磷化氢熏蒸对米象抗性相关基因表达的影响

[目的]考察磷化氢熏蒸处理后米象抗性相关基因的表达量情况,为基因功能研究提供参考。[方法]对米象成虫进行磷化氢熏蒸后,使用荧光定量PCR对谷胱甘肽硫转移酶基因家族、乙酰胆碱酯酶基因等10个特异性靶标酶基因的表达变化进行分析。[结果]磷化氢熏蒸处理后,米象的谷胱甘肽硫转移酶相关基因的表达量总体呈上升趋势;抗性相关靶标基因的表达量总体呈现下降的趋势。[结论]谷胱甘肽硫转移酶等基因均与抗性相关,研究可为今后明确米象抗性分子机制提供参考。     

Detection and Analysis of Magnaporthe oryzae Avirulence Genes AVR-Pib,AVR-Pik and AvrPiz-t in Heilongjiang Province

【Objective】The objective of this study is to investigate the distribution and variation mechanism of avirulence genes AVR-Pib, AVR-Pik and AvrPiz-t in Magnaporthe oryzae strains from different regions and years in Heilongjiang Province, to understand the pathogenic phenotypes of different avirulence gene alleles, and to provide a reference for utilization and distribution of resistance cultivars in Heilongjiang Province.【Method】Based on the avirulence gene sequences published in NCBI, specific primers were designed to amplify full length and the coding sequence (CDS) regions of three genes, respectively. From 2016 to 2017, 335 M. oryzae strains in different regions of Heilongjiang Province were collected and isolated, and their DNA was PCR-amplified using avirulence genes primers and analyzed by agarose gel electrophoresis. The PCR products with different band patterns and from representative strains of different regions were selected for sequencing. The sequencing results were compared with the corresponding avirulence gene sequences for base and amino acid. The pathogenic phenotype of M. oryzae strains with different variant types was determined based on the rice resistance to single-gene lines.【Result】The specific bands of AVR-Pib, AVR-Pik and AvrPiz-t were detected in PCR detection and appeared in different distribution frequencies and mutation types, indicating that these 3 avirulence genes were all distributed in Heilongjiang Province. The average amplification frequency of the 3 avirulence genes was 75.52%, 87.16% and 85.67%, respectively. Among them, 4 types of band (bandless, high band, mid to high band and low band) of AVR-Pib were detected by electrophoresis analysis and 5 variant types AVR-Pib (1-1, 1-2, 2, 3, 3-1) were detected by PCR product sequencing. The genotypes AVR-Pib-1-1, AVR-Pib-1-2, AVR-Pib-2 and AVR-Pib-3-1 are newly discovered variant types, of which genotypes AVR-Pib-1-1 and AVR-Pib-1-2 are insertions of transposon Pot2 but with different insertion sites. The genotype AVR-Pib-2 has a small fragment insertion in the upstream of CDS region. The genotype AVR-Pib-3-1 base sequence has 4 differences from the original sequence, namely 32 (C/G) 35 (T/A) 36 (T/A) 38 (T/A), and the amino acid translation was terminated prematurely. Pathogenic analysis showed that except for the normal genotype AVR-Pib-3, the other alleles lost their avirulence functions. Seven AVR-Pik alleles (D, A, B, C, E, F, F2) were detected after PCR product sequencing, and the alterations in the nucleotide sequences of these alleles all resulted in amino acid missense mutations. The 7 AVR-Pik alleles have been reported previously. The avirulence gene AvrPiz-t was analyzed by electrophoresis and sequencing of PCR products, and 2 types of band (high band and normal band type) and 4 genotypes of AvrPiz-t (A, B, C, D) were revealed. Among them, AvrPiz-t-A is the original genotype, while AvrPiz-t-B has a base A insertion at position 191, causing premature termination of amino acid translation. Genotype AvrPiz-t-C is a newly discovered allelic type, characterized by the presence of a nucleotide variation at position 17 (T/C) and the insertion of base C at position 19 compared with type A, leading to the frameshift mutation and premature translation termination. The high band type avirulent genotype AvrPiz-t-D was sequenced and verified as having an insertion of the Pot3 transposon. Rice single-gene lines infection showed that the strains with AvrPiz-t-A were avirulent to Piz-t line due to Piz-t recognition, whereas the strains with AvrPiz-t (B, C, D) were virulent to Piz-t line due to lost the ability recognized by Piz-t.【Conclusion】The avirulence genes AVR-Pib, AVR-Pik and AvrPiz-t of M. oryzae in Heilongjiang Province are widely distributed and the types of variation are abundant. The results of this study can provide a reference for breeding and popularizing rice cultivars with corresponding disease-resistance genes.     

MoHRD3基因参与调控稻瘟病菌的生长发育和致病力

 稻瘟病菌(Magnaporthe oryzae)作为主要的农业病原微生物,其引起的稻瘟病严重威胁着水稻等谷类作物的生产安全。内质网相关蛋白质降解途径(Endoplasmic reticulum-associated protein degradation, ERAD)是生物体应答内质网压力的主要方式之一,其在机体生长发育过程中具有重要作用。而HRD(HMG-CoA reductase degradation)复合物作为ERAD的关键组分,主要由Hrd1、Hrd3、以及凝集素Yos9等蛋白组成,负责内质网中错误折叠蛋白的识别、转运以及泛素化过程,最终由蛋白酶体降解,从而有效缓解内质网压力,保证细胞的正常生理活动。有研究表明,Hrd3属于单次跨膜蛋白,在内质网腔中与Hrd1、Yos9相结合,负责底物的识别并起着稳定Hrd1的作用。目前Hrd3在稻瘟病菌中的生物学功能尚不清楚。本研究通过基因敲除及互补试验获得了稻瘟病菌的ΔMohrd3突变体和ΔMohrd3-C回补菌株,并以野生型为对照,对突变体的生物学表型进行了分析。结果显示,ΔMohrd3突变体的生长速率、产孢量明显下降;对大麦和水稻的致病力显著减弱。进一步胁迫试验表明,MoHRD3的缺失导致稻瘟病菌对外界盐胁迫、渗透压胁迫的耐受性增强,对内质网胁迫耐受性减弱,而对细胞壁胁迫无明显变化。同时,MoHRD3基因的缺失激活了未折叠蛋白响应途径(Unfolded protein response, UPR)。上述结果表明,MoHRD3参与调控稻瘟病菌的营养生长、无性繁殖、致病及对不同环境胁迫的响应过程。     

4种微量元素对水稻白叶枯病菌生长的影响

设计０、０．００１、０．０１、０．１、１、５、１０ｍｍｏｌ／Ｌ７种浓度,研究了Ｃｕ、Ｆｅ、Ｚｎ、Ｂ４种微量元素对水稻白叶枯病（Ｘａｎｔｈｏｍｏｎａｓｏ－ｒｙｚａｅｐｖ．ｏｒｙｘａｅ,简称（Ｘｏｏ）生长的影响,结果表明：不同微量元素在不同浓度对Ｘｏｏ生长具有不同的影响效果。Ｃｕ抑制效果最强,在０．１ｍｍｏｌ／Ｌ时即有较强的抑制作用。５ｍｍｏｌ／Ｌ时完全抑制Ｘｏ的生长;其次为Ｆｅ、在０．１和１ｍｍｏｌ／Ｌ时,只对Ｘｏ的生长后期有抑制作用,对生长初期有些刺激作用,但到５ｍｍｏｌ／Ｌ时则有很好的抑制效果;再次为Ｚｎ,在１和５ｍｍｏｌ／Ｌ时,只对Ｘｏ的生长初期略有抑制作用,对生长后期有些刺激作用,高达１０ｍｍｏｌ／Ｌ时才表现明显的抑制作用;Ｂ在低浓度时对Ｘｏ的生长无影响,而在５和１０ｍｍｏｌ／Ｌ时则表现为刺激作用。     

武粳4号抗稻瘟病基因分析

将太湖稻区高抗稻瘟病的主栽品种武粳４号与感病品种丽江新团黑谷和苏御糯杂交并回交,获得Ｆ１,Ｆ２,ＢＣ和部分Ｆ３世代,３－４叶期采用３个有代表性的稻瘟病菌生理小种ＺＧ,ＺＥ３和ＺＡ４９对不同世代材料进行接种鉴定,根据抗,感分离情况分析抗病亲本的抗病基因组成。结果表明,武粳４号对ＺＧ１,ＺＥ３和ＺＡ４９３个生理小种的抗性分别由一对显性基因控制。     

Effect of infection by Rhizopus oryzae on biochemical composition of stored potato tubers

The carbohydrate fractions, protein and lipid contents of two cultivars of potato namely, Irish Cobbler and Red Pontiac, were altered quantitatively by Rhizopus oryzae during the 10-day incubation period. Glucose content increased during the incubation period for both healthy and inoculated tubers. Starch, maltose, sucrose, protein and lipid contents decreased more rapidly in inoculated tubers than in healthy tubers in both cultivars. The depletion in starch and protein in the infected tubers appeared to be greater for Irish Cobbler than in Red Pontiac; decreases for other constituents seemed fairly comparable.     

晚稻细菌性条斑病流行趋势预测模型的研究

本文在分析万安县１９８９～１９９４年晚稻细菌性条斑病发生的历史资料和相关气象资料的基础上,应用灰色关联度选择预报因子,根据模糊综合评判原理组建模型,预报晚稻细菌性条斑病的流行趋势,经回测符合率达１００％,并在１９９５年、１９９６年和１９９７年应用该模型指导万安县晚稻细菌性条斑病的防治,取得了显著的经济效益。     

稻瘟病菌的拮抗细菌筛选

自稻叶及其它禾本科杂草叶片表面分离纯化９８５株细菌分离物,分别培养在ＮＢ液体培养基中,经８０００ｒ／ｍｉｎ离心１０ｍｉｎ后取上清液,加入稻瘟病菌分生孢子悬液中,测定对稻瘟病菌分生孢子发芽,附着胞形成的抑制作用,从而筛选出１３株对稻瘟病菌孢子芽管生长或附着胞形成具有抑制作用的拮抗细菌．采用离体稻叶接种测定,进一步筛选得到３株拮抗细菌,初步鉴定为：Ｐｓｅｕｄｏｍｏｎａｓｆｌｕｏｒｅｓｃｅｎｓ（Ｘｈ２１６）,Ｂａｃｉｌｕｓｓｐ．（Ｘｈ２２２）及Ｐｓｅｕｄｏｍｏｎａｓｓｐ．（Ｘｈ２４０）．在离体条件下,Ｘｈ２１６,Ｘｈ２２２和Ｘｈ２４０对叶瘟的防治效果分别为７２．３％,７０．５％和６７．４％．     

稻瘟菌诱导的稻叶脂氧合酶的纯化及部分氨基酸序列测定

 作者对已报道的稻瘟菌诱导稻叶脂氧合酶CM-LOX 1和CM-LOX 2的纯化方法作了进一步改进和完善。改进后的纯化方法可缩短该脂氧合酶的纯化时间,并提高纯化倍数。利用改进的方法,作者从20 g非亲和性稻瘟菌小种接种稻叶中获得了电泳纯的酶蛋白CM-L OX 1 133 μ g和CM-LOX 2 208μg。在此基础上,采用特异性的蛋白酶endoproteinase Lys-C和溴化氰对该酶蛋白进行了裂解,回收其肽段,得到了CM-LOX 1的3个不同肽段共30个氨基酸和CM-L OX 2的7个不同肽段共87个氨基酸的序列,为最终克隆这2个酶的基因奠定了基础。