BTH诱导橡胶树对白粉病的抗性研究
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摘要
橡胶白粉病是橡胶的重要病害之一,由橡胶粉孢属(Oidium heveae B. A. Steinm)侵染引起,严重影响橡胶树生长和产量。诱导植物抗性是用生物和非生物因子激发植物的自身免疫力,达到防病的效果。利用诱导植物抗性来防治病虫草害已成为植保技术的新途径。
本研究通过盆栽试验,以橡胶品种热研7-33-97和白粉菌为试验材料,对BTH(苯并噻二唑)、PHDC(调环酸钙)、禾甲安和碧护4种诱导剂进行筛选,并选用有效诱导剂BTH对诱导间隔期、诱导次数和诱导抗性相关酶的活性进行了系列试验。同时利用SSH(抑制消减杂交)技术构建了BTH诱导的橡胶抗白粉病的叶片cDNA差减文库,分析BTH诱导后橡胶抗白粉病的基因表达情况,试图从分子水平揭示橡胶的抗病机制,为橡胶抗白粉病的防治和研究提供新方法。
研究结果如下：
1. BTH、PHDC、禾甲安和碧护4种诱导剂对橡胶白粉病均有一定的防治效果,其中BTH250mg/L和禾甲安500倍液的效果较好,分别为57.8%和51.4%,但BTH显著优于禾甲安和其它处理。
2.用BTH 250mg/L处理橡胶芽接苗,不同接种时间间隔实验结果表明,最佳诱导间隔期为5-11天,防治效果在33-57%,持续期至少11d。诱导后,随着接种间隔期的延长,诱导抗病性的效果也在减弱。
3. BTH 250mg/L处理并接种病原菌后,POD(过氧化物酶)和PAL(苯丙氨酸解氨酶)的活性比对照显著升高;当酶活性下降至与对照无差异时,进行第2次施药,POD、PAL的活性会再次升高,抗病性增强。
4.通过试剂盒RNAiso Reagent法、改良SDS法和改良CTAB-PVP法3种方法对橡胶叶片总RNA的提取进行比较后发现,采用改良SDS法能去除叶片中含有的胶乳,有利于抑制消减杂交文库的构建。
5.选用Clontech公司的PCR-select cDNA subtraction kit,构建了BTH诱导的橡胶抗白粉病叶片cDNA文库,并对SSH的各个关键步骤如接头连接效率、消减效率、转化效率等的检测结果显示,差减文库有较高的质量。
6.从文库中随机挑取42个阳性克隆进行测序,并在Genbank上对核酸序列比较后发现,在已知功能的核酸序列中,有能量和基础代谢类、信号转导类、膜和转运类和次生代谢类共21条序列和2条重复序列,未知功能的有14条序列,数据库中无显著匹配的有2条序列。
BTH通过诱导橡胶树叶片防御相关基因的表达,提高抗性相关酶的活性,有效控制橡胶树白粉病,为橡胶树白粉病防治提供了新的途径,也为进一步研究橡胶树防御基因的表达和信号转导等打下基础。
Rubber powdery mildew is one of the most important diseases on rubber trees, which is caused by Oidium heveae B.A.Steinm. It affects seriously the growth and yield of rubber trees. Induced resistance is the use of biotic or abiotic inducers to stimulate plant defense genes to achieve the effects of disease prevention. Induction of plant resistance has become a new way of plant protection to control plant diseases, pests and parasitic weeds.
In this experiment, four inducers of BTH (benzothiadiazole), PHDC (Prohexadione calcium), Chitosan and Comcat(?) were screened with rubber clone Reyan 7-33-97 and Oidium in pot tests. The effective inducer BTH was used to conduct a series of tests on induction intervals, the numbers of induction and the activity of enzymes related. At the same time, the cDNA library induced by BTH to powdery mildew of rubber tree leaves was established using suppression subtractive hybridization (SSH) technology. Gene expression of induced resistance to powdery mildew was analysed. It tries to explain the mechanism of BTH-induced resistance to rubber tree powdery mildew in molecular level and provide a new control method for the disease. The results of the study included the following six aspects:
1. Four inducers of BTH, PHDC, Chitosan and Comcat could induce rubber tree resistance to powdery mildew. BTH at 250mg/L and Chitosan at 500 times had better control of 57.8% and 51.4%, respectively. But BTH was significant better than Chitosan and other treatments.
2. The optimal inoculation intervals were from 5 to 11 days after induction of BTH at 250mg/L, those had control efficacy of 33 to 57%. The duration was at least more than 11d. After induction, with the inoculation interval increasing, the effect of induced resistance was also reduced.
3. Activities of POD (peroxidase) and PAL (phenylalanine ammonia lyase) were significantly higher than control after BTH at 250mg/L treatment and the pathogen inoculation. Activities of the enzymes were increased again after BTH twice treatment when the enzyme activities declined to control level.
4. The modified SDS method was the best method for extraction of total rubber RNA, which could effectively remove the leaves latex and polysaccharides and conductively construct cDNA library with suppression subtractive hybridization (SSH), after comparison of RNAiso Reagent Kit, modified SDS and modified CTAB-PVP method.
5. cDNA library of BTH induced rubber tree resistance to powdery mildew was constructed by Clontech's PCR-select cDNA subtraction kit. so in the case of small amount of RNA synthesis of cDNA has high efficiency, the smooth development of SSH basis. The subtractive library had high quality after detection of all key processes of SSH, such as joint connection efficiency, subtractive efficiency and conversion efficiency.
6. There were 21 cDNA sequences and 2 repeats known of function as energy and basic metabolism, signal transduction, membrane and transport and secondary metabolism,14 sequences unknown of function and 2 sequence no significant match in nucleic acid sequences database after detection of sequences of 42 positive clones randomly selected from the cDNA library and comparison on nucleic acid sequences in Genbank.
BTH could effectively induced rubber tree resistance to powdery mildew through increasing expresses of defense-related genes and activities of resistance-related enzymes in leaves of rubber tree. It provides a new approach for rubber tree powdery mildew control and lays a good foundation for further study on expresses of defense-related genes and signal translocation in rubber trees.
本研究通过盆栽试验,以橡胶品种热研7-33-97和白粉菌为试验材料,对BTH(苯并噻二唑)、PHDC(调环酸钙)、禾甲安和碧护4种诱导剂进行筛选,并选用有效诱导剂BTH对诱导间隔期、诱导次数和诱导抗性相关酶的活性进行了系列试验。同时利用SSH(抑制消减杂交)技术构建了BTH诱导的橡胶抗白粉病的叶片cDNA差减文库,分析BTH诱导后橡胶抗白粉病的基因表达情况,试图从分子水平揭示橡胶的抗病机制,为橡胶抗白粉病的防治和研究提供新方法。
研究结果如下：
1. BTH、PHDC、禾甲安和碧护4种诱导剂对橡胶白粉病均有一定的防治效果,其中BTH250mg/L和禾甲安500倍液的效果较好,分别为57.8%和51.4%,但BTH显著优于禾甲安和其它处理。
2.用BTH 250mg/L处理橡胶芽接苗,不同接种时间间隔实验结果表明,最佳诱导间隔期为5-11天,防治效果在33-57%,持续期至少11d。诱导后,随着接种间隔期的延长,诱导抗病性的效果也在减弱。
3. BTH 250mg/L处理并接种病原菌后,POD(过氧化物酶)和PAL(苯丙氨酸解氨酶)的活性比对照显著升高;当酶活性下降至与对照无差异时,进行第2次施药,POD、PAL的活性会再次升高,抗病性增强。
4.通过试剂盒RNAiso Reagent法、改良SDS法和改良CTAB-PVP法3种方法对橡胶叶片总RNA的提取进行比较后发现,采用改良SDS法能去除叶片中含有的胶乳,有利于抑制消减杂交文库的构建。
5.选用Clontech公司的PCR-select cDNA subtraction kit,构建了BTH诱导的橡胶抗白粉病叶片cDNA文库,并对SSH的各个关键步骤如接头连接效率、消减效率、转化效率等的检测结果显示,差减文库有较高的质量。
6.从文库中随机挑取42个阳性克隆进行测序,并在Genbank上对核酸序列比较后发现,在已知功能的核酸序列中,有能量和基础代谢类、信号转导类、膜和转运类和次生代谢类共21条序列和2条重复序列,未知功能的有14条序列,数据库中无显著匹配的有2条序列。
BTH通过诱导橡胶树叶片防御相关基因的表达,提高抗性相关酶的活性,有效控制橡胶树白粉病,为橡胶树白粉病防治提供了新的途径,也为进一步研究橡胶树防御基因的表达和信号转导等打下基础。
Rubber powdery mildew is one of the most important diseases on rubber trees, which is caused by Oidium heveae B.A.Steinm. It affects seriously the growth and yield of rubber trees. Induced resistance is the use of biotic or abiotic inducers to stimulate plant defense genes to achieve the effects of disease prevention. Induction of plant resistance has become a new way of plant protection to control plant diseases, pests and parasitic weeds.
In this experiment, four inducers of BTH (benzothiadiazole), PHDC (Prohexadione calcium), Chitosan and Comcat(?) were screened with rubber clone Reyan 7-33-97 and Oidium in pot tests. The effective inducer BTH was used to conduct a series of tests on induction intervals, the numbers of induction and the activity of enzymes related. At the same time, the cDNA library induced by BTH to powdery mildew of rubber tree leaves was established using suppression subtractive hybridization (SSH) technology. Gene expression of induced resistance to powdery mildew was analysed. It tries to explain the mechanism of BTH-induced resistance to rubber tree powdery mildew in molecular level and provide a new control method for the disease. The results of the study included the following six aspects:
1. Four inducers of BTH, PHDC, Chitosan and Comcat could induce rubber tree resistance to powdery mildew. BTH at 250mg/L and Chitosan at 500 times had better control of 57.8% and 51.4%, respectively. But BTH was significant better than Chitosan and other treatments.
2. The optimal inoculation intervals were from 5 to 11 days after induction of BTH at 250mg/L, those had control efficacy of 33 to 57%. The duration was at least more than 11d. After induction, with the inoculation interval increasing, the effect of induced resistance was also reduced.
3. Activities of POD (peroxidase) and PAL (phenylalanine ammonia lyase) were significantly higher than control after BTH at 250mg/L treatment and the pathogen inoculation. Activities of the enzymes were increased again after BTH twice treatment when the enzyme activities declined to control level.
4. The modified SDS method was the best method for extraction of total rubber RNA, which could effectively remove the leaves latex and polysaccharides and conductively construct cDNA library with suppression subtractive hybridization (SSH), after comparison of RNAiso Reagent Kit, modified SDS and modified CTAB-PVP method.
5. cDNA library of BTH induced rubber tree resistance to powdery mildew was constructed by Clontech's PCR-select cDNA subtraction kit. so in the case of small amount of RNA synthesis of cDNA has high efficiency, the smooth development of SSH basis. The subtractive library had high quality after detection of all key processes of SSH, such as joint connection efficiency, subtractive efficiency and conversion efficiency.
6. There were 21 cDNA sequences and 2 repeats known of function as energy and basic metabolism, signal transduction, membrane and transport and secondary metabolism,14 sequences unknown of function and 2 sequence no significant match in nucleic acid sequences database after detection of sequences of 42 positive clones randomly selected from the cDNA library and comparison on nucleic acid sequences in Genbank.
BTH could effectively induced rubber tree resistance to powdery mildew through increasing expresses of defense-related genes and activities of resistance-related enzymes in leaves of rubber tree. It provides a new approach for rubber tree powdery mildew control and lays a good foundation for further study on expresses of defense-related genes and signal translocation in rubber trees.
引文
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