利用cDNA-AFLP技术分析橡胶树叶片组织受多主棒孢侵染后的基因差异表达
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摘要
橡胶树棒孢霉落叶病是东南亚和中非植胶区最具破坏性的橡胶树叶部病害之一,是危害世界天然橡胶产业健康发展的重要限制性生物因子,其病原菌为多主棒孢(Corynespora cassiicola)。该病自2006年首次在国内发现为害,至今已在除广东外的主要植胶区的橡胶苗圃和部分开割胶园发生为害,且蔓延迅速,业已成为我国天然橡胶产业健康发展的潜在威胁之一。了解并利用好橡胶树抗性机制来综合防治该病已成为目前橡胶生产亟待解决的现实问题。
本研究利用cDNA-AFLP技术和常规生理生化分析手段,通过比较具有一定抗性的橡胶树品种热研7-33-97受多主棒孢病菌(HCCGD01)侵染前后的基因表达情况,筛选抗性相关的特异表达序列；并且比较不同寄主(橡胶、木薯、番木瓜和黄瓜)多主棒孢病菌侵染橡胶树叶片后防御酶活性的变化情况。研究结果发现,利用优化的cDNA-AFLP分析体系,96对选择性扩增引物的扩增,共获得372条差异谱带；其中的265条谱带表现为上调表达,107条谱带表现为下调表达。选取其中的81条差异谱带进行克隆与序列分析,经Blastx序列同源性比对发现,20条差异序列与未知功能蛋白相关,61条差异序列与已知功能蛋白具有一定的同源性；45条差异表达序列可能参与多主棒孢与橡胶树的相互作用,其功能涉及新陈代谢、蛋白质合成、定位与贮存、信号转导、物质运输和抗病等生理生化过程；其中的EY712和FI732差异表达序列功能与抗病性相关。进一步选取其中的6条差异谱带进行荧光定量PCR验证分析,结果与cDNA-AFLP技术分析获得的结果保持一致。这不仅验证了本研究利用cDNA-AFLP技术来分析橡胶树叶片组织受多主棒孢侵染后的基因差异表达的可行性,也可推测出所分析的差异表达序列可能是属于诱导表达型的基因。
不同寄主多主棒孢病菌侵染橡胶树叶片后防御酶活性变化测定结果发现,来自不同寄主的多主棒孢侵染橡胶树叶片组织所引起的四种防御酶活性变化有所不同。其中,分离自橡胶树的两株多主棒孢菌株HCCGD01和HCCHN42侵染橡胶树叶片组织后引起的防御酶活性变化最大,均表现出明显增强；来自木薯的MaCCGD02菌株侵染后防御酶活性变化次之；而来自番木瓜的CpCCYN01菌株和黄瓜的PaCCSD04菌株侵染后,除苯丙氨酸解氨酶(PAL)有所变化外,其余的(?)-1,3-葡聚糖酶、过氧化物酶(POD)和多酚氧化酶(PPO)活性变化很小。
本研究结果为进一步克隆和分析橡胶树叶片组织受多主棒孢侵染后的差异基因奠定了一定的研究基础,为阐明多主棒孢病菌与橡胶树互作机理,以及开展橡胶树抗棒孢霉落叶病分子辅助育种提供理论依据。
Corynespora Leaf Fall, caused by Corynespora cassiicola, is one of the most destructive diseases of Hevea brasiliensis in South East Asian and Central African countries. It has been a biological constraint for the healthy development of the world's natural rubber industry. The first report of Corynespora cassiicola on Hevea rubber in China was in 2006. Nowadays, the disease has occurred in some plantations on tapping and rubber nurseries of main rubber cultivation areas except Guangdong Province. The endangered areas threated by the disease have been expanded rapidly. The Corynespora Leaf Fall disease has emerged as a potential threat to healthy development of natural rubber industry in China. In order to prevent the disease, it has become an urgent problem to understand and make a good use of host resistance mechanisms in natural rubber industry.
To selecte defense-related genes we analyzed the differentially expressed genes from the leaves of CATAS 7-33-97 after inoculated with Corynespora cassiicola (HCCGD01) by cDNA-AFLP technique. At the same time, we also measured the changes of defensive ezymes activities in the leaves of CATAS 7-33-97 after inoculated by Corynespora cassiicola, which were isolated from different hosts (Hevea brasiliensis, Cassava, Papaya and Cucumber). The result showed that the 372 transcript derived fragments (TDFs) displayed altered-expression patterns after inoculation by 96 primer pairs, with 265 of which are showing up-regulated and 107 down-regulated. The 81 TDFs were cloned and sequenced, of which 61 had known functions and 21 were related with unknown protein through Blastx searching the GenBank database. The 45 TDFs may have an effect on the interactions of Hevea brasiliensis with Corynespora cassiicola, which were homologous to genes involved in some means of physiological and biochemical such as metabolism, protein synthesis, signal transduction, disease defense, protein destination and storage, transporters et al. EY712 and FI732 were related to disease defense. The 6 interesting TDFs were further selected for validation of cDNA-AFLP expression patterns by means of qRT-PCR analysis. It not noly confirmed the acuracy of the cDNA-AFLP technique, but also sepeculated that the differential expressioned sequences may be induced.
The influence of Corynespora cassiicola isolated from 4 different hosts on defensive enzymes activities (β-1,3-glucanase, POD, PAL, PPO) in the leaves of CATAS 7-33-97 was studied in the paper. The results showed that the changes of 4 kinds of defensive ezymes activities in the leaves of CATAS 7-33-97 were different with inoculated Corynespora cassiicola isolated from different hosts. The activities of these defensive enzymes quickly enhanced after inoculation by the pathogens (HCCGD01 and HCCHN42) isloated from Heavea brasiliensis, and slowly increased in leaves inoculated by MaCCGD02 isolated from cassava. However, the activities ofβ-1,3-glucanase, POD, PPO almost unchanged when the leaves were infected by PaCCSD04 from cucumber and CpCCYN01 from papaya.
The result of this study should be helpful for further cloning and analysis of the different expressed genes in the leaves of CATAS 7-33-97, which inoculated by Corynespora cassiicola. Moreover, the molecular mechanism of the interactions about Heavea brasiliensis and Corynespora cassiicola was elucidated. And it has established the theoretical basis for advanced Heavea brasiliensis molecular breeding resistant to Corynespora cassiicola.
本研究利用cDNA-AFLP技术和常规生理生化分析手段,通过比较具有一定抗性的橡胶树品种热研7-33-97受多主棒孢病菌(HCCGD01)侵染前后的基因表达情况,筛选抗性相关的特异表达序列；并且比较不同寄主(橡胶、木薯、番木瓜和黄瓜)多主棒孢病菌侵染橡胶树叶片后防御酶活性的变化情况。研究结果发现,利用优化的cDNA-AFLP分析体系,96对选择性扩增引物的扩增,共获得372条差异谱带；其中的265条谱带表现为上调表达,107条谱带表现为下调表达。选取其中的81条差异谱带进行克隆与序列分析,经Blastx序列同源性比对发现,20条差异序列与未知功能蛋白相关,61条差异序列与已知功能蛋白具有一定的同源性；45条差异表达序列可能参与多主棒孢与橡胶树的相互作用,其功能涉及新陈代谢、蛋白质合成、定位与贮存、信号转导、物质运输和抗病等生理生化过程；其中的EY712和FI732差异表达序列功能与抗病性相关。进一步选取其中的6条差异谱带进行荧光定量PCR验证分析,结果与cDNA-AFLP技术分析获得的结果保持一致。这不仅验证了本研究利用cDNA-AFLP技术来分析橡胶树叶片组织受多主棒孢侵染后的基因差异表达的可行性,也可推测出所分析的差异表达序列可能是属于诱导表达型的基因。
不同寄主多主棒孢病菌侵染橡胶树叶片后防御酶活性变化测定结果发现,来自不同寄主的多主棒孢侵染橡胶树叶片组织所引起的四种防御酶活性变化有所不同。其中,分离自橡胶树的两株多主棒孢菌株HCCGD01和HCCHN42侵染橡胶树叶片组织后引起的防御酶活性变化最大,均表现出明显增强；来自木薯的MaCCGD02菌株侵染后防御酶活性变化次之；而来自番木瓜的CpCCYN01菌株和黄瓜的PaCCSD04菌株侵染后,除苯丙氨酸解氨酶(PAL)有所变化外,其余的(?)-1,3-葡聚糖酶、过氧化物酶(POD)和多酚氧化酶(PPO)活性变化很小。
本研究结果为进一步克隆和分析橡胶树叶片组织受多主棒孢侵染后的差异基因奠定了一定的研究基础,为阐明多主棒孢病菌与橡胶树互作机理,以及开展橡胶树抗棒孢霉落叶病分子辅助育种提供理论依据。
Corynespora Leaf Fall, caused by Corynespora cassiicola, is one of the most destructive diseases of Hevea brasiliensis in South East Asian and Central African countries. It has been a biological constraint for the healthy development of the world's natural rubber industry. The first report of Corynespora cassiicola on Hevea rubber in China was in 2006. Nowadays, the disease has occurred in some plantations on tapping and rubber nurseries of main rubber cultivation areas except Guangdong Province. The endangered areas threated by the disease have been expanded rapidly. The Corynespora Leaf Fall disease has emerged as a potential threat to healthy development of natural rubber industry in China. In order to prevent the disease, it has become an urgent problem to understand and make a good use of host resistance mechanisms in natural rubber industry.
To selecte defense-related genes we analyzed the differentially expressed genes from the leaves of CATAS 7-33-97 after inoculated with Corynespora cassiicola (HCCGD01) by cDNA-AFLP technique. At the same time, we also measured the changes of defensive ezymes activities in the leaves of CATAS 7-33-97 after inoculated by Corynespora cassiicola, which were isolated from different hosts (Hevea brasiliensis, Cassava, Papaya and Cucumber). The result showed that the 372 transcript derived fragments (TDFs) displayed altered-expression patterns after inoculation by 96 primer pairs, with 265 of which are showing up-regulated and 107 down-regulated. The 81 TDFs were cloned and sequenced, of which 61 had known functions and 21 were related with unknown protein through Blastx searching the GenBank database. The 45 TDFs may have an effect on the interactions of Hevea brasiliensis with Corynespora cassiicola, which were homologous to genes involved in some means of physiological and biochemical such as metabolism, protein synthesis, signal transduction, disease defense, protein destination and storage, transporters et al. EY712 and FI732 were related to disease defense. The 6 interesting TDFs were further selected for validation of cDNA-AFLP expression patterns by means of qRT-PCR analysis. It not noly confirmed the acuracy of the cDNA-AFLP technique, but also sepeculated that the differential expressioned sequences may be induced.
The influence of Corynespora cassiicola isolated from 4 different hosts on defensive enzymes activities (β-1,3-glucanase, POD, PAL, PPO) in the leaves of CATAS 7-33-97 was studied in the paper. The results showed that the changes of 4 kinds of defensive ezymes activities in the leaves of CATAS 7-33-97 were different with inoculated Corynespora cassiicola isolated from different hosts. The activities of these defensive enzymes quickly enhanced after inoculation by the pathogens (HCCGD01 and HCCHN42) isloated from Heavea brasiliensis, and slowly increased in leaves inoculated by MaCCGD02 isolated from cassava. However, the activities ofβ-1,3-glucanase, POD, PPO almost unchanged when the leaves were infected by PaCCSD04 from cucumber and CpCCYN01 from papaya.
The result of this study should be helpful for further cloning and analysis of the different expressed genes in the leaves of CATAS 7-33-97, which inoculated by Corynespora cassiicola. Moreover, the molecular mechanism of the interactions about Heavea brasiliensis and Corynespora cassiicola was elucidated. And it has established the theoretical basis for advanced Heavea brasiliensis molecular breeding resistant to Corynespora cassiicola.
引文
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