平榛WRKY转录因子的克隆与功能鉴定
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摘要
在农业生产上，低温（冻）寒害是一种严重自然灾害，世界上每年因此造成的损失巨大。因此，植物抗寒性机理研究不仅在理论上具有十分重要的意义，在生产上也具有广泛的应用价值。平榛是我国特有的抗寒种质资源，能够耐受-48℃的低温，但是关于平榛抗寒转录因子方面的研究基本上没有报道。WRKY转录因子作为一个重要的响应非生物胁迫的转录因子家族，在植物抗寒性中也发挥着十分重要的作用。本研究中就着重研究了平榛冬季花芽转录组库中筛选得到的WRKY转录因子成员及相关基因，分析了平榛中WRKY转录因子在自然条件及人工逆境胁迫下的表达情况。通过研究获得了平榛中的抗寒关键WRKY转录因子，这对于今后采取分子辅助育种手段和转基因育种工程培育抗寒榛子新品种具有重要的意义。主要研究结果如下：
1.从平榛花芽的转录组库中共筛选得到49条WRKY转录因子的Unigene序列，其中有26条Unigene片段的序列与杨树比对上，表明平榛转录组中的WRKY序列与公共数据库中同为木本植物的杨树的WRKY转录因子家族关系较近。根据目标WRKY的Unigene序列设计3’RACE与5’RACE的引物，共克隆得到14个WRKY转录因子，分别命名为：ChWRKY1、ChWRKY2、ChWRKY28、ChWRKY7、ChWRKY13、ChWRKY10、ChWRKY11、ChWRKY18、ChWRKY32、ChWRKY9、ChWRKY33、ChWRKY20、ChWRKY4、ChWRKY6，其中13个为全长序列。获得的这14个平榛WRKY转录因子中有5个为WRKY分类中的第Ⅰ类型，有8个基因为第Ⅱ类型，ChWRKY6的结构最特殊，它属于WRKY家族类型中的第Ⅲ类型，锌指结构为C2-HC。
2.以ChActin为内参，对平榛7个ChWRKY基因在自然越冬条件下花芽中的表达情况作了初步的研究。结果表明在自然条件下ChWRKY7、ChWRKY6、ChWRKY13在11月达到最高的表达量，ChWRKY1、ChWRKY2、ChWRKY28、ChWRKY20则在12月份达到了最高的表达量。随后ChWRKY1、ChWRKY2、ChWRKY28、ChWRKY7表达量随着季节变化表达量开始逐渐降低，但ChWRKY20、ChWRKY6、ChWRKY13这3个基因在3月份的表达量略有升高。
3.对平榛ChWRKY基因在人工4℃低温及盐、旱处理下的表达情况进行了分析，结果表明ChWRKY2、ChWRKY28、ChWRKY7、ChWRKY6、ChWRKY13这5个基因均受4℃低温、盐及旱的诱导而上升表达，其中响应低温和旱处理的时间较早，响应盐胁迫的时间相对较晚。另外，ChWRKY1受低温胁迫的诱导，而ChWRKY20则受盐和旱的诱导而高表达，受低温胁迫的诱导作用较小。
4.平榛WRKY转录因子的核定位分析表明，构建好的p35S-WRKY28-GFP的重组质粒侵染细胞后，只有在细胞核上有荧光信号，而对照在细胞质、细胞核中都有绿色荧光信号。说明WRKY28是核定位蛋白，与通过软件预测的结果相一致，WRKY28作为转录因子是在细胞核上起作用。
5.构建植物表达载体PBI121-WRKY28，将构建好的表达载体通过农杆菌介导，花粉管通道法转化拟南芥，获得了WRKY28的转基因拟南芥株系。将野生型与转基因拟南芥进行4℃处理，发现野生型拟南芥在4℃处理12h后失水萎蔫现象十分明显，而转基因的拟南芥则失水萎蔫相对较少。
6.通过RACE技术获得了平榛MAP激酶级联系统中的两个基因ChMAPKKK和ChMAPKK基因。ChMAPKKK全长2396bp，包含1个1704bp的开放阅读框，编码568个氨基酸，分子量为61.68kD，理论等电点（pI）为4.75。ChMAPKK全长1665bp，包含1个1062bp的开放阅读框，编码354个氨基酸，分子量为39.32kD，理论等电点（pI）为5.82，含有磷酸一致化序列SLADIDS。在研究中发现平榛ChMAPKKK和ChMAPKK基因在旱、盐及低温胁迫处理后的总体表达趋势与平榛WRKY在相同处理下的趋势基本上相一致。
7.通过对生长季节平榛在低温、旱及盐胁迫下叶片中的可溶性蛋白、SOD、POD酶活性以及MDA、脯氨酸含量的分析，发现在不同逆境处理下这些生化物质具有不同的变化趋势。在低温胁迫下，上述指标在处理2h后均有不同程度的上升并达到最大值（除脯氨酸在4h、POD在8h达到最大值外），随后开始出现整体下降趋势，但SOD的活性有略微的上升。在旱胁迫处理下可溶性蛋白、POD酶活性、MDA和脯氨酸含量在处理2h后均上升，并达到最大值，脯氨酸在处理24h后达到最大值。在盐胁迫处理4h后MDA含量达到最大值，POD酶活性、可溶性蛋白和脯氨酸含量则在盐处理24h后达到最大值。SOD酶的活性在旱及盐胁迫处理下的变化趋势不明显。对野生型和转基因拟南芥在低温处理12h后叶片中的相对电导率、可溶性蛋白含量、SOD、CAT、POD酶活性及MDA的含量情况进行了对比分析，结果发现转基因拟南芥在低温处理12h后的相对电导率、MDA含量的增加程度小于野生型拟南芥，MDA含量越多，表明膜脂过氧化损伤程度越严重。转基因株系中的可溶性蛋白含量、SOD酶活性在处理12小时后的增加量要高于野生型拟南芥。转基因拟南芥中这些生化物质及指标的变化，提高了其在低温胁迫下的抗性。
In agricultural production, low temperature (frozen) cold harm is a kind of serious naturaldisasters, which give rise to gigantic loss every year in the world. Therefore, plant hardinessmechanism study not only plays an important role in the production in theory, but also have thewide application value in production. Corylus heterophylla Fisch. is the peculiar germplasmresources in China, which can tolerate low temperature-48℃, however, the research related toits transcription factors of cold resistance has been rarely reported. WRKY transcription factorsas an important transcription factor family responding to the abiotic stress, plays a fundamentalrole in plant hardiness. In this study, we focused on the members of WRKY transcriptionfactors screened from transcription library of Corylus heterophylla Fisch. winter bud, andrelated genes. Meanwhile, we analyzed its expression under natural conditions and artificialadversity stress, respectively. Finally, we obtained the key transcription factors of WRKYrelated to cold resistance from Corylus heterophylla Fisch., which possess an significantmeaning for the future breeding of new varieties resistant to cold by molecular assistedbreeding method and transgenic breeding engineering. The main results are as follows:
1. Getting the Unigene sequence of49WRKY transcription factors, totally, in whichthere were26sequence of Unigene fragments that can be campared to poplar, indicating thatWRKY sequence of transcription library from Corylus heterophylla Fisch. has an quite closerelation with the WRKY transcription factor family of public database in poplar as woodyplants. Designing the3'RACE and5' RACE primers according to the target WRKY Unigenesequence, we cloned and obtained14WRKY transcription factors, named ChWRKY1,ChWRKY2, ChWRKY28, ChWRKY7, ChWRKY13, ChWRKY10, ChWRKY11, ChWRKY18,ChWRKY32, ChWRKY9, ChWRKY33, ChWRKY20, ChWRKY4, ChWRKY6respectively. Thereare5genes of the14WRKY transcription factors from Corylus heterophylla Fisch. belong tothe Ⅰt ype of WRKY,8genes belong to the Ⅱ type, especially theChWRKY6belongs to theⅢ type in WRKY family, moreover, zinc finger structure of which is the C2-HC.
2. We took the ChActin as internal parameter and made a preliminary study for the expression of7ChWRKY genes in bud during the winter. The results indicate that under naturalconditions,ChWRKY7, ChWRKY6, ChWRKY13in November has the highest expression,ChWRKY1, ChWRKY2, ChWRKY28, ChWRKY20is in December.Then, ChWRKY1, ChWRKY2,ChWRKY28, ChWRKY7expression began to decrease gradually with the season change,ChWRKY20, ChWRKY6, ChWRKY13in March has a slightly increase instead.
3. Expression of ChWRKY genes from Corylus heterophylla Fisch. were analyzed underartificial4℃low temperature, salt, drought condition, respectively. The results showed thatthe expression of ChWRKY2, ChWRKY28, ChWRKY7, ChWRKY6, ChWRKY13were allincreased induced by4℃low temperature, salt and drought stress, what’s more, the responseto low temperature and drought treatment is relative earlier, and later to salt stress. In addition,ChWRKY1expression rise induced by the low temperature stress, while ChWRKY20inducedby salt and drought stress, lesser induced by the former stress.
4. Nucleus location analysis of WRKY transcription factor from Corylus heterophyllaFisch. showed that after infecting cells, p35S-WRKY28-GFP restructured plasmid only hadgreen fluorescent signals in the nucleus,while in both cytoplasm and nucleus in the control,which indicating WRKY28is an Nucleus location protein that is consistent to forecastingresults through the software. In other words, WRKY28as transcription factors, play part in thenucleus.
5. Build plant expression vector PBI121-WRKY28, will build a good expression vectorby agrobacterium mediated, pollen tube channel method into arabidopsis, won the WRKY28transgenic arabidopsis strain. Will the wild type and genetically modified arabidopsis4℃processing, found that wild type arabidopsis at4℃processing after12h wilting phenomena arevery obvious, and genetically modified arabidopsis wilting is relatively small.
6. Obtained the two genes ChMAPKKK and ChMAPKK of MAP kinase cascade systemfrom Corylus heterophylla Fisch. through the RACE technology.The overall length ofChMAPKKK was2396bp, including one1704bp open reading frame, coding568amino acids,and the molecular weight was61.68kD, and theory isoelectric point (pI) was4.75. The overalllength of ChMAPKK was1665bp, including one1062bp open reading box, coding354aminoacids, and the molecular weight was39.32kD, and theory isoelectric point (pI) was5.82. The typical phosphoric acid consistent sequence of ChMAPKK was the SLADIDS. In our study wealso found that the general expression trends of ChMAPKKK and ChMAPKK was basicallyconsistent with WRKY of Corylus heterophylla Fisch. under drought,salt,and low temperaturestress.
7. The soluble protein, SOD and POD enzyme activity, MDA, proline content in leaf ofCorylus heterophylla Fisch. under low temperature, drought and salt stress were analysesed,which had different variation trend under different adeversity treatment. Analysing the relativeelectric conductivity, soluble protein content, SOD, CAT, POD enzyme activity and the contentof MDA in leaf of the wild type and transgenetic arabidopsis after12h by low temperaturetreatment, the results showed that the relative electrical conductivity, the increase of the contentof MDA in transgenetic arabidopsis were less than wild type.The more the content of MDA,the more serious the degree of Lipid peroxidation in membrane. The incrase in soluble proteincontent, SOD enzyme activity in transgenetic arabidopsis were higher than wild typearabidopsis thaliana after12hours of treatment. The different changes of biochemical indicatorhappened in transgenetic arabidopsis so that to improve the resistance to low temperaturestress.
1.从平榛花芽的转录组库中共筛选得到49条WRKY转录因子的Unigene序列，其中有26条Unigene片段的序列与杨树比对上，表明平榛转录组中的WRKY序列与公共数据库中同为木本植物的杨树的WRKY转录因子家族关系较近。根据目标WRKY的Unigene序列设计3’RACE与5’RACE的引物，共克隆得到14个WRKY转录因子，分别命名为：ChWRKY1、ChWRKY2、ChWRKY28、ChWRKY7、ChWRKY13、ChWRKY10、ChWRKY11、ChWRKY18、ChWRKY32、ChWRKY9、ChWRKY33、ChWRKY20、ChWRKY4、ChWRKY6，其中13个为全长序列。获得的这14个平榛WRKY转录因子中有5个为WRKY分类中的第Ⅰ类型，有8个基因为第Ⅱ类型，ChWRKY6的结构最特殊，它属于WRKY家族类型中的第Ⅲ类型，锌指结构为C2-HC。
2.以ChActin为内参，对平榛7个ChWRKY基因在自然越冬条件下花芽中的表达情况作了初步的研究。结果表明在自然条件下ChWRKY7、ChWRKY6、ChWRKY13在11月达到最高的表达量，ChWRKY1、ChWRKY2、ChWRKY28、ChWRKY20则在12月份达到了最高的表达量。随后ChWRKY1、ChWRKY2、ChWRKY28、ChWRKY7表达量随着季节变化表达量开始逐渐降低，但ChWRKY20、ChWRKY6、ChWRKY13这3个基因在3月份的表达量略有升高。
3.对平榛ChWRKY基因在人工4℃低温及盐、旱处理下的表达情况进行了分析，结果表明ChWRKY2、ChWRKY28、ChWRKY7、ChWRKY6、ChWRKY13这5个基因均受4℃低温、盐及旱的诱导而上升表达，其中响应低温和旱处理的时间较早，响应盐胁迫的时间相对较晚。另外，ChWRKY1受低温胁迫的诱导，而ChWRKY20则受盐和旱的诱导而高表达，受低温胁迫的诱导作用较小。
4.平榛WRKY转录因子的核定位分析表明，构建好的p35S-WRKY28-GFP的重组质粒侵染细胞后，只有在细胞核上有荧光信号，而对照在细胞质、细胞核中都有绿色荧光信号。说明WRKY28是核定位蛋白，与通过软件预测的结果相一致，WRKY28作为转录因子是在细胞核上起作用。
5.构建植物表达载体PBI121-WRKY28，将构建好的表达载体通过农杆菌介导，花粉管通道法转化拟南芥，获得了WRKY28的转基因拟南芥株系。将野生型与转基因拟南芥进行4℃处理，发现野生型拟南芥在4℃处理12h后失水萎蔫现象十分明显，而转基因的拟南芥则失水萎蔫相对较少。
6.通过RACE技术获得了平榛MAP激酶级联系统中的两个基因ChMAPKKK和ChMAPKK基因。ChMAPKKK全长2396bp，包含1个1704bp的开放阅读框，编码568个氨基酸，分子量为61.68kD，理论等电点（pI）为4.75。ChMAPKK全长1665bp，包含1个1062bp的开放阅读框，编码354个氨基酸，分子量为39.32kD，理论等电点（pI）为5.82，含有磷酸一致化序列SLADIDS。在研究中发现平榛ChMAPKKK和ChMAPKK基因在旱、盐及低温胁迫处理后的总体表达趋势与平榛WRKY在相同处理下的趋势基本上相一致。
7.通过对生长季节平榛在低温、旱及盐胁迫下叶片中的可溶性蛋白、SOD、POD酶活性以及MDA、脯氨酸含量的分析，发现在不同逆境处理下这些生化物质具有不同的变化趋势。在低温胁迫下，上述指标在处理2h后均有不同程度的上升并达到最大值（除脯氨酸在4h、POD在8h达到最大值外），随后开始出现整体下降趋势，但SOD的活性有略微的上升。在旱胁迫处理下可溶性蛋白、POD酶活性、MDA和脯氨酸含量在处理2h后均上升，并达到最大值，脯氨酸在处理24h后达到最大值。在盐胁迫处理4h后MDA含量达到最大值，POD酶活性、可溶性蛋白和脯氨酸含量则在盐处理24h后达到最大值。SOD酶的活性在旱及盐胁迫处理下的变化趋势不明显。对野生型和转基因拟南芥在低温处理12h后叶片中的相对电导率、可溶性蛋白含量、SOD、CAT、POD酶活性及MDA的含量情况进行了对比分析，结果发现转基因拟南芥在低温处理12h后的相对电导率、MDA含量的增加程度小于野生型拟南芥，MDA含量越多，表明膜脂过氧化损伤程度越严重。转基因株系中的可溶性蛋白含量、SOD酶活性在处理12小时后的增加量要高于野生型拟南芥。转基因拟南芥中这些生化物质及指标的变化，提高了其在低温胁迫下的抗性。
In agricultural production, low temperature (frozen) cold harm is a kind of serious naturaldisasters, which give rise to gigantic loss every year in the world. Therefore, plant hardinessmechanism study not only plays an important role in the production in theory, but also have thewide application value in production. Corylus heterophylla Fisch. is the peculiar germplasmresources in China, which can tolerate low temperature-48℃, however, the research related toits transcription factors of cold resistance has been rarely reported. WRKY transcription factorsas an important transcription factor family responding to the abiotic stress, plays a fundamentalrole in plant hardiness. In this study, we focused on the members of WRKY transcriptionfactors screened from transcription library of Corylus heterophylla Fisch. winter bud, andrelated genes. Meanwhile, we analyzed its expression under natural conditions and artificialadversity stress, respectively. Finally, we obtained the key transcription factors of WRKYrelated to cold resistance from Corylus heterophylla Fisch., which possess an significantmeaning for the future breeding of new varieties resistant to cold by molecular assistedbreeding method and transgenic breeding engineering. The main results are as follows:
1. Getting the Unigene sequence of49WRKY transcription factors, totally, in whichthere were26sequence of Unigene fragments that can be campared to poplar, indicating thatWRKY sequence of transcription library from Corylus heterophylla Fisch. has an quite closerelation with the WRKY transcription factor family of public database in poplar as woodyplants. Designing the3'RACE and5' RACE primers according to the target WRKY Unigenesequence, we cloned and obtained14WRKY transcription factors, named ChWRKY1,ChWRKY2, ChWRKY28, ChWRKY7, ChWRKY13, ChWRKY10, ChWRKY11, ChWRKY18,ChWRKY32, ChWRKY9, ChWRKY33, ChWRKY20, ChWRKY4, ChWRKY6respectively. Thereare5genes of the14WRKY transcription factors from Corylus heterophylla Fisch. belong tothe Ⅰt ype of WRKY,8genes belong to the Ⅱ type, especially theChWRKY6belongs to theⅢ type in WRKY family, moreover, zinc finger structure of which is the C2-HC.
2. We took the ChActin as internal parameter and made a preliminary study for the expression of7ChWRKY genes in bud during the winter. The results indicate that under naturalconditions,ChWRKY7, ChWRKY6, ChWRKY13in November has the highest expression,ChWRKY1, ChWRKY2, ChWRKY28, ChWRKY20is in December.Then, ChWRKY1, ChWRKY2,ChWRKY28, ChWRKY7expression began to decrease gradually with the season change,ChWRKY20, ChWRKY6, ChWRKY13in March has a slightly increase instead.
3. Expression of ChWRKY genes from Corylus heterophylla Fisch. were analyzed underartificial4℃low temperature, salt, drought condition, respectively. The results showed thatthe expression of ChWRKY2, ChWRKY28, ChWRKY7, ChWRKY6, ChWRKY13were allincreased induced by4℃low temperature, salt and drought stress, what’s more, the responseto low temperature and drought treatment is relative earlier, and later to salt stress. In addition,ChWRKY1expression rise induced by the low temperature stress, while ChWRKY20inducedby salt and drought stress, lesser induced by the former stress.
4. Nucleus location analysis of WRKY transcription factor from Corylus heterophyllaFisch. showed that after infecting cells, p35S-WRKY28-GFP restructured plasmid only hadgreen fluorescent signals in the nucleus,while in both cytoplasm and nucleus in the control,which indicating WRKY28is an Nucleus location protein that is consistent to forecastingresults through the software. In other words, WRKY28as transcription factors, play part in thenucleus.
5. Build plant expression vector PBI121-WRKY28, will build a good expression vectorby agrobacterium mediated, pollen tube channel method into arabidopsis, won the WRKY28transgenic arabidopsis strain. Will the wild type and genetically modified arabidopsis4℃processing, found that wild type arabidopsis at4℃processing after12h wilting phenomena arevery obvious, and genetically modified arabidopsis wilting is relatively small.
6. Obtained the two genes ChMAPKKK and ChMAPKK of MAP kinase cascade systemfrom Corylus heterophylla Fisch. through the RACE technology.The overall length ofChMAPKKK was2396bp, including one1704bp open reading frame, coding568amino acids,and the molecular weight was61.68kD, and theory isoelectric point (pI) was4.75. The overalllength of ChMAPKK was1665bp, including one1062bp open reading box, coding354aminoacids, and the molecular weight was39.32kD, and theory isoelectric point (pI) was5.82. The typical phosphoric acid consistent sequence of ChMAPKK was the SLADIDS. In our study wealso found that the general expression trends of ChMAPKKK and ChMAPKK was basicallyconsistent with WRKY of Corylus heterophylla Fisch. under drought,salt,and low temperaturestress.
7. The soluble protein, SOD and POD enzyme activity, MDA, proline content in leaf ofCorylus heterophylla Fisch. under low temperature, drought and salt stress were analysesed,which had different variation trend under different adeversity treatment. Analysing the relativeelectric conductivity, soluble protein content, SOD, CAT, POD enzyme activity and the contentof MDA in leaf of the wild type and transgenetic arabidopsis after12h by low temperaturetreatment, the results showed that the relative electrical conductivity, the increase of the contentof MDA in transgenetic arabidopsis were less than wild type.The more the content of MDA,the more serious the degree of Lipid peroxidation in membrane. The incrase in soluble proteincontent, SOD enzyme activity in transgenetic arabidopsis were higher than wild typearabidopsis thaliana after12hours of treatment. The different changes of biochemical indicatorhappened in transgenetic arabidopsis so that to improve the resistance to low temperaturestress.
引文
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