黄瓜低霜霉威残留性的生理生化及分子基础研究
摘要
黄瓜（Cucumis sativus L.）是一种重要的可鲜食类蔬菜。蔬菜不同于粮食作物，内吸性较强，农药喷洒后容易被蔬菜组织内部吸收，残留的农药在人体内蓄积会造成慢性中毒，有的农药还有致DNA损伤、致畸和致癌的作用。更为重要的是，黄瓜等蔬菜作物因存在鲜食特性，其农药残留问题尤为突出，已成为政府、市场和消费者都非常关注的热点问题。霜霉威是一种氨基甲酸酯类杀菌剂，我国有近80%的农民使用它来防治黄瓜霜霉病。由于生产上的大剂量使用，黄瓜霜霉威残留是已经是一个影响黄瓜品质的重要问题。
本研究首先利用气相色谱法对32份不同生态类型的黄瓜种质资源进行了霜霉威残留量的测定，从中筛选出低残留资源3份和高残留资源3份。并以低残留资源D0351和高残留资源D9320为材料，从生理生化、基因组学、蛋白质组学等角度，开展了对黄瓜果实低霜霉威残留性的生理和分子机理的研究。在此基础上利用RT-PCR技术克隆出1个与黄瓜低霜霉威残留相关的ABC转运蛋白基因（ABCA19），并进行了组织特异性表达。
试验结果如下：
1.通过对32份不同生态类型的黄瓜种质资源果实进行的霜霉威残留量的测定，采用主观评价体系和按照8%的入选率，最终筛选出低霜霉威残留品系2份，残留量均在0.2mg/kg以下，依次是D0351（腌渍型）和D0406-15（欧洲温室型）；高霜霉威残留品系2份，残留量均在2.0mg/kg以上，依次是津研4号（华北型）和D9320（华南型）。
2.利用Solexa技术和比较基因组学的方法对霜霉威胁迫下黄瓜果实差异表达基因进行鉴定，结果发现黄瓜嫩果果实在霜霉威胁迫下差异表达的基因共有14530个，符合FDR≤0.001且倍数差异在2倍以上的基因共731个，上调表达的有546个，占总数的74.7%,下调表达的有185个，占总数的25.3%。在农药霜霉威胁迫下，黄瓜果实差异表达的基因主要有四大类，第一类主要为农药解毒相关的基因，包括多向耐药性蛋白基因（PDR），多药耐药性蛋白基因（MRP）,谷胱甘肽转移酶基因（GST），ABC转动蛋白基因（ABCT），细胞色素P450（CYP），苯丙氨酸解氨酶基因（PAL）等。第二类主要包括一些与转录调节相关的转录因子，涉及TCP转录因子，GATA转录因子，WRKY转录因子30，DREB1转录因子等。第三类主要包括转运与信号相关基因，蔗糖转动基因（SUT），氨基转移酶（AMT），琥珀酸脱氢酶（SUD）等。第四类主要包括胁迫与刺激相关的基因，过氧化物酶基因（POD），几丁质酶Ⅳ基因（CHI4），半胱氨酸合酶（CYS），70kDa热休克蛋白基因（HSP）等。
3、GO分析显示差异表达基因涉及了多个生物学过程，在细胞成分,分子功能和生物学进程分类中，细胞组成（cell part）,催化活性（catalytic activity）和代谢过程（metabolic process）占据各自最主要的部分。
4、在霜霉威胁迫下，黄瓜果实差异表达基因显著性分析显示差异表达基因共涉及90个Pathway。其中P≤0.05且Q≤0.5的Pathway共有8个。其中谷胱甘肽代谢通路（Glutathionemetabolism）是一条显著性富集Pathway，进一步分析表明，其中的6个谷胱甘肽代谢基因在农药霜霉威处理后均表现出上调表达趋势。
5、利用MALDI-TOF-TOF技术，获得差异蛋白的质谱图谱，用Mascot软件搜索NCBI数据库，鉴定出了18个差异表达的高丰度蛋白点。其中13个上调表达蛋白，为Spot1、6、8、10、11、12、13、14、15、16、17、18和19；5个下调表达蛋白，为Spot2、3、5、7和9。差异蛋白质点的主要功能是关于解毒、能量和转运、蛋白合成及抵抗外界胁迫反应。
6、克隆了黄瓜ABC转运蛋白基因（ABCA19），并对其蛋白质结构进行了预测。该基因序列的全长为921bp，编码306个氨基酸。黄瓜ABCA19氨基酸序列与与拟南芥的同源性为56%。该基因编码的蛋白含特有的ATP/GTP结合区A序列[ATP/GTP-binding site motif A(P-loop)]。基因表达分析表明，黄瓜ABCA19在果实中受霜霉威胁迫的响应迅速，主要在处理的前期响应表达。同时，该基因在黄瓜叶、果和茎的表达量高低顺序稳定，在叶片中是表达量最高，在茎中是表达量最低。
Cucumber (Cucumis sativus L.) is a kind of important fresh vegetable. Vegetables are differentfrom food crops, and its attractivity is stronger. Pesticides are easily absorbed by the vegetableorganization after spraying. The accumulation of pesticide residues in the human body can causechronic poisoning, and some pesticides cause DNA damage, teratogenic and carcinogenic effects.More importantly, cucumber and other vegetable crops due to the presence of fresh features,pesticide residues have become the hot issues that the government, the market, and consumers arevery concerned. Propamocarb is a carbamate fungicide, and nearly80percent of the farmers inChina use it against cucumber downy mildew. Due to the production of high-dose use,propamocarb residue of cucumber has been an important issue of cucumber quality. Study aboutlow pesticide residues of cucumber has an important theoretical and practical significance to reduce the use of pesticides, cucumber production costs, improve the quality of cucumber products, protectthe safety of the diet of the people, and enhance the international competitiveness of vegetableproducts in China.
In this study, propamocarb residues of32cucumber germplasms of different ecological typeswere determined, three low residual resources and three high residual resources were screened.With low residual resource (D0351) and high residual resource (D9320) as materials, and thecucumber fruit propamocarb residual physiological and molecular mechanism is carried out fromthe point of view of physiology and biochemistry, genomics and proteomics. On this basis, an ABCtransporter protein gene (ABCA19) related to cucumber low propamocarb residue was cloned byRT-PCR, and tissue-specific expression.
Test results are as follows:
1. This test was carried out for determination of propamocarb residue in32cucumbergermplasm resources of different ecological types. The use of subjective evaluation system andaccording to8%of the rate of selection, the final selection of low propamocarb residue strain2,followed by D0351(pickled) and D0406-15(European greenhouse type); high propamocarb residue strain2, followed by Jinyan No.4(North China type) and D9320(South China type).
2. Using Solexa technology, differentially expressed genes were identified under propamocarbtreatment of cucumber fruit through the method of comparative genomics. The results found thatdifferentially expressed genes had a total of14,530, in line with FDR≤0.001and differences in2times more than a total of731genes.546genes were up-regulated expression, accounting for74.7%, and185genes were down-regulated expression, accounting for25.3%. Under propamocarbstress, differentially expressed genes are mainly four categories, the first category is mainly relatedto pesticide detoxification genes, including the multidirectional resistance protein gene (PDR),multidrug resistance protein gene (MRP), glutathione transferase gene (GST), ABC protein gene(ABCT), cytochrome P450(CYP), phenylalanine ammonia solution enzyme gene (PAL), etc. Thesecond type mainly includes some transcription factors, including TCP transcription factors, GATAtranscription factor, WRKY transcription factor30etc. Third category mainly includes transportand signal related genes, including sucrose transport genes (SUT), transaminase (AMT), succinatedehydrogenase (SUD), etc. Fourth category mainly includes stress stimulus related genes, includingperoxidase gene (POD), cysteine synthase gene (CYS),70kDa heat shock protein gene (HSP), etc.
3. GO analysis showed that differentially expressed genes involved in several biologicalprocesses, including cellular components, molecular function and biological processes. Cell part,catalytic activity and metabolic process occupy mainpart respectively.
4. In propamocarb stress, differentially expressed genes involved90Pathway. The Pathway ofP≤0.05and Q≤0.5had a total of eight. Glutathione metabolism pathway (Glutathione metabolism)is a significant enrichment pathway among them, and further analysis showed that six genes ofglutathione metabolism were all up-regulated expression.
5. Using MALDI-TOF-TOF technology,18differentially expressed proteins of highabundance were identified with Mascot software.13protein spots showed up-regulated expression,which were Spot1,6,8,10,11,12,13,14,15,16,17,18and19;5protein spots showeddown-regulated, Spot2,3,5,7and9. The main function of the differential protein spots wasdetoxification, energy and transport, protein synthesis, and resistance to stress reaction.
6. In this study, the cucumber ABC transporter gene (ABCA19) was cloned, and its proteinstructure was predicted. The entire length sequence of this gene was921bp encoding306aminoacids. The homology of ABCA19amino acid sequence between cucumber and arabidopsis was 56%. Encoding the protein of this gene contained the ATP/GTP binding region [ATP/GTP-bindingsite motif A (P-loop)]. Gene expression analysis showed that, ABCA19responded rapidly underpropamocarb stress, mainly expressed in the early treatment. At the same time, the gene expressionfound that the leaf had the highest expression level, and stem had the lowest expression.
本研究首先利用气相色谱法对32份不同生态类型的黄瓜种质资源进行了霜霉威残留量的测定，从中筛选出低残留资源3份和高残留资源3份。并以低残留资源D0351和高残留资源D9320为材料，从生理生化、基因组学、蛋白质组学等角度，开展了对黄瓜果实低霜霉威残留性的生理和分子机理的研究。在此基础上利用RT-PCR技术克隆出1个与黄瓜低霜霉威残留相关的ABC转运蛋白基因（ABCA19），并进行了组织特异性表达。
试验结果如下：
1.通过对32份不同生态类型的黄瓜种质资源果实进行的霜霉威残留量的测定，采用主观评价体系和按照8%的入选率，最终筛选出低霜霉威残留品系2份，残留量均在0.2mg/kg以下，依次是D0351（腌渍型）和D0406-15（欧洲温室型）；高霜霉威残留品系2份，残留量均在2.0mg/kg以上，依次是津研4号（华北型）和D9320（华南型）。
2.利用Solexa技术和比较基因组学的方法对霜霉威胁迫下黄瓜果实差异表达基因进行鉴定，结果发现黄瓜嫩果果实在霜霉威胁迫下差异表达的基因共有14530个，符合FDR≤0.001且倍数差异在2倍以上的基因共731个，上调表达的有546个，占总数的74.7%,下调表达的有185个，占总数的25.3%。在农药霜霉威胁迫下，黄瓜果实差异表达的基因主要有四大类，第一类主要为农药解毒相关的基因，包括多向耐药性蛋白基因（PDR），多药耐药性蛋白基因（MRP）,谷胱甘肽转移酶基因（GST），ABC转动蛋白基因（ABCT），细胞色素P450（CYP），苯丙氨酸解氨酶基因（PAL）等。第二类主要包括一些与转录调节相关的转录因子，涉及TCP转录因子，GATA转录因子，WRKY转录因子30，DREB1转录因子等。第三类主要包括转运与信号相关基因，蔗糖转动基因（SUT），氨基转移酶（AMT），琥珀酸脱氢酶（SUD）等。第四类主要包括胁迫与刺激相关的基因，过氧化物酶基因（POD），几丁质酶Ⅳ基因（CHI4），半胱氨酸合酶（CYS），70kDa热休克蛋白基因（HSP）等。
3、GO分析显示差异表达基因涉及了多个生物学过程，在细胞成分,分子功能和生物学进程分类中，细胞组成（cell part）,催化活性（catalytic activity）和代谢过程（metabolic process）占据各自最主要的部分。
4、在霜霉威胁迫下，黄瓜果实差异表达基因显著性分析显示差异表达基因共涉及90个Pathway。其中P≤0.05且Q≤0.5的Pathway共有8个。其中谷胱甘肽代谢通路（Glutathionemetabolism）是一条显著性富集Pathway，进一步分析表明，其中的6个谷胱甘肽代谢基因在农药霜霉威处理后均表现出上调表达趋势。
5、利用MALDI-TOF-TOF技术，获得差异蛋白的质谱图谱，用Mascot软件搜索NCBI数据库，鉴定出了18个差异表达的高丰度蛋白点。其中13个上调表达蛋白，为Spot1、6、8、10、11、12、13、14、15、16、17、18和19；5个下调表达蛋白，为Spot2、3、5、7和9。差异蛋白质点的主要功能是关于解毒、能量和转运、蛋白合成及抵抗外界胁迫反应。
6、克隆了黄瓜ABC转运蛋白基因（ABCA19），并对其蛋白质结构进行了预测。该基因序列的全长为921bp，编码306个氨基酸。黄瓜ABCA19氨基酸序列与与拟南芥的同源性为56%。该基因编码的蛋白含特有的ATP/GTP结合区A序列[ATP/GTP-binding site motif A(P-loop)]。基因表达分析表明，黄瓜ABCA19在果实中受霜霉威胁迫的响应迅速，主要在处理的前期响应表达。同时，该基因在黄瓜叶、果和茎的表达量高低顺序稳定，在叶片中是表达量最高，在茎中是表达量最低。
Cucumber (Cucumis sativus L.) is a kind of important fresh vegetable. Vegetables are differentfrom food crops, and its attractivity is stronger. Pesticides are easily absorbed by the vegetableorganization after spraying. The accumulation of pesticide residues in the human body can causechronic poisoning, and some pesticides cause DNA damage, teratogenic and carcinogenic effects.More importantly, cucumber and other vegetable crops due to the presence of fresh features,pesticide residues have become the hot issues that the government, the market, and consumers arevery concerned. Propamocarb is a carbamate fungicide, and nearly80percent of the farmers inChina use it against cucumber downy mildew. Due to the production of high-dose use,propamocarb residue of cucumber has been an important issue of cucumber quality. Study aboutlow pesticide residues of cucumber has an important theoretical and practical significance to reduce the use of pesticides, cucumber production costs, improve the quality of cucumber products, protectthe safety of the diet of the people, and enhance the international competitiveness of vegetableproducts in China.
In this study, propamocarb residues of32cucumber germplasms of different ecological typeswere determined, three low residual resources and three high residual resources were screened.With low residual resource (D0351) and high residual resource (D9320) as materials, and thecucumber fruit propamocarb residual physiological and molecular mechanism is carried out fromthe point of view of physiology and biochemistry, genomics and proteomics. On this basis, an ABCtransporter protein gene (ABCA19) related to cucumber low propamocarb residue was cloned byRT-PCR, and tissue-specific expression.
Test results are as follows:
1. This test was carried out for determination of propamocarb residue in32cucumbergermplasm resources of different ecological types. The use of subjective evaluation system andaccording to8%of the rate of selection, the final selection of low propamocarb residue strain2,followed by D0351(pickled) and D0406-15(European greenhouse type); high propamocarb residue strain2, followed by Jinyan No.4(North China type) and D9320(South China type).
2. Using Solexa technology, differentially expressed genes were identified under propamocarbtreatment of cucumber fruit through the method of comparative genomics. The results found thatdifferentially expressed genes had a total of14,530, in line with FDR≤0.001and differences in2times more than a total of731genes.546genes were up-regulated expression, accounting for74.7%, and185genes were down-regulated expression, accounting for25.3%. Under propamocarbstress, differentially expressed genes are mainly four categories, the first category is mainly relatedto pesticide detoxification genes, including the multidirectional resistance protein gene (PDR),multidrug resistance protein gene (MRP), glutathione transferase gene (GST), ABC protein gene(ABCT), cytochrome P450(CYP), phenylalanine ammonia solution enzyme gene (PAL), etc. Thesecond type mainly includes some transcription factors, including TCP transcription factors, GATAtranscription factor, WRKY transcription factor30etc. Third category mainly includes transportand signal related genes, including sucrose transport genes (SUT), transaminase (AMT), succinatedehydrogenase (SUD), etc. Fourth category mainly includes stress stimulus related genes, includingperoxidase gene (POD), cysteine synthase gene (CYS),70kDa heat shock protein gene (HSP), etc.
3. GO analysis showed that differentially expressed genes involved in several biologicalprocesses, including cellular components, molecular function and biological processes. Cell part,catalytic activity and metabolic process occupy mainpart respectively.
4. In propamocarb stress, differentially expressed genes involved90Pathway. The Pathway ofP≤0.05and Q≤0.5had a total of eight. Glutathione metabolism pathway (Glutathione metabolism)is a significant enrichment pathway among them, and further analysis showed that six genes ofglutathione metabolism were all up-regulated expression.
5. Using MALDI-TOF-TOF technology,18differentially expressed proteins of highabundance were identified with Mascot software.13protein spots showed up-regulated expression,which were Spot1,6,8,10,11,12,13,14,15,16,17,18and19;5protein spots showeddown-regulated, Spot2,3,5,7and9. The main function of the differential protein spots wasdetoxification, energy and transport, protein synthesis, and resistance to stress reaction.
6. In this study, the cucumber ABC transporter gene (ABCA19) was cloned, and its proteinstructure was predicted. The entire length sequence of this gene was921bp encoding306aminoacids. The homology of ABCA19amino acid sequence between cucumber and arabidopsis was 56%. Encoding the protein of this gene contained the ATP/GTP binding region [ATP/GTP-bindingsite motif A (P-loop)]. Gene expression analysis showed that, ABCA19responded rapidly underpropamocarb stress, mainly expressed in the early treatment. At the same time, the gene expressionfound that the leaf had the highest expression level, and stem had the lowest expression.
引文
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