritance of the transgene. No effect on plant morphology was observed in the afp -expressing rice lines. The inhibitory activity of protein extracts prepared from leaves of afp plants on the in vitro growth of M. grisea indicated that the AFP protein produced by the trangenic rice plants was biologically active. Several of the T(2) homozygous afp lines were challenged with M. grisea in a detached leaf infection assay. Transformants exhibited resistance to rice blast at various levels. Altogether, the results presented here indicate that AFP can be functionally expressed in rice plants for protection against the rice blast fungus M. grisea.     

Molecular characterization and expression analysis of <Emphasis Type="Italic">OsBISERK1</Emphasis>, a gene encoding a leucine-rich repeat receptor-like kinase,during disease resistance responses in rice

A rice gene, OsBISERK1, encoding a protein belonging to SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) type of leucine-rich repeat receptor-like kinases (LRR-RLKs) was identified. The OsBISERK1 encodes a 624 aa protein with high level of identity to known plant SERKs. OsBISERK1 contains a hydrophobic signal peptide, a leucine zipper, and five leucine-rich repeat motifs in the extracellular domain; the cytoplasmic region carries a proline-rich region and a single transmembrane domain, as well as a conserved intracellular serine/threonine protein kinase domain. OsBISERK1 has a low level of basal expression in leaf tissue. However, expression of OsBISERK1 was induced by treatment with benzothiadiazole (BTH), which is capable of inducing disease resistance in rice, and also up-regulated after inoculation with Magnaporthe grisea in BTH-treated rice seedlings and during incompatible interaction between a blast-resistant rice genotype and M. grisea. The results suggest that OsBISERK1 may be involved in disease resistance responses in rice.