‘嘎拉’苹果及其杂交后代对炭疽叶枯病的抗性机制分析

为解析苹果对炭疽叶枯病的抗性机制,该研究以‘嘎拉’、‘藤牧1号’苹果及其杂交后代等87个苹果品种(系)为试验材料,进行田间调查及人工接种病菌并检测不同品种(系)中病菌生物量,利用SSR分子标记进行基因型鉴定,分析各品种(系)对炭疽叶枯病的抗性差异,利用荧光定量PCR及酶活检测比较分析水杨酸相关基因、抗性酶基因及酶活性水平差异。结果表明：(1)87个苹果品种(系)对苹果炭疽叶枯病的抗性差异明显,‘嘎拉’、‘2 5’、‘19 19’等品种(系)叶片发病严重,表现出对炭疽叶枯病的高感性,‘藤牧1号’、‘40 9’及‘16 16’等品种(系)叶片无病斑或病斑极少,炭疽叶枯病菌含量显著低于感病性品种(系),其抗性显著。(2)SSR标记S0405127和S0304673的基因型鉴定结果与田间表型调查结果相比,准确率分别为93.10%和91.95%,与人工接种结果相比,准确率分别为91.95%和95.40%。(3)SA相关基因的表达模式结果表明,接种炭疽叶枯病菌4 d后,‘藤牧1号’、‘40 9’及‘16 16’等抗性品种(系)中SA合成相关基因MdEDS1、MdPAD4和MdPAL被强烈诱导表达;同时,SA信号转导相关基因MdNPR1、MdPR1、MdPR5的表达显著高于‘嘎拉’等感病品种(系)。(4)接种炭疽叶枯病菌4 d后,‘藤牧1号’、‘40 9’及‘16 16’等抗性品种(系)的MdSOD、MdPOD酶基因表达水平及酶活性显著高于‘嘎拉’、‘2 5’、‘19 19’等感病品种(系)。研究认为,‘藤牧1号’、‘40 9’及‘16 16’等品种(系)通过调控水杨酸合成和信号转导通路及氧化还原相关反应等提高对炭疽叶枯病的抗性,为挖掘抗性基因以及利用优良种质选育抗病品种奠定了基础。     

矮牵牛PhTPS5基因的克隆与表达分析

海藻糖 6 磷酸合成酶（Trehalose 6 phosphate synthase）基因TPS是海藻糖生物合成途径中的关键基因之一。该研究从矮牵牛 (Petunia hybrida)中分离了TPS5的同源基因PhTPS5。该基因开放阅读框（ORF）为2 595 bp,编码864个氨基酸。推测PhTPS5蛋白的分子式为C⁴³⁶³H⁶⁸²⁵N¹¹⁷³O¹²⁸⁹S³⁷。组织特异性表达分析显示：PhTPS5基因在根中表达量最高,叶片中的表达量最低;去顶6 h能够显著促进PhTPS5基因的表达,但24 h后表达量明显下降;去顶后施加生长素则能够有效抑制去顶对PhTPS5基因表达的调节;施加细胞分裂素6 h后PhTPS5基因的表达水平显著上调,但随着处理时间的增加,其表达水平有所下降。该研究为进一步揭示TPS途径在矮牵牛分枝发育中的调控机制奠定了理论基础。     

中国野生毛葡萄VqTLP15基因的克隆与抗病性功能研究

该研究以抗病品种中国野生毛葡萄‘商 24’和感病品种欧洲葡萄‘红地球’为材料,利用 RT PCR 方法克隆TLP15基因,分别命名为 VqTLP15 和 VvTLP15(GSVIVT01018769001),对其进行生物信息学分析、亚细胞定位、转化拟南芥,并接种不同病原菌观察分析转基因株系的抗性,采用qRT PCR 检测SA 和 JA/Eth信号途径以及调控气孔运动的相关基因表达。结果显示：(1)成功克隆获得 VqTLP15 基因的开放阅读框 (ORF);氨基酸序列比对显示,VqTLP15基因与葡萄基因组网站欧洲葡萄‘黑比诺’VvTLP15 和‘红地球’克隆的 VvTLP15基因的同源性分别为 98.99％ 和 99.66％。 (2)亚细胞定位表明,VqTLP15 定位于细胞质。(3)成功获得 VqTLP15 转基因拟南芥株系(L1、 L2、 L3)。(4)接种观察发现：白粉菌处理 7 d后转基因株系对白粉菌的抗性较野生型(Col 0)提高,且其叶片的白粉菌孢子浓度显著低于Col 0;灰霉菌诱导的叶片坏死性损伤在转基因株系(L1、L2 和L3病斑面积>40%的比例分别为 71%、62%和67%)中显著大于 Col 0(43%);接种 PstDC3000后转基因株系叶片的病害表型没有 Col 0 明显,叶片孔径减小程度大于 Col 0,且细菌浓度低于 Col 0。(5)组织化学染色分析表明：白粉菌处理后转基因株系叶片胼胝质沉积、细胞死亡率和 O₂^-·水平都显著大于 Col 0;灰霉菌处理后转基因株系的细胞死亡率、H₂O₂和 O₂^-·水平均高于 Col 0; PstDC3000 处理后细胞死亡率和 O₂^-·积累水平都高于 Col 0。(6)qRT PCR 检测显示：接种白粉菌后,转基因株系中PR1 和 ICS1 的表达水平均升高,PR1 表达在接种72 h时达到峰值,而 ICS1 在 接种120 h时达到峰值,LOX3 的表达水平逐渐降低,并于接种120 h时降至最低水平,但仍高于 Col 0;接种灰霉菌后,转基因株系中 PR1、NPR1 和 PDF1.2 基因的表达均上调,并在接种 48 h时达到峰值,LOX3 基因的表达水平下降,但仍高于 Col 0;接种 PstDC3000 后,转基因株系中 PR1、PDF1.2 和 NHL10的表达均高于 Col 0,但WRKY53的表达低于Col 0, L1 中 COI1、FRK1、ATPPC2、FLS2、OST1 的表达水平高于 Col 0;接种flg22 或 LPS后, L1 中 COI1基因的表达低于 Col 0,但ATPPC2、FLS2、OST1基因的表达水平高于 Col 0。研究表明,过量表达 VqTLP15 基因降低了对白粉菌和 PstDC3000 的敏感性,增加了对灰霉菌的敏感性。 VqTLP15 基因可能通过介导水杨酸 (SA) 和茉莉酸/乙烯 (JA/Eth) 信号转导途径以及气孔免疫反应来参与植物的抗病防御反应,为葡萄抗病分子育种提供了一个可能的候选基因。     

不结球白菜金属硫蛋白基因的克隆及表达分析

通过RACE技术,从不结球白菜抗病品种‘苏州青’叶片克隆到金属硫蛋白(metallothionein)基因的全长cDNA序列(BcMT2)。序列分析结果表明,BcMT2基因的cDNA全长为528 bp,其中开放阅读框长度为243 bp,共编码80个氨基酸,相对分子质量为8.02×10³ Da,理论等电点是4.61。氨基酸同源系统进化分析表明,不结球白菜BcMT2基因属于Ⅱ类植物MT2基因家族,且与同科植物的进化关系相近,其中与大白菜第5号染色体的基因(Bra029765)相似性最高(100%)。qRT PCR分析表明,BcMT2基因在不结球白菜叶中表达最强;霜霉病菌诱导后,BcMT2基因在抗病品种‘苏州青’中的表达量于48 h达到峰值,而在感病品种‘矮脚黄’中的表达量于72 h达到峰值;盐处理条件下,BcMT2基因在抗病品种‘苏州青’中的表达量于12 h达到峰值,而在感病品种‘矮脚黄’中的表达量于24 h达到峰值;ABA处理下,‘苏州青’中其表达量于24 h达到峰值,且在‘矮脚黄’中BcMT2基因的表达趋势与‘苏州青’类似;干旱处理条件下BcMT2基因的表达在两材料中均受到抑制。BcMT2原核表达分析发现,在37 ℃、1.0 mmol·L^-1IPTG诱导2、4 、6 h后,均检测到了一条蛋白分子质量约为8×10³ Da的表达特异条带,这与预期融合蛋白的相对分子质量的理论值8.02×10³ Da相符。研究表明,不结球白菜BcMT2基因在受到生物胁迫和非生物胁迫等逆境条件下均发挥着重要作用,且BcMT2在大肠杆菌中成功实现融合表达,为进一步进行蛋白水平和转基因功能研究奠定了基础,也为选育高产、优质的不结球白菜新品种提供重要的理论依据。     

沙棘种子形成发育过程中油脂合成积累关键基因的表达分析

为探讨沙棘种子油脂积累与相关基因表达的关系,以不同发育期间（6月25日、7月6日、7月17日、7月28日、8月8日和8月19日）的近缘高油品系‘新俄3号’和低油品系‘绥棘1号’种子为试材,利用氯仿甲醇法测定含油率,采用实时荧光定量PCR方法分析限速酶基因的表达模式,验证各基因在天然高低油种子间的表达差异。结果表明：（1）除7月17日外,其他时期‘新俄3号’的种子含油率均高于‘绥棘1号’;7月6日~7月28日期间油脂迅速积累,而且‘新俄3号’种子含油率增速大于‘绥棘1号’。（2）油脂迅速积累期的GPD1基因高表达,可能通过促进3 磷酸甘油合成进而加速‘新俄3号’种子油脂高积累;油脂积累过程中DGAT1和DGAT2基因在‘新俄3号’种子中的表达量一直高于‘绥棘1号’。研究认为,GPD1、DGAT1和DGAT2基因可能与‘新俄3号’种子油脂的相对高积累有关。本研究结果为进一步深入验证沙棘油脂合成限速酶基因功能奠定基础。     

Pi-ta+基因和几丁质酶基因双价基因导入水稻的研究

为了提高‘云资粳41’和‘云资粳43’的抗稻瘟病能力,利用农杆菌介导法将二价表达载体pCAMBIA1300-Pi-ta⁺-Bchi转化到水稻愈伤组织中。经组织培养获得再生苗,再通过氯酚红显色法、PCR检测和抗稻瘟病鉴定法获得抗稻瘟病的转基因植株,为进一步创建持久、广谱抗稻瘟病水稻新材料奠定基础。结果显示:(1)抗性愈伤组织经分化和生根培养后,共获得T⁰代水稻再生苗137株,其中‘云资粳41’14株,‘中花11’82株,‘云资粳43’41株。(2)经氯酚红显色法和PCR对再生苗检测,‘中花11’、‘云资粳41’、‘云资粳43’的转化苗阳性率分别为66%、43%和55%。证明2个外源基因已经整合到水稻基因组中。(3)对转化阳性植株温室接种稻瘟病病菌66b鉴定结果显示,转基因植株较非转基因植株对稻瘟病的抗性明显增强,而且转Pi-ta⁺基因和几丁质酶基因双价的水稻植株比转单价Pi-ta⁺基因或几丁质酶基因的水稻植株抗稻瘟病能力强。（4）氯酚红检测结果存在一定的假阳性,PCR检测结果更真实可靠,但氯酚红显色法方便、快速,结果观察直观,可对大量的转基因植株进行初步筛选。研究表明,转Pi-ta⁺基因和几丁质酶基因双价基因的水稻植株具有更高的抗稻瘟病能力。     

茶树SBP box转录因子的比较基因组学与遗传分析

SQUAMOSA启动子结合蛋白(SBP box)是植物特异性转录因子,在植物生长发育中发挥重要作用。该研究利用生物信息学分析方法,对不同‘铁观音’、‘黄棪’、‘舒茶早’和‘龙井43’茶树基因组的SBP基因进行鉴定和比较,通过qRT PCR技术分析CsTGY_SBP家族成员在不同茶树品种中的表达模式,为探究SBP基因在茶树杂种和亲本上的遗传规律提供参考。结果显示：(1)在茶树品种‘铁观音’、‘黄棪’、‘舒茶早’和‘龙井43’基因组中分别鉴定出21个、25个、24个和23个SBP家族基因。(2)系统进化树将其分为8个亚家族,共线性分析发现茶树和拟南芥、葡萄的SBP基因共线性关系与水稻相比更强,同物种内发现‘铁观音’与‘黄棪’的共线性关系更显著。(3)qRT PCR结果表明,CsTGY_SBP5、CsTGY_SBP9和CsTGY_SBP14在F₁‘金观音’中呈中亲表达的模式;大部分CsTGY_SBPs基因在F₁‘黄观音’呈低于双亲表达模式,CsTGY_SBP5和CsTGY_SBP8在F₁‘金牡丹’中显著高于亲本;CsTGY_SBP5、CsTGY_SBP7、CsTGY_SBP12、CsTGY_SBP16和CsTGY_SBP18在F₁‘紫玫瑰’中的表达量显著高于亲本,呈超高亲表达的模式;CsTGY_SBPs基因在F₁‘紫牡丹’中整体表达趋于亲本铁观音,在F₁‘瑞香’中整体呈现低于亲本‘黄棪’的表达模式。研究表明,CsTGY_SBP5在F₁‘金牡丹’和F₁‘紫玫瑰’中的表达均显著高于亲本,推测CsTGY_SBP5可能是茶树杂种优势的重要调控因子。     

黄毛草莓FnMYB24转录因子基因和启动子的克隆及表达分析

该研究以黄毛草莓(Fragaria nilgerrensis Schltdl.)为材料,采用RT PCR技术克隆了黄毛草莓FnMYB24基因的cDNA和启动子序列。生物信息学分析表明,FnMYB24的cDNA序列长为1 033 bp(GenBank登录号为MN879283),其开放阅读框(ORF)长为609 bp,编码202个氨基酸,含有1个保守的MYB_DNA binding结构域。同源分析结果显示,黄毛草莓FnMYB24基因编码的氨基酸序列与森林草莓(Fragaria vesca)编码的氨基酸相似性较高;同时进一步克隆了该基因编码起始位点上游长度为718 bp启动子序列(GenBank登录号为MN879285),预测该序列包含激素响应元件、光调控元件等多个顺式作用元件。通过构建pFnMYB24∷GUS表达载体进行烟草瞬时转化,发现pFnMYB24启动子具有转录活性且能够驱动FnMYB24基因表达。实时荧光定量PCR结果显示：抗病品种黄毛草莓和易感病栽培品种‘妙香3号’的叶片接种胶孢炭疽菌(Colletotrichum gloeosporioides)后MYB24基因表达量均有上调,但‘妙香3号’的MYB24表达量始终低于黄毛草莓的表达量;SA处理后2个草莓品种的MYB24表达量均高于对照组,表明MYB24基因受水杨酸(SA)的诱导表达。研究表明,草莓MYB24基因可能参与调控抗炭疽病,为进一步研究MYB24基因在草莓抗炭疽病中的功能奠定了基础。     

荷兰鸢尾突变株‘紫韵’的花色突变相关机理分析

为了探索荷兰鸢尾蓝紫色花及突变紫色花的显色分子机制及色素沉积差异,该研究以蓝紫色野生型‘展翅’和紫色突变株‘紫韵’为材料,通过花色素苷测定、转录组测序和qRT PCR方法对花色变异进行分析。结果表明：(1) 紫色突变株‘紫韵’花旗瓣中的总花色素苷含量(392.7 μg·g^-1)显著低于野生型‘展翅’(543.5 μg·g^-1);与‘展翅’相比,‘紫韵’有3种花色素苷含量显著下降［矢车菊素 3 芸香糖苷含量由144.42 μg·g^-1降为46.39 μg·g^-1,矮牵牛素 3 (6 鼠李糖基 2 木糖基葡萄糖苷)含量由61.86 μg · g^-1降为31.67 μg · g^-1,矢车菊素 3 (2G 木糖基芸香糖苷)含量由25.22 μg·g^-1降为7.65 μg·g^-1］,但6 羟基矢车菊素 3 葡萄糖苷含量由5.88 μg·g^-1升为10.34 μg·g^-1。(2)RNA seq分析共获得46 530个unigenes,与‘展翅’相比,‘紫韵’有43个基因上调表达,73个基因下调表达; 层次聚类分析发现,花色素苷途径中共有2个差异表达基因——查尔酮合成酶(CHS)基因(IhCHS1)和花青素 3 O葡萄糖转移酶(UFGT)基因(IhUFGT1),且二者均下调表达。(3)qRT PCR分析表明,随着花的发育,IhUFGT1在2个品种中表达量均上升,始花期达到最高,且在‘紫韵’花中的表达明显低于‘展翅’。研究认为,4种花色素苷含量的显著变化,可能是导致花色由野生型‘展翅’蓝紫色向突变株‘紫韵’紫色方向转变的主要原因;IhUFGT1基因在紫色‘紫韵’花中表达量比‘展翅’相对大幅度的降低,致使花色素苷含量相应变化,最终导致花色由蓝紫色转为紫色。     

杜鹃花TPS基因家族鉴定及与萜类物质代谢的关系分析

萜烯合成酶（terpene synthase,TPS）能催化不同的前体物质生成不同的萜类化合物,是合成萜类物质的关键酶。为探究杜鹃花TPS基因家族成员在萜类物质代谢过程中的表达模式,本文基于杜鹃花基因组数据库,利用生物信息学方法对杜鹃花TPS基因（TPS）进行家族成员鉴定;通过云锦杜鹃和诺娃杜鹃两种不同种高山杜鹃的转录组测序结果,结合qRT-PCR、顶空固相微萃取和气相色谱-质谱联用技术,分析两种杜鹃不同发育时期花瓣中TPS家族成员表达水平和代谢物含量变化关系。结果表明,从杜鹃花基因组数据库中共鉴定获得47个RsTPS成员,RsTPS家族成员长度在591-2 634 bp之间,含有3-12个外显子不等,编码196-877个氨基酸;RsTPS家族成员主要分布在叶绿体和细胞质;系统进化分析结果显示RsTPS基因分为5个亚组。通过分析转录组数据得到7个功能注释为TPS的基因家族成员,发现TPS1、TPS10、TPS12和TPS13的表达量在4个时期中呈现出先上升,到盛开期达到顶峰后再下降的趋势。对基因表达量变化与萜类物质含量变化进行相关性分析,发现TPS1、TPS4、TPS9、TPS10、TPS12和TPS13表达量与云锦杜鹃不同时期花瓣中萜类物质含量变化呈显著性正相关,推测这6个基因家族成员可能是参与云锦杜鹃花香调控的关键基因。     

叶片涂施茉莉酸对玉米幼苗防御反应的时间和浓度效应

茉莉酸是环境胁迫下植物产生防御反应的重要信号物质, 但它发挥生理作用的时间和浓度效应以及该效应在叶片和根系中差异性并不清楚。该文以‘高油115’玉米(Zea mays)为材料, 采用4种浓度(1、2.5、5和10 mmol·L^-1)的外源茉莉酸溶液涂施玉米幼苗叶片, 在3~48 h的不同时间内跟踪测定叶片和根系中的直接防御物质(丁布(DIMBOA)和总酚)含量及其合成调控基因(Bx1、Bx9和PAL)、直接防御蛋白调控基因(PR-1、PR-2a和MPI)和间接防御物质挥发物调控基因(FPS和TPS)表达的动态变化。结果表明, 外源茉莉酸处理对玉米叶和根系的化学防御反应具有显著的时间和浓度效应。茉莉酸处理玉米叶片后3~6 h就能诱导叶片中Bx9和PAL基因的表达, 使得丁布和总酚的含量显著增加, 且与处理浓度有呈正比的趋势, 随后诱导作用逐渐减弱; 茉莉酸处理还能明显诱导叶片中PR-2a和MPI基因的表达, 诱导作用分别持续到24和48 h; 在处理后3~6 h内, 高浓度茉莉酸处理对挥发物调控基因FPS表达起诱导作用, 而低浓度茉莉酸则对TPS基因的表达起诱导作用。此外, 茉莉酸处理玉米叶片还能间接影响到根系的防御反应, 但大部分检测指标表明间接诱导作用主要出现在处理后期(24~48 h)。例如, 在处理后48 h, 茉莉酸能系统增加根系中直接防御物质丁布和总酚的含量, 增强根系中防御相关基因PR-2a、MPI、FPS和TPS的表达, 并有随茉莉酸处理浓度的增加而增强的趋势。可见, 外源茉莉酸叶片涂施玉米幼苗对根系的间接诱导作用不如对叶片的直接诱导作用强; 叶片启动防御反应的时间较根系早; 随着处理浓度的增加, 茉莉酸对叶片和根系中防御反应的诱导作用有增强的趋势。     

Rice OsPAD4 functions differently from Arabidopsis AtPAD4 in host‐pathogen interactions

The extensively studied Arabidopsis phytoalexin deficient 4 (AtPAD4) gene plays an important role in Arabidopsis disease resistance; however, the function of its sequence ortholog in rice is unknown. Here, we show that rice OsPAD4 appears not to be the functional ortholog of AtPAD4 in host‐pathogen interactions, and that the OsPAD4 encodes a plasma membrane protein but that AtPAD4 encodes a cytoplasmic and nuclear protein. Suppression of OsPAD4 by RNA interference (RNAi) increased rice susceptibility to the biotrophic pathogen Xanthomonas oryzae pv. oryzae (Xoo), which causes bacteria blight disease in local tissue. OsPAD4‐RNAi plants also show compromised wound‐induced systemic resistance to Xoo. The increased susceptibility to Xoo was associated with reduced accumulation of jasmonic acid (JA) and phytoalexin momilactone A (MOA). Exogenous application of JA complemented the phenotype of OsPAD4‐RNAi plants in response to Xoo. The following results suggest that OsPAD4 functions differently than AtPAD4 in response to pathogen infection. First, OsPAD4 plays an important role in wound‐induced systemic resistance, whereas AtPAD4 mediates systemic acquired resistance. Second, OsPAD4‐involved defense signaling against Xoo is JA‐dependent, but AtPAD4‐involved defense signaling against biotrophic pathogens is salicylic acid‐dependent. Finally, OsPAD4 is required for the accumulation of terpenoid‐type phytoalexin MOA in rice‐bacterium interactions, but AtPAD4‐mediated resistance is associated with the accumulation of indole‐type phytoalexin camalexin.     

Allene oxide cyclase dependence of the wound response and vascular bundle-specific generation of jasmonates in tomato - amplification in wound signalling

The allene oxide cyclase (AOC)-catalyzed step in jasmonate (JA) biosynthesis is important in the wound response of tomato. As shown by treatments with systemin and its inactive analog, and by analysis of 35S::prosysteminsense and 35S::prosysteminantisense plants, the AOC seems to be activated by systemin (and JA) leading to elevated formation of JA. Data are presented on the local wound response following activation of AOC and generation of JA, both in vascular bundles. The tissue-specific occurrence of AOC protein and generation of JA is kept upon wounding or other stresses, but is compromised in 35S::AOCsense plants, whereas 35S::AOCantisense plants exhibited residual AOC expression, a less than 10% rise in JA, and no detectable expression of wound response genes. The (i). activation of systemin-dependent AOC and JA biosynthesis occurring only upon substrate generation, (ii). the tissue-specific occurrence of AOC in vascular bundles, where the prosystemin gene is expressed, and (iii). the tissue-specific generation of JA suggest an amplification in the wound response of tomato leaves allowing local and rapid defense responses.     

Jasmonate biosynthesis and the allene oxide cyclase family of Arabidopsis thaliana

In biosynthesis of octadecanoids and jasmonate (JA), the naturally occurring enantiomer is established in a step catalysed by the gene cloned recently from tomato as a single-copy gene (Ziegler et al., 2000). Based on sequence homology, four full-length cDNAs were isolated from Arabidopsis thaliana ecotype Columbia coding for proteins with AOC activity. The expression of AOCgenes was transiently and differentially up-regulated upon wounding both locally and systemically and was induced by JA treatment. In contrast, AOC protein appeared at constitutively high basal levels and was slightly increased by the treatments. Immunohistochemical analyses revealed abundant occurrence of AOC protein as well as of the preceding enzymes in octadecanoid biosynthesis, lipoxygenase (LOX) and allene oxide synthase (AOS), in fully developed tissues, but much less so in 7-day old leaf tissues. Metabolic profiling data of free and esterified polyunsaturated fatty acids and lipid peroxidation products including JA and octadecanoids in wild-type leaves and the jasmonate-deficient mutant OPDA reductase 3 (opr3) revealed preferential activity of the AOS branch within the LOX pathway. 13-LOX products occurred predominantly as esterified derivatives, and all 13-hydroperoxy derivatives were below the detection limits. There was a constitutive high level of free 12-oxo-phytodienoic acid (OPDA) in untreated wild-type and opr3 leaves, but an undetectable expression of AOC. Upon wounding opr3 leaves exhibited only low expression of AOC, wounded wild-type leaves, however, accumulated JA and AOC mRNA. These and further data suggest regulation of JA biosynthesis by OPDA compartmentalization and a positive feedback by JA during leaf development.     

外源茉莉酸处理地下部对玉米化学防御反应影响的时间和浓度效应

在“高油115”玉米幼苗地下部施用4种不同浓度（10、50、100、200 μmol·L^-1）的茉莉酸3～48 h后,采用生化成分分析和基因表达分析方法,检测了处理部位（根系）和非处理部位（叶片）中防御物质及防御相关基因的表达,以探讨茉莉酸诱导玉米地下部对化学防御反应影响的时间和浓度效应.结果表明：茉莉酸处理玉米地下部对处理部位（根系）和非处理部位（叶片）化学防御反应的影响均与茉莉酸作用的时间和浓度相关.茉莉酸处理玉米地下部后,在3～12 h内就能直接诱导处理部位（根系）Bx9、PAL、PR-2a、MPI和FPS基因的表达,使得根中的丁布含量增加而总酚含量下降,其中100 μmol·L^-1茉莉酸处理浓度产生的诱导作用最强,其次是50 μmol·L^-1,再次是10 μmol·L^-1;后期诱导作用则逐渐减弱.另外,茉莉酸处理玉米地下部还能间接影响到非处理部位（叶片）的化学防御反应,50 μmol·L^-1茉莉酸在处理后3 h就能系统诱导叶片中Bx9和FPS基因的表达,使叶片的丁布含量增加;在6～24 h内则使叶片Bx9、PAL、PR-1、MPI和TPS基因表达量增加,丁布和总酚含量降低.可见,对于大部分检测指标来说,茉莉酸处理玉米地下部对地上部的间接诱导作用不如对地下部的直接诱导作用强,根系启动防御反应的时间较叶片早,同时,随着处理浓度的增加,茉莉酸对根系和叶片防御反应的诱导作用有增强趋势.     

OsGLP3-7 positively regulates rice immune response by activating hydrogen peroxide,jasmonic acid,and phytoalexin metabolic pathways

Although germin-like proteins (GLPs) have been demonstrated to participate in plant biotic stress responses, their specific functions in rice disease resistance are still largely unknown. Here, we report the identification and characterization of OsGLP3-7, a member of the GLP family in rice. Expression of OsGLP3-7 was significantly induced by pathogen infection, jasmonic acid (JA) treatment, and hydrogen peroxide (H₂O₂) treatment. OsGLP3-7 was highly expressed in leaves and sublocalized in the cytoplasm. Overexpression of OsGLP3-7 increased plant resistance to leaf blast, panicle blast, and bacterial blight, whereas disease resistance in OsGLP3-7 RNAi silenced plants was remarkably compromised, suggesting this gene is a positive regulator of disease resistance in rice. Further analysis showed that OsGLP3-7 has superoxide dismutase (SOD) activity and can influence the accumulation of H₂O₂ in transgenic plants. Many genes involved in JA and phytoalexin biosynthesis were strongly induced, accompanied with elevated levels of JA and phytoalexins in OsGLP3-7-overexpressing plants, while expression of these genes was significantly suppressed and the levels of JA and phytoalexins were reduced in OsGLP3-7 RNAi plants compared with control plants, both before and after pathogen inoculation. Moreover, we showed that OsGLP3-7-dependent phytoalexin accumulation may, at least partially, be attributed to the elevated JA levels observed after pathogen infection. Taken together, our results indicate that OsGLP3-7 positively regulates rice disease resistance by activating JA and phytoalexin metabolic pathways, thus providing novel insights into the disease resistance mechanisms conferred by GLPs in rice.     

Rapid induction of phenylalanine ammonia-lyase and chalcone synthase mRNAs during fungus infection of soybean (Glycine max L.) roots or elicitor treatment of soybean cell cultures at the onset of phytoalexin synthesis

The differential regulation of the activities and amounts of mRNAs for two enzymes involved in isoflavonoid phytoalexin biosynthesis in soybean was studied during the early stages after inoculation of primary roots with zoospores from either race 1 (incompatible, host resistant) or race 3 (compatible, host susceptible) of Phytophthora megasperma f.sp. glycinea, the causal fungus of root rot disease. In the incompatible interaction, cloned cDNAs were used to demonstrate that the amounts of phenylalanine ammonia-lyase and chalcone synthase mRNAs increased rapidly at the time of penetration of fungal germ tubes into epidermal cell layers (1–2 h after inoculation) concomitant with the onset of phytoalxxin accumulation; highest levels were reached after about 7 h. In the compatible interaction, only a slight early enhancement of mRNA levels was found and no further increase occurred until about 9 h after inoculation. The time course for changes in the activity of chalcone synthase mRNA also showed major differences between the incompatible and compatible interaction. The observed kinetics for the stimulation of mRNA expression related to phytoalexin synthesis in soybean roots lends further support to the hypothesis that phytoalexin production is an early defense response in the incompatible plant-fungus interaction. The kinetics for the enhancement of mRNA expression after treatment of soybean cell suspension cultures with a glucan elicitor derived from P. megasperma cell walls was similar to that measured during the early stages of the resistant response of soybean roots.Abbreviations cDNA copy DNA - CHS chalcone synthase - PAL phenylalanine ammonia-lyase     

OsNPR1 negatively regulates herbivore‐induced JA and ethylene signaling and plant resistance to a chewing herbivore in rice

NPR1 (a non‐expressor of pathogenesis‐related genes1) has been reported to play an important role in plant defense by regulating signaling pathways. However, little to nothing is known about its function in herbivore‐induced defense in monocot plants. Here, using suppressive substrate hybridization, we identified a NPR1 gene from rice, OsNPR1, and found that its expression levels were upregulated in response to infestation by the rice striped stem borer (SSB) Chilo suppressalis and rice leaf folder (LF) Cnaphalocrocis medinalis, and to mechanical wounding and treatment with jasmonic acid (JA) and salicylic acid (SA). Moreover, mechanical wounding induced the expression of OsNPR1 quickly, whereas herbivore infestation induced the gene more slowly. The antisense expression of OsNPR1 (as‐npr1), which reduced the expression of the gene by 50%, increased elicited levels of JA and ethylene (ET) as well as of expression of a lipoxygenase gene OsHI‐LOX and an ACC synthase gene OsACS2. The enhanced JA and ET signaling in as‐npr1 plants increased the levels of herbivore‐induced trypsin proteinase inhibitors (TrypPIs) and volatiles, and reduced the performance of SSB. Our results suggest that OsNPR1 is an early responding gene in herbivore‐induced defense and that plants can use it to activate a specific and appropriate defense response against invaders by modulating signaling pathways.     

Salicylate-mediated suppression of jasmonate-responsive gene expression in Arabidopsis is targeted downstream of the jasmonate biosynthesis pathway

Jasmonates (JAs) and salicylic acid (SA) are plant hormones that play pivotal roles in the regulation of induced defenses against microbial pathogens and insect herbivores. Their signaling pathways cross-communicate providing the plant with a regulatory potential to finely tune its defense response to the attacker(s) encountered. In Arabidopsis thaliana, SA strongly antagonizes the jasmonic acid (JA) signaling pathway, resulting in the downregulation of a large set of JA-responsive genes, including the marker genes PDF1.2 and VSP2. Induction of JA-responsive marker gene expression by different JA derivatives was equally sensitive to SA-mediated suppression. Activation of genes encoding key enzymes in the JA biosynthesis pathway, such as LOX2, AOS, AOC2, and OPR3 was also repressed by SA, suggesting that the JA biosynthesis pathway may be a target for SA-mediated antagonism. To test this, we made use of the mutant aos/dde2, which is completely blocked in its ability to produce JAs because of a mutation in the ALLENE OXIDE SYNTHASE gene. Mutant aos/dde2 plants did not express the JA-responsive marker genes PDF1.2 or VSP2 in response to infection with the necrotrophic fungus Alternaria brassicicola or the herbivorous insect Pieris rapae. Bypassing JA biosynthesis by exogenous application of methyl jasmonate (MeJA) rescued this JA-responsive phenotype in aos/dde2. Application of SA suppressed MeJA-induced PDF1.2 expression to the same level in the aos/dde2 mutant as in wild-type Col-0 plants, indicating that SA-mediated suppression of JA-responsive gene expression is targeted at a position downstream of the JA biosynthesis pathway.