小麦TaCBL和TaWRKY基因家族的克隆及功能分析
摘要
作物在干旱、高温、低温或高盐等非生物胁迫后其生长发育会受到影响,并最终导致产量降低。研究表明,植物中CBL和WRKY两个基因家族在应答胁迫反应中具有重要的调控功能。本研究以小麦为研究对象,从全基因组水平对TaCBL和TaWRKY两个基因家族进行了生物信息学分析,最终克隆鉴定了6个TaCBL基因及7个TaWRKY基因,完成了小麦逆境胁迫前后基因表达水平分析和转基因功能鉴定,以期为小麦抗逆分子机理积累有价值的信息和资料。主要结果如下：
1.克隆了6个小麦TaCBL基因,分别命名为TaCBL1、TaCBL2、TaCBL3、TaCBL4、TaCBL5、 TaCBL6、通过序列分析发现TaCBL基因分别含有292、213、226、225、225、215个氨基酸残基,蛋白质结构分析表明,6个TaCBL基因都含有4个典型的钙结合蛋白必须的EF-hand结构域,TaCBL2含有2个CBL基因必须的N-豆蔻酰化位点,其它TaCBL基因含有1个N-豆蔻酰化位点。通过对小麦、水稻、拟南芥的CBL基因进行遗传进化分析,发现CBL基因共分成4组,其中TaCBL3、TaCBL4和TaCBL5归入了其中1组,其它3个基因分别归入其它3组,预示TaCBL基因可能有不同的生物学功能。
2.对6个TaCBL基因在不同组织器官的表达分析表明,TaCBL基因的表达具有组织特异性,TaCBL3和TaCBL4基因在雄蕊中高丰度表达而在其它器官中表达量极低。对6个TaCBL基因在胁迫处理下的表达分析表明,TaCBL基因对胁迫的响应较慢,暗示这些基因不是处于调控的最上游；同一TaCBL基因对不同胁迫处理有着相似的应答模式,如TaCBL1在高温、ABA、氧化、高盐、干旱胁迫中,胁迫初期其表达量微量下调,但到12h时,其表达量出现了剧烈上调,说明同一CBL基因可能参与了植物的多种胁迫应答反应；同一胁迫处理,不同的TaCBL基因也有着相似的应答反应,如高温处理时,6个TaCBL基因都出现了上调表达,干旱胁迫时,TaCBL5和TaCBL6基因都出现了持续剧烈的上调表达,说明不同的TaCBL基因可能存在功能上的冗余；同一TaCBL基因对不同的逆境处理有的也存在着截然不同的应答机制,例如TaCBL4基因在高温处理时,其出现了持续上调表达的应答模式,但在ACC、MeJA处理时却出现了持续下调的表达模式。
3.通过对6个TaCBL基因在拟南芥中超表达对其功能进行了分析,结果表明,超表达TaCBL1、 TaCBL2、TaCBL5、TaCBL6基因的拟南芥植株其耐盐性降低,超表达TaCBL4基因的拟南芥植株耐盐性和抗旱性得到了提高；超表达TaCBL1、TaCBL2基因的拟南芥植株其抗旱能力反而下降；TaCBL2、TaCBL4基因可能参与了小麦对钾离子的吸收和转运；TaCBL2、TaCBL3基因在小麦的高温应答反应中起作用。
4.在实验室前期工作基础上,我们对7个小麦TaWRKY基因进行了功能分析。结果表明,超表达TaWRKY10基因促进拟南芥提前开花,超表达TaWRKY53a基因拟南芥侧根增多。TaWRKY53a、TaWRKY68a、TaWRKY71、TaWRKY72b基因的超表达提高了拟南芥的抗旱能力。TaWRKY10基因的超表达提高了拟南芥的耐热性。
Abiotic stress is one of the major constrains for crop production. The crops are challenged by different kinds of abiotic stresses such as drought, high and low temperatures and salinity in different developmental stages, resulting in yield reduction. The calcineurin B-like (CBL) and WRKY gene families were found to be an important player in stress response. In this study, the CBL and WRKY gene families were analyzed in whole genome scale using bioinformatic tools in high temperature resistant wheat (Triticum aestivum L.) lines. Temporal and spatial expression patterns of the TaCBL and TaWRKY genes and their biological function were characterized. The main results are as follows:
1. Six TaCBL genes, TaCBLl, TaCBL2, TaCBL3, TaCBL4, TaCBL5and TaCBL6, were identified from the hexaploid wheat genome. Protein structure analyses showed that these genes belong to calcineurin B-like proteins with EF-hand motifs and N-myristoylation domains. Phylogenetic analysis indicated thatthe CBL genes were classified into four different groups in wheat, rice and Arabidopsis, indicating different biological functions of the TaCBL genes.
2. Tissue-specific expression patterns of the six TaCBL genes were observed. The real time PCR analyses showed that the TaCBL genes response slowly to stress, indicating that they were not likely involved in the upstream of stress regulation pathway. The TaCBL genes showed similar expression pattern to different stress treatment, relative low expression level in the initial stress treatments and an intense up-regulation after12h of the TaCBLl was recorded after high-temperature, ABA, oxidation, high salt and drought treatments, indicating the TaCBL genes were involved in various stress response. Meanwhile, different TaCBL genes expressed similar stress responses to the same stress treatment, for example, all the six TaCBL genes were up regulated by high temperature treatment, both TaCBL5gene and TaCBL6were dramatically up-regulated by drought treatment, suggesting that they may have functional redundancy. Distinct response mechanism for different stress treatments was also observed. The TaCBL4showed steady up-regulation to high-temperature treatment, but down-regulation to ACC and Me JA treatments.
3. The biological functions of the six TaCBL genes were characterized by overexpression analyses in Arabidopsis. The results indicated that overexpression of TaCBLl, TaCBL2, TaCBL4, TaCBL5and TaCBL6reduced salt tolerance while overexpression of TaCBL4enhanced salt tolerance in Arabidopsis. Overexpression of TaCBLl and TaCBL2reduced drought tolerance, but overexpression of TaCBL4increased the drought tolerance of Arabidopsis. We proposed that TaCBL2and TaCBL4might involve in the potassium ionic absorption and transportation pathway, leading to stress responses.
4. Further more, the expression patterns of seven TaWRKY genes were analyzed by Real time PCR and overexpressed in Arabidopsis. The results showed that TaWRKY10and TaWRKY53a might be involved in wheat morphogenesis. Overexpression of TaWRKY10shorten the flowering time and overexpression of TaWRKY53a increased the lateral root number of Arabidopsis. The results also indicated that TaWRKY19a, TaWRKY53a, TaWRKY68a, TaWRKY11and TaWRKY72b played positive regulatory role in salt stress response, while TaWRKY53a, TaWRKY68a, TaWRKY71and TaWRKY72b enhanced drought tolerance in Arabidopsis. The TaWRKY10also enhanced heat tolerance in Arabidopsiand. TaWRKY46, TaWRKY71, TaWRKY72b might be involved in the ABA regulatory pathway.
1.克隆了6个小麦TaCBL基因,分别命名为TaCBL1、TaCBL2、TaCBL3、TaCBL4、TaCBL5、 TaCBL6、通过序列分析发现TaCBL基因分别含有292、213、226、225、225、215个氨基酸残基,蛋白质结构分析表明,6个TaCBL基因都含有4个典型的钙结合蛋白必须的EF-hand结构域,TaCBL2含有2个CBL基因必须的N-豆蔻酰化位点,其它TaCBL基因含有1个N-豆蔻酰化位点。通过对小麦、水稻、拟南芥的CBL基因进行遗传进化分析,发现CBL基因共分成4组,其中TaCBL3、TaCBL4和TaCBL5归入了其中1组,其它3个基因分别归入其它3组,预示TaCBL基因可能有不同的生物学功能。
2.对6个TaCBL基因在不同组织器官的表达分析表明,TaCBL基因的表达具有组织特异性,TaCBL3和TaCBL4基因在雄蕊中高丰度表达而在其它器官中表达量极低。对6个TaCBL基因在胁迫处理下的表达分析表明,TaCBL基因对胁迫的响应较慢,暗示这些基因不是处于调控的最上游；同一TaCBL基因对不同胁迫处理有着相似的应答模式,如TaCBL1在高温、ABA、氧化、高盐、干旱胁迫中,胁迫初期其表达量微量下调,但到12h时,其表达量出现了剧烈上调,说明同一CBL基因可能参与了植物的多种胁迫应答反应；同一胁迫处理,不同的TaCBL基因也有着相似的应答反应,如高温处理时,6个TaCBL基因都出现了上调表达,干旱胁迫时,TaCBL5和TaCBL6基因都出现了持续剧烈的上调表达,说明不同的TaCBL基因可能存在功能上的冗余；同一TaCBL基因对不同的逆境处理有的也存在着截然不同的应答机制,例如TaCBL4基因在高温处理时,其出现了持续上调表达的应答模式,但在ACC、MeJA处理时却出现了持续下调的表达模式。
3.通过对6个TaCBL基因在拟南芥中超表达对其功能进行了分析,结果表明,超表达TaCBL1、 TaCBL2、TaCBL5、TaCBL6基因的拟南芥植株其耐盐性降低,超表达TaCBL4基因的拟南芥植株耐盐性和抗旱性得到了提高；超表达TaCBL1、TaCBL2基因的拟南芥植株其抗旱能力反而下降；TaCBL2、TaCBL4基因可能参与了小麦对钾离子的吸收和转运；TaCBL2、TaCBL3基因在小麦的高温应答反应中起作用。
4.在实验室前期工作基础上,我们对7个小麦TaWRKY基因进行了功能分析。结果表明,超表达TaWRKY10基因促进拟南芥提前开花,超表达TaWRKY53a基因拟南芥侧根增多。TaWRKY53a、TaWRKY68a、TaWRKY71、TaWRKY72b基因的超表达提高了拟南芥的抗旱能力。TaWRKY10基因的超表达提高了拟南芥的耐热性。
Abiotic stress is one of the major constrains for crop production. The crops are challenged by different kinds of abiotic stresses such as drought, high and low temperatures and salinity in different developmental stages, resulting in yield reduction. The calcineurin B-like (CBL) and WRKY gene families were found to be an important player in stress response. In this study, the CBL and WRKY gene families were analyzed in whole genome scale using bioinformatic tools in high temperature resistant wheat (Triticum aestivum L.) lines. Temporal and spatial expression patterns of the TaCBL and TaWRKY genes and their biological function were characterized. The main results are as follows:
1. Six TaCBL genes, TaCBLl, TaCBL2, TaCBL3, TaCBL4, TaCBL5and TaCBL6, were identified from the hexaploid wheat genome. Protein structure analyses showed that these genes belong to calcineurin B-like proteins with EF-hand motifs and N-myristoylation domains. Phylogenetic analysis indicated thatthe CBL genes were classified into four different groups in wheat, rice and Arabidopsis, indicating different biological functions of the TaCBL genes.
2. Tissue-specific expression patterns of the six TaCBL genes were observed. The real time PCR analyses showed that the TaCBL genes response slowly to stress, indicating that they were not likely involved in the upstream of stress regulation pathway. The TaCBL genes showed similar expression pattern to different stress treatment, relative low expression level in the initial stress treatments and an intense up-regulation after12h of the TaCBLl was recorded after high-temperature, ABA, oxidation, high salt and drought treatments, indicating the TaCBL genes were involved in various stress response. Meanwhile, different TaCBL genes expressed similar stress responses to the same stress treatment, for example, all the six TaCBL genes were up regulated by high temperature treatment, both TaCBL5gene and TaCBL6were dramatically up-regulated by drought treatment, suggesting that they may have functional redundancy. Distinct response mechanism for different stress treatments was also observed. The TaCBL4showed steady up-regulation to high-temperature treatment, but down-regulation to ACC and Me JA treatments.
3. The biological functions of the six TaCBL genes were characterized by overexpression analyses in Arabidopsis. The results indicated that overexpression of TaCBLl, TaCBL2, TaCBL4, TaCBL5and TaCBL6reduced salt tolerance while overexpression of TaCBL4enhanced salt tolerance in Arabidopsis. Overexpression of TaCBLl and TaCBL2reduced drought tolerance, but overexpression of TaCBL4increased the drought tolerance of Arabidopsis. We proposed that TaCBL2and TaCBL4might involve in the potassium ionic absorption and transportation pathway, leading to stress responses.
4. Further more, the expression patterns of seven TaWRKY genes were analyzed by Real time PCR and overexpressed in Arabidopsis. The results showed that TaWRKY10and TaWRKY53a might be involved in wheat morphogenesis. Overexpression of TaWRKY10shorten the flowering time and overexpression of TaWRKY53a increased the lateral root number of Arabidopsis. The results also indicated that TaWRKY19a, TaWRKY53a, TaWRKY68a, TaWRKY11and TaWRKY72b played positive regulatory role in salt stress response, while TaWRKY53a, TaWRKY68a, TaWRKY71and TaWRKY72b enhanced drought tolerance in Arabidopsis. The TaWRKY10also enhanced heat tolerance in Arabidopsiand. TaWRKY46, TaWRKY71, TaWRKY72b might be involved in the ABA regulatory pathway.
引文
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