OsDCL1基因沉默突变体诱导稻瘟病抗性的转录组分析

稻瘟病是严重危害水稻生长的真菌病害,每年给水稻生产带来重大经济损失。与野生型日本晴NPB相比,OsDCL1基因沉默突变体增强了水稻对稻瘟菌的广谱抗病性。为了解水稻OsDCL1-RNAi突变体的抗病机制,我们分析和比较了它和野生型日本晴NPB在稻瘟菌侵染前后转录组水平的变化。响应稻瘟菌侵染的7 129个差异表达基因(DEGs)中,NPB和OsDCL1-RNAi突变体分别有5 382和5 180个基因表达发生了变化,参与生物胁迫反应、信号通路、蛋白代谢和RNA调控4个通路的基因明显被富集。以野生型未受稻瘟菌侵染的基因表达为对照,在OsDCL1-RNAi突变体中共发现1 318个DEGs,且超过70%是上调的,其中很多基因参与了生物胁迫反应(26.9%)和信号通路(11%),上调表达基因中与生物胁迫反应相关的主要为PR基因和细胞壁相关基因。另外,还发现10个茉莉酸相关基因和11个水杨酸相关基因分别在OsDCL1-RNAi突变体中显著下调和上调表达。在OsDCL1-RNAi突变体中还发现WRKY和MYB等一些转录因子家族以及部分受体类激酶被诱导表达。用qRT-PCR方法验证部分差异表达的基因,其结果与DEGs基本一致。OsDCL1-RNAi突变体中转录组变化的分析,为深入了解该突变体抗瘟性增强的机制奠定了基础。     

沉默干旱应答元件结合蛋白基因OsDREB1A对高温下水稻中褐飞虱卵孵化的影响

干旱应答元件结合蛋白(dehydration responsive element binding protein,DREB)广泛参与调控植物抗非生物胁迫反应,如低温、高温等逆境(文锦芬等,2019),但是否参与植物抗虫反应目前尚无报道。水稻中该家族基因成员OsDREB1A在低温下被诱导表达,能增加水稻在低温、高盐和干旱胁迫下的存活率(Ito et al.,2006);本课题组前期研究发现水稻中OsDREB1A基因在害虫为害后会被诱导表达。     

MircoRNA156家族在小麦非生物胁迫中的表达分析

MircoRNA (miRNA)作为一类20碱基左右的非编码RNA,它在转录水平调控基因的表达,在植物抗逆境胁迫的调节网络中发挥了重要作用.通过对小麦miRNAs的研究,可以扩展小麦抵抗自然灾害的途径,减少损失.有报道表明miR156作为保守的miRNA在多种植物中参与到抗逆反应[1],Xin等[2]发现不同小麦品种的miR156在不同时间点分别受到白粉菌和热胁迫的诱导,尽管对miR156家族个别成员的研究已经有了相关报道,但对小麦miR156的整个家族的报道较少,对它们在小麦受到非生物胁迫网络中发挥的作用还不清楚.本研究拟明确小麦中miR156家族成员,并选取机械伤害、冷害和对植物损害较大的UV-B三种处理方法,探索miR156家族成员在不同的非生物胁迫中的作用.     

玉米MYB转录因子靶基因的全基因组预测及验证

MYB转录因子参与调控植物的生长发育及逆境应答过程。为预测MYB靶基因,本研究将Hex DIFF算法与支持向量机结合,根据已验证的MYB识别位点的核心序列及其侧翼序列构建分类模型,在玉米全基因组范围内预测MYB靶基因及其启动子。结果表明,共预测到435个MYB结合位点,其下游的424个基因判定为MYB靶基因,涉及众多生长发育过程。为验证预测结果,经凝胶迁移实验检测玉米MYB-IF25蛋白与30个预测位点的体外结合,结果有27个预测位点可以与MYB-IF25蛋白互作。GUS瞬时表达实验表明,渗透胁迫下,预测的MYB逆境相关靶基因启动子能够启动GUS基因表达,说明其具有启动活性。以上结果证明,此预测方法可靠性高,可为预测转录因子靶基因提供借鉴。     

OsHsp18.0-CI调控水稻对白叶枯病的抗性

 热激蛋白广泛存在于各种生物中,响应多种生物胁迫和非生物胁迫。前期研究结果显示,在水稻中超量表达小热激蛋白OsHsp18.0-CI基因,能通过增强水稻的基础防卫反应提高对水稻细菌性条斑病的抗性。本研究发现,OsHsp18.0-CI基因也能够受到白叶枯病菌的诱导表达,超量表达OsHsp18.0-CI的转基因水稻也增强了对多个白叶枯病致病菌株的抗性。转录组测序分析发现,OsHsp18.0-CI基因介导的水稻对白叶枯病的抗病信号途径有部分类似于其介导的对细菌性条斑病的抗性路径,同时也有大量新的未知功能基因的参与。以上结果表明,OsHsp18.0-CI基因受到病原菌的侵染时,也能通过增强水稻的基础防卫反应来提高对白叶枯病的抗性。     

玉米NF-YC亚基基因的鉴定与表达规律分析

0 引言 NF-Y(nuclear factor-Y,NF-Y)是一类与CCAAT基序结合的转录因子,由NF-YA、NF-YB和NF-YC 3个亚基组成[1-2].NF-YC参与并调控植物生长发育的进程以及在生物和非生物胁迫响应过程中起作用[3].AtNF-YC1通过与AtXTH21基因启动子区的CCAAT-box结合...     

白桦TGA基因家族全基因组鉴定及表达特性分析

TGA转录因子是bZIP转录因子(basic region-leucine zippers)家族成员，是植物中最早鉴定得到的一类转录因子，可以结合基因启动子的TGACG序列，调节靶基因的转录水平，在植物防御反应及花器官发育中发挥重要作用。本研究从白桦的基因组数据库中筛选出6条TGA基因，分别命名为BpTGA1～BpTGA6。生物信息分析结果表明：白桦TGA家族蛋白主要富含酸性氨基酸，亚细胞定位在细胞核，无信号肽结构，二级结构以α-螺旋为主，均属于亲水性蛋白，无跨膜结构。白桦TGA家族基因启动子区存在10～40个不等的增强启动元件(CAAT-box)和核心启动子元件(TATA-box),还具有非生物胁迫和逆境胁迫响应相关元件。系统进化分析结果表明：白桦TGA家族基因与拟南芥TGA家族基因亲缘关系较近。RT-qPCR结果表明，白桦TGA家族基因在根、茎、叶中的表达各有不同，BpTGA3在根中的表达量较高；BpTGA1、BpTGA2、BpTGA4和BpTGA6在茎中的表达量较高；BpTGA5在叶片中表达量较高。该家族成员中BpTGA2、BpTGA4、BpTGA5响应链格孢霉菌和立枯丝核菌侵染...     

本氏烟NbWRKY40亚家族转录因子抗病相关功能研究

 植物WRKY类转录因子的IIa亚类广泛参与调控植物的生物胁迫和非生物胁迫过程。本研究根据拟南芥和普通烟中具有抗病和抗胁迫功能的IIa亚类WRKY40转录因子的序列,利用基因同源克隆法,获得本氏烟中NbWRKY40基因。序列分析表明在本氏烟中有5个属于IIa亚类的基因,其序列之间具有高度相似性。qRT-PCR检测发现NbWRKY40基因是受寄生疫霉侵染诱导上调表达的基因,通过VIGS技术研究该类基因在本氏烟中的抗病相关功能,用半活体营养型寄生疫霉进行抗病性试验,结果表明NbWRKY40基因的沉默降低了本氏烟对寄生疫霉的抗性,且在侵染点的细胞坏死程度增强,过氧化氢积累和胼胝质沉积都明显减少。NbWRKY40基因沉默同样降低了本氏烟对死体营养型灰霉菌的抗性。检测NbWRKY40基因沉默后4个不同抗病信号通路下游基因的表达,发现基因沉默导致参与抗病和SA途径的PR1b、PR2b基因受疫霉侵染诱导表达的水平明显下降。综上,推测NbWRKY40基因可能依靠SA介导的信号通路参与调控本氏烟抗病性。     

可可毛色二孢效应子LT_397基因的克隆及转录分析

可可毛色二孢Lasiodiplodia theobromae是引起葡萄溃疡病的主要致病菌之一。病原菌会分泌许多效应子抑制植物的防御反应。本试验对可可毛色二孢特有的效应子LT_397基因进行克隆,开放阅读框(open reading frame,ORF)全长1140 bp,含有11个半胱氨酸。生物信息学软件预测显示:效应子LT_397信号肽为位于N端的1~16个氨基酸。qRT-PCR结果表明:LT_397受可可毛色二孢诱导下,在侵染24 h的基因相对转录水平最高;在逆境胁迫处理方面:LT_397在营养、氧化、离子、酸碱和UV胁迫下基因的相对转录水平都上升。烟草瞬时表达表明,效应子LT_397可抑制水稻细菌性谷枯病菌Burkholderia glumae引发的过敏反应(hypersensitive response,HR)。以上结果为进一步分析效应子对寄主的致病机制奠定了基础。     

Identification of differentially expressed genes in the mutualistic association of tall fescue with Neotyphodium coenophialum

Tall fescue (Lolium arundinaceum), an agronomically important forage grass, is typically associated with a mutualistic asexual fungus Neotyphodium coenophialum. Plant colonization is endophytic with no symptoms, and fungal growth is confined to the intercellular spaces. The endophyte enhances host fitness by providing protection from various abiotic and biotic stresses and by improving nutrient acquisition. By suppression subtractive hybridization (SSH) we identified 29 genes that are up-regulated or down-regulated in endophyte-infected tall fescue as compared to endophyte-free tall fescue. Of the genes that had matches to known genes present in the NCBI databases (approximately 50%), several had roles related to plant defense and stress tolerance. Differential expression of these genes was confirmed by semi-quantitative RT-PCR, competitive RT-PCR, and northern hybridization. Endophyte-associated changes in gene expression patterns were consistent among cultivars of tall fescue but differed in some other grass–endophyte associations. Our results indicate that both partners in this symbiosis are active participants, and that the endophyte may be suppressing at least one plant defense gene (putatively encoding PR-10). Further analyses of the differentially expressed genes should aid in understanding the fundamental nature of this mutualistic symbiosis and provide insight into the mechanisms of documented endophyte-enhanced plant improvements.     

The effect of salt stress on Arabidopsis thaliana and Phelipanche ramosa interaction

Salinity and Orobanche or Phelipanche spp. infection are important crop stress factors in agricultural areas. In this study, we investigated the effect of salt stress on Phelipanche ramosa seed germination and its attachment onto Arabidopsis thaliana roots. We also evaluated the effect of both stresses on the expression of genes regulated by abiotic and biotic stresses. According to our results, high concentration of NaCl delayed P. ramosa seed germination in the presence of a strigolactone analogue (GR24). A similar pattern was observed in the presence of A. thaliana plants. Furthermore, we found that salt‐treated A. thaliana seedlings were more sensitive to P. ramosa attachment compared with the untreated plants, indicating that there was a positive correlation between salt sensitivity and the ability of P. ramosa to infect A. thaliana plants. At the molecular level, a synergystic effect of both salt and P. ramosa stresses was observed on the cold‐regulated (COR) gene expression profile of treated A. thaliana seedlings. Our data clarify the interaction between parasitic plants and their hosts under abiotic stress conditions.     

Erwinia carotovora Infection Enhances the Expression of Two Novel Abiotic Stress-inducible Genes in Potato

In this study, cDNAs of two Erwinia carotovora-induced potato genes, designated Solanum tuberosum–Erwinia-induced-1 and 2 (Stei1 and Stei2) were isolated by differential display technique. Stei1 and Stei2 were detected in low copy number in the potato genome and found to encode putative proteins with no significant homology to any genes with known function. Treatment of the leaves with salicylic acid, methyl jasmonate and ethylene elevated neither Stei1 nor Stei2 mRNA levels. However, Stei1 and Stei2 expression were induced not only by E. carotovora but also by infiltration of water in leaves, albeit to a lesser extent. In addition, Stei2 was up-regulated by NaCl, wounding, dehydration and abscisic acid. Thus Stei1 and Stei2 define novel genes belonging to the family of those pathogenesis-related genes whose expression can be induced both by biotic and abiotic stresses.     

植物内生真菌作为生防因子的研究进展

本文对内生真菌作为生防因子的研究进展进行了综述,主要包括内生真菌在植物抗病性、抗虫性、抗逆性等方面的研究情况及其在生产中应用存在的问题.     

Identification and characterization of a serine protease from wheat leaves

A putative serine protease with a potential role in the plant biotic and abiotic stress response was purified from wheat leaf apoplastic fluid and partially characterized. Following two-dimensional electrophoresis a protein of M_r = 75 k and a pI of 4.2 to 4.5 was observed. This protein displayed in-gel protease activity and was specifically inhibited by phenylmethanesulfonyl fluoride and partially inhibited by Ca²⁺ and Zn²⁺, but not by E-64 or leupeptin. An internal tryptic fragment of 13 amino acids was identified by MALDI QqTOF MS/MS, and this peptide showed a high level of homology (85–100 % identity) to a highly conserved region of known plant subtilisin-like proteases. We demonstrated that the protease activity increased until a late stage of wheat leaf development and increased in response to heat shock. In both cases Rubisco large subunit was degraded with time. Protease activity was also increased during biotic stress. Leaves challenged with leaf rust (Puccinia triticina), showed an approximately three fold increase in protease activity during an incompatible interaction, compared to activity in mock-inoculated leaves and to leaves in a compatible leaf rust interaction. These results suggest that the expression of this serine protease could be involved in the defense response against both abiotic and biotic stresses.     

我国农业害虫性诱监测技术的开发和应用

通过多年的实验室研究,结合从2009年开始的多地、多作物、多年田间试验示范,集中研究了与重要农业害虫性诱监测密切相关的性信息素配比和剂量、种专一性、微量组分、缓释、诱捕器结构及其应用技术,解决了性信息素种间细微差别、种内地理区系差异、信息素化合物的纯化以及稳定保护和缓慢均匀释放等影响测报精准度的关键问题,开发了适应昆虫飞行行为、利于捕获收集的系列诱捕器,配合组装了成套的田间应用技术,开发了性诱自动计数系统,简化了害虫诱获鉴定操作,促进了虫情自动化记载传递,丰富了弱光性害虫监测手段,从而在一定程度上实现了害虫监测预警轻简化、标准化、自动化。分析提出为促进性诱监测技术的发展,要建立性诱监测技术的科学评价体系,并在迁飞性害虫、同种害虫的不同地理种群监测中开辟性诱技术应用的新领域。     

向日葵3个谷胱甘肽-S-转移酶GST基因的克隆及表达模式分析

从向日葵转录组测序结果中获得了3个对盐胁迫有响应的谷胱甘肽-S-转移酶（GST,EC2.5.1.18）GST基因,构建了系统进化树并进行分析,得知这3个基因属于Tau型GST,并将它们命名为HaGSTU26（HanXRQChr04g0127901）、HaGSTU8（HanXRQChr06g0177581）、HaGSTU27（HanXRQChr10g0316331）,然后以向日葵Sk02R为试验材料,克隆这3个基因,并进行了不同组织和不同胁迫条件下的表达分析。序列分析表明：HaGSTU26基因组为1674bp,CDS（编码蛋白序列）长666bp,编码221个氨基酸,由2个外显子和1个内含子组成;HaGSTU8基因组为2271bp,CDS长654bp,编码217个氨基酸,由2个外显子和1个内含子组成; HaGSTU27基因组为663bp,CDS长663bp,编码220个氨基酸,由1个外显子组成。实时荧光定量PCR分析表明：向日葵HaGSTU26、HaGSTU8、HaGSTU27基因在不同组织（根、幼叶、成熟叶、茎、幼茎、苞叶）中表达量不同,其中,HaGSTU26基因和HaGSTU27基因在根中表达量最高,而HaGSTU8基因在苞叶中表达量最高,但这3个基因均在成熟茎中的表达量最低。在不同胁迫条件下,测定这3个基因在向日葵幼苗中的表达量,结果表明在盐及ABA胁迫下,基因表达量均随着处理时间的增加而呈现先增加后下降的趋势。其中,在盐胁迫下,HaGSTU26基因在12 h后相对表达量最高,HaGSTU8及HaGSTU27基因表达量在3h后相对表达量最高。在ABA胁迫后,HaGSTU26及HaGSTU27基因表达量在12 h后相对表达量最高,HaGSTU8在24 h后相对表达量最高。在冷胁迫下,HaGSTU26和HaGSTU27基因上调表达,它们分别在3、24 h后相对表达量最高,HaGSTU8基因下调表达,其相对表达量随处理时间的延长呈现逐渐减少的趋势。在热胁迫条件下,这3个基因的相对表达量随着胁迫时间的延长而增加,均在24 h后表达量最高。以上结果说明这3个基因对不同非生物胁迫（盐、ABA、冷、热胁迫）均有响应。