花生抗黄曲霉（Aspergillus flavus Link）侵染和产毒机制以及抗性遗传规律的研究
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摘要
培育抗黄曲霉品种是解决花生黄曲霉毒素污染最有效、最经济和最安全的方法，而明确抗性品种的抗性机制以及它的遗传规律是培育抗黄曲霉品种所必需的前提条件。
本文通过1)比较抗-感黄曲霉品种种子所固有的抗性组分的差异；2)比较抗-感种子感染A.flavus后所诱导的防卫反应的差异；3)分析抗性和感病品种杂交后代抗性的表现，探讨花生抗黄曲霉侵染和产毒的机制和抗性的遗传规律。
结果表明：
一、种皮的蜡质和角质层，以及种子所含有抗菌蛋白是花生抗黄曲霉侵染和产毒所固有的抗性机制。
1、种皮破损的花生种子黄曲霉毒素含量显著高于种皮完整的种子，表明花生种皮在抗黄曲霉侵染和产毒方面起着重要的屏障作用。用氯仿去除种皮蜡质、KOH或角质酶去除种皮角质层后均能显著提高种子黄曲霉感染率和黄曲霉毒素含量。同时去除种皮蜡质和角质层的种子与种皮破损种子的黄曲霉感染率和毒素含量差异不显著，表明种皮的抗性成份主要是蜡质和角质层。种皮蜡质含量测定和种皮表面扫描电镜观察表明，种皮表面蜡质的含量和角质层的厚度与品种的抗性有关。抗性品种种皮蜡质含量显著高于感病品种，且其表面覆盖蜡质的面积较感病品种广。种皮蜡质提取物在体外抑菌效果不显著，说明蜡质的抗性作用主要是物理性阻止黄曲霉菌的穿透。
2、不同pH值缓冲液提取蛋白抑菌试验表明花生种子pH2.8缓冲液提取蛋白中含有能抑制黄曲霉生长的有效蛋白组份。抗-感品种pH2.8缓冲液提取蛋白质的PAGE电泳图谱没有明显差异，而SDS-PAGE电泳图谱显示所有抗性品种38kDa蛋白的相对含量均比感病品种高。通过硫酸铵分级沉淀、分子筛和离子交换柱层析方法，从pH2.8缓冲液提取蛋白中分离纯化出在体外能显著地抑制A.flavus孢子萌发和菌丝生长的26.3kDa和14.2kDa蛋白。经肽质量指纹谱分析和蛋白质数据库检索，表明26.3kDa和14.2kDa蛋白可能是目前尚未发现的新蛋白质。
3、通过丙酮分级沉淀和DEAE-Sephadex A50离子交换柱，从花生种子醋酸提取液中分离纯化的具有胰蛋白酶抑制剂活性的胰蛋白酶抑制剂。SDS-PAGE电泳图谱显示，纯化的胰蛋白酶抑制剂是以二聚体形式存在，分子量分别为17.1和10.3kDa，纯化的胰蛋白酶抑制剂在体外能显著地抑制A.flavus孢子的萌发和菌丝的生长，表明胰蛋白抑制剂是花生种子抗黄曲霉的有效成分。种子胰蛋白抑制剂含量的多少、活性的高低与品种抗黄曲霉侵染的能力有关。5个抗性品种胰蛋白抑制剂的含量和活性均高于感病品种，且抗性品种胰蛋白抑制剂粗提液体外抑菌效果也较感病品种显著，表明花生种子胰蛋白抑制剂含量和活性可作为花生抗黄曲霉侵染的标记性状之一。
二、花生对黄曲霉入侵作出的主动防卫反应是多方面的，各种防下反应
具有时序性、协同性和多样性，而且在花生抗黄曲霉伎染利产迈中发拼巫要
的作川。受贝曲霉侵染后，抗－感品种防卫反应之间存在明显的斧异。
l、受黄曲霉感染后，抗－感花生种于过氧化物酶（POD）、多酚氧化酶
（PO）和苯丙氨酸解氨酶（PA L）活性以及木质素含量等均发生显义变化。
接种后POD活性随感染大数增加而增加，但抗病品种比感病品种POD活性
增幅人，接种后1～3d抗性品种PPO t性达峰值，而感病品种d才达峰
值。抗性品种PAL活性在栓种后M活性急增，并达峰值，而感病品种在4—sd
才达峰值。按种后种于木质素的含量陋着黄曲霉由侵染时间延K而增加，但
抗病品种按种5－7d木质素的增K速率和累积量明显高丁感病品种。由丁PAL
和叩D等酶活性的提高，加速了苯丙烷衍生物如木质索的人挝合成，从而加
强防御黄曲霉侵染的能力。
2、花生对茧曲霉的抗性与其受侵染后膜质过氧化的动态变化有X。受
黄曲霉侵染后，5个抗性品种的脂氧合酶活性升高的速度快丁感病品种，且
活性氧爆发较早且量人。抗性品种受侵染后屿＄1；Ho；产生速度较快，并有
两个高峰拙，第二个高峰期量人且持续时间较K，而感病品种产生速度权慢，
且仅在接种后45d有一个高峰期。相反，感染后抗性品种活性氧消除酶CAT
PJ SOD活性变化不明显，而感病品种在感染后 id急剧升高并达峰值。由丁
脂氧合酶活性提高和活性氧爆发的甲且量多，但体内的 SOD＊ICATffi性没有
变化，未能消除累积的活性氧，从而使抗性品种膜质过氧发生较甲且积度高，
表皮细胞发生及时性的过敏性反应坏死，抵御黄曲霉的穿透，达到抗黄曲霉
侵染的目的。
3、白黎芦醇是花生植保素中的一种。人土活力的花生种十不能产生白
基芦醇。受黄曲霉侵染后种子白黎卢醇产生的速度与抗性有关。抗性品种在
受侵染后3d人量累积，含量提高30倍，就达到峰值。而感病品种则要在侵
染后4d才达到此水平。放线菌素酮和放线菌素D处理后按种试验表明，种
子本身含有合成白螫芦‘醇的mRNA，当受外源Aj7Uv。。侵染时能檄活与合成
白黎芦醇相关酶的活性，合成大量白葵芦醇。但种十体山与一黎卢醇合成有
关的mRNA的数量似乎不多。
4、花生种子受黄曲霉侵染后可诱导产生相应的病程相关蛋白（PR－蛋
白），抗性品种PR－蛋白产生的速度较感病品种早，而且产生的m－蛋白?
Aflatoxins, the high carcinogenic secondary metabolites of Aspergillus flavus Link, is possess of serious health hazards to human and domestic animals because it frequently contaminates agricultural commodities.
Peanut (Arachics hypogaea L.) is one of most susceptible host crops to A.flavus invasion and subsequently aflatoxin production. Development of host resistance to A.flavus invasion and aflatoxin producing is the best and most widely explored strategy to control aflatoxin contamination. About 20 resistance genotypes have been identified since 1970's, but progress toward developing resistance variety is the slow and the mechanism and inheritance of resistance were not clear, especially the specifics markers responsible for resistance.
The objectives of this thesis were: ?to identified the main factors responsible for resistance in peanut seed; (2) to detect the difference of defense response between resistant and susceptible peanut seed after A.flavus invaded; (D to analyze the performance of resistant characters in progeny of resistant X susceptible genotype.
To gain these informations above mentioned, 5 resistant genotypes (J-ll, VRR245, PI337494-F, UF71513 and Zhangqui 48) and 4 susceptible genotypes (Yueyou 5, Yueyou 114, Shanyou 523 and Zhanyou 30) were used. The experiment results are outlined below.
1. The aflatoxin content of broken seeds was higher than intact seeds after inoculation with A.flavus. The account, distribution, bioassays in vitro of wax and cutin layers in peanut seeds was investigated. The datum revealed that there were significant differences between genotypes tested. The kernel wax contents of resistant genotypes were higher than susceptible genotypes. The bioassays in vitro showed that no significant test-by-treatment interactions were detected for wax bioassay data. Scanning electron microscopy of kernel wax revealed distinct differences between seed of resistant and susceptible genotypes. Most of resistant kernels were thick and coarse in appearance and had rich wax deposits on seed surfaces. Thin and a small amount of surface wax deposits were observed on most susceptible seeds.
Removing of wax with chloroform, and removing of cutin with KOH and cutinase or both can increase the susceptibility of peanut seeds but don't reducing their germination. The infected rate of intact seeds after removing both wax and cutin were nearly equal to those in wounded control seeds in both genotypes.
These results indicated wax and cutin layers of peanut seed might play a role in resistance to A.flavus invasion and colonization.
2. In attempt to search for zn\\-A.flavus protein, peanut seeds proteins extracted at pH2.8 and pH7.5 were analyzed with PAGE and SDS-PAGE. Consistent differences in protein profiles were detected only in proteins extracted at pH2.8 buffer among genotypes. A protein of 38kDa was unique or presented in higher concentration in resistant genotypes, whereas absent or in low concentration in susceptible genotypes. Protein extracted at pH2.8 from seed showed markedly antifungal activity against A.flavus in vitro. By means of (NH)4SO4 precipitation and chromatography, 14.2 and 26.3 kDa proteins were isolated and purified. 14.2 and 26.3 kDa proteins can inhibit spores germination and hypha extending of A.flavus in vitro. The peptide mass fingerprinting and database search showed 14.2 and 26.3 kDa proteins may be new proteins.
3. A trypsin inhibitor in peanut seeds was purified by acetone fractionation, followed by passing through DEAE-Sephadex A50 ion-exchange column. The purified inhibitor consisted of two subunits with molecular weight of 10.3 andlTkDs respectively. The inhibition of germination and hypha growth of A.flavus were observed in protein concentration 10 u g on Czapek medium. The content and activity of trypsin inhibitor were relative to genotypes resistance. The content and activity of trypsin inhibitor in resistant genotypes were significantly higher than that in susceptible genotypes.
4. The activities of perox
本文通过1)比较抗-感黄曲霉品种种子所固有的抗性组分的差异；2)比较抗-感种子感染A.flavus后所诱导的防卫反应的差异；3)分析抗性和感病品种杂交后代抗性的表现，探讨花生抗黄曲霉侵染和产毒的机制和抗性的遗传规律。
结果表明：
一、种皮的蜡质和角质层，以及种子所含有抗菌蛋白是花生抗黄曲霉侵染和产毒所固有的抗性机制。
1、种皮破损的花生种子黄曲霉毒素含量显著高于种皮完整的种子，表明花生种皮在抗黄曲霉侵染和产毒方面起着重要的屏障作用。用氯仿去除种皮蜡质、KOH或角质酶去除种皮角质层后均能显著提高种子黄曲霉感染率和黄曲霉毒素含量。同时去除种皮蜡质和角质层的种子与种皮破损种子的黄曲霉感染率和毒素含量差异不显著，表明种皮的抗性成份主要是蜡质和角质层。种皮蜡质含量测定和种皮表面扫描电镜观察表明，种皮表面蜡质的含量和角质层的厚度与品种的抗性有关。抗性品种种皮蜡质含量显著高于感病品种，且其表面覆盖蜡质的面积较感病品种广。种皮蜡质提取物在体外抑菌效果不显著，说明蜡质的抗性作用主要是物理性阻止黄曲霉菌的穿透。
2、不同pH值缓冲液提取蛋白抑菌试验表明花生种子pH2.8缓冲液提取蛋白中含有能抑制黄曲霉生长的有效蛋白组份。抗-感品种pH2.8缓冲液提取蛋白质的PAGE电泳图谱没有明显差异，而SDS-PAGE电泳图谱显示所有抗性品种38kDa蛋白的相对含量均比感病品种高。通过硫酸铵分级沉淀、分子筛和离子交换柱层析方法，从pH2.8缓冲液提取蛋白中分离纯化出在体外能显著地抑制A.flavus孢子萌发和菌丝生长的26.3kDa和14.2kDa蛋白。经肽质量指纹谱分析和蛋白质数据库检索，表明26.3kDa和14.2kDa蛋白可能是目前尚未发现的新蛋白质。
3、通过丙酮分级沉淀和DEAE-Sephadex A50离子交换柱，从花生种子醋酸提取液中分离纯化的具有胰蛋白酶抑制剂活性的胰蛋白酶抑制剂。SDS-PAGE电泳图谱显示，纯化的胰蛋白酶抑制剂是以二聚体形式存在，分子量分别为17.1和10.3kDa，纯化的胰蛋白酶抑制剂在体外能显著地抑制A.flavus孢子的萌发和菌丝的生长，表明胰蛋白抑制剂是花生种子抗黄曲霉的有效成分。种子胰蛋白抑制剂含量的多少、活性的高低与品种抗黄曲霉侵染的能力有关。5个抗性品种胰蛋白抑制剂的含量和活性均高于感病品种，且抗性品种胰蛋白抑制剂粗提液体外抑菌效果也较感病品种显著，表明花生种子胰蛋白抑制剂含量和活性可作为花生抗黄曲霉侵染的标记性状之一。
二、花生对黄曲霉入侵作出的主动防卫反应是多方面的，各种防下反应
具有时序性、协同性和多样性，而且在花生抗黄曲霉伎染利产迈中发拼巫要
的作川。受贝曲霉侵染后，抗－感品种防卫反应之间存在明显的斧异。
l、受黄曲霉感染后，抗－感花生种于过氧化物酶（POD）、多酚氧化酶
（PO）和苯丙氨酸解氨酶（PA L）活性以及木质素含量等均发生显义变化。
接种后POD活性随感染大数增加而增加，但抗病品种比感病品种POD活性
增幅人，接种后1～3d抗性品种PPO t性达峰值，而感病品种d才达峰
值。抗性品种PAL活性在栓种后M活性急增，并达峰值，而感病品种在4—sd
才达峰值。按种后种于木质素的含量陋着黄曲霉由侵染时间延K而增加，但
抗病品种按种5－7d木质素的增K速率和累积量明显高丁感病品种。由丁PAL
和叩D等酶活性的提高，加速了苯丙烷衍生物如木质索的人挝合成，从而加
强防御黄曲霉侵染的能力。
2、花生对茧曲霉的抗性与其受侵染后膜质过氧化的动态变化有X。受
黄曲霉侵染后，5个抗性品种的脂氧合酶活性升高的速度快丁感病品种，且
活性氧爆发较早且量人。抗性品种受侵染后屿＄1；Ho；产生速度较快，并有
两个高峰拙，第二个高峰期量人且持续时间较K，而感病品种产生速度权慢，
且仅在接种后45d有一个高峰期。相反，感染后抗性品种活性氧消除酶CAT
PJ SOD活性变化不明显，而感病品种在感染后 id急剧升高并达峰值。由丁
脂氧合酶活性提高和活性氧爆发的甲且量多，但体内的 SOD＊ICATffi性没有
变化，未能消除累积的活性氧，从而使抗性品种膜质过氧发生较甲且积度高，
表皮细胞发生及时性的过敏性反应坏死，抵御黄曲霉的穿透，达到抗黄曲霉
侵染的目的。
3、白黎芦醇是花生植保素中的一种。人土活力的花生种十不能产生白
基芦醇。受黄曲霉侵染后种子白黎卢醇产生的速度与抗性有关。抗性品种在
受侵染后3d人量累积，含量提高30倍，就达到峰值。而感病品种则要在侵
染后4d才达到此水平。放线菌素酮和放线菌素D处理后按种试验表明，种
子本身含有合成白螫芦‘醇的mRNA，当受外源Aj7Uv。。侵染时能檄活与合成
白黎芦醇相关酶的活性，合成大量白葵芦醇。但种十体山与一黎卢醇合成有
关的mRNA的数量似乎不多。
4、花生种子受黄曲霉侵染后可诱导产生相应的病程相关蛋白（PR－蛋
白），抗性品种PR－蛋白产生的速度较感病品种早，而且产生的m－蛋白?
Aflatoxins, the high carcinogenic secondary metabolites of Aspergillus flavus Link, is possess of serious health hazards to human and domestic animals because it frequently contaminates agricultural commodities.
Peanut (Arachics hypogaea L.) is one of most susceptible host crops to A.flavus invasion and subsequently aflatoxin production. Development of host resistance to A.flavus invasion and aflatoxin producing is the best and most widely explored strategy to control aflatoxin contamination. About 20 resistance genotypes have been identified since 1970's, but progress toward developing resistance variety is the slow and the mechanism and inheritance of resistance were not clear, especially the specifics markers responsible for resistance.
The objectives of this thesis were: ?to identified the main factors responsible for resistance in peanut seed; (2) to detect the difference of defense response between resistant and susceptible peanut seed after A.flavus invaded; (D to analyze the performance of resistant characters in progeny of resistant X susceptible genotype.
To gain these informations above mentioned, 5 resistant genotypes (J-ll, VRR245, PI337494-F, UF71513 and Zhangqui 48) and 4 susceptible genotypes (Yueyou 5, Yueyou 114, Shanyou 523 and Zhanyou 30) were used. The experiment results are outlined below.
1. The aflatoxin content of broken seeds was higher than intact seeds after inoculation with A.flavus. The account, distribution, bioassays in vitro of wax and cutin layers in peanut seeds was investigated. The datum revealed that there were significant differences between genotypes tested. The kernel wax contents of resistant genotypes were higher than susceptible genotypes. The bioassays in vitro showed that no significant test-by-treatment interactions were detected for wax bioassay data. Scanning electron microscopy of kernel wax revealed distinct differences between seed of resistant and susceptible genotypes. Most of resistant kernels were thick and coarse in appearance and had rich wax deposits on seed surfaces. Thin and a small amount of surface wax deposits were observed on most susceptible seeds.
Removing of wax with chloroform, and removing of cutin with KOH and cutinase or both can increase the susceptibility of peanut seeds but don't reducing their germination. The infected rate of intact seeds after removing both wax and cutin were nearly equal to those in wounded control seeds in both genotypes.
These results indicated wax and cutin layers of peanut seed might play a role in resistance to A.flavus invasion and colonization.
2. In attempt to search for zn\\-A.flavus protein, peanut seeds proteins extracted at pH2.8 and pH7.5 were analyzed with PAGE and SDS-PAGE. Consistent differences in protein profiles were detected only in proteins extracted at pH2.8 buffer among genotypes. A protein of 38kDa was unique or presented in higher concentration in resistant genotypes, whereas absent or in low concentration in susceptible genotypes. Protein extracted at pH2.8 from seed showed markedly antifungal activity against A.flavus in vitro. By means of (NH)4SO4 precipitation and chromatography, 14.2 and 26.3 kDa proteins were isolated and purified. 14.2 and 26.3 kDa proteins can inhibit spores germination and hypha extending of A.flavus in vitro. The peptide mass fingerprinting and database search showed 14.2 and 26.3 kDa proteins may be new proteins.
3. A trypsin inhibitor in peanut seeds was purified by acetone fractionation, followed by passing through DEAE-Sephadex A50 ion-exchange column. The purified inhibitor consisted of two subunits with molecular weight of 10.3 andlTkDs respectively. The inhibition of germination and hypha growth of A.flavus were observed in protein concentration 10 u g on Czapek medium. The content and activity of trypsin inhibitor were relative to genotypes resistance. The content and activity of trypsin inhibitor in resistant genotypes were significantly higher than that in susceptible genotypes.
4. The activities of perox
引文
1、梁炫强、潘瑞炽、宾金华，影响花生收获前黄曲霉感染因素的研究概况．中国油料作物学报，2000，22(4)：
2、梁炫强、潘瑞炽、宾金华，花生抗黄曲霉侵染机理的研究进展．中国油料作物学报，2000，22(3)：77-80．
3、梁炫强、周桂元、潘瑞炽，花生种子受黄曲霉菌侵染后若干生化成份的变化及其与抗性的关系，中国油料作物学报，2001，23(2)：26-31。
4、Liang X Q(梁炫强)and Pan R C(潘瑞炽), Studies on the mechanism of resistance to aflatoxin contamination and development of host resistance in peanut, New Millennium International Groundnut Workshop, Qingda China. 2001,pp: 66-70.
5、周桂元，梁炫强．花生种子抗黄曲霉侵染性状遗传控制的研究，花生学报，2001，30(3)：13-16。
6、周桂元，梁炫强．抗黄曲霉花生种皮纹理超微结构的研究，中国油料作物学报1999，21(1)
7、王金生编著，分子植物病理学，中国农业出版社，1998年。
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