慈竹CBF1基因克隆及其耐寒性研究
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摘要
CBF/DREB1是一个布冷驯化过程中起关键作用的转录因了家族,该转录因子操控很多抗逆相关基因的表达,是抗寒相关基因启动表达的总开关。本研究通过RACE技术,克隆了丛生经济竹种之一慈竹的CBF1基因全长序列克隆,同时构建慈竹CBF1基因的过表达载体和RNA干涉载体,并通过农杆菌介导法转入模式植物烟草,研究慈竹CBF1基因功能,为进一步研究慈竹耐寒机理奠定基础。主要研究结果如下：
1.以慈竹叶片为材料,根据已报道的CBF1(CRT-Binding factor1)基因的cDNA序列设计简并引物,通过TD-PCR技术,得到1条新的CBF1基因保守区片段。以这条保守区片段为模板,结合5'RACE和3'RACE得到一条新的CBF1基因全长序列,命名为NaCBF1,并提交Genbank注册,注册号为JN896707.生物信息学分析结果表明,该序列编码区(coding sequence, CDS)全长为663bp,编码221个氨基酸,其序列中存在PKRRAGRTKFKETRHP和LNFADS两大CBF1氨基酸保守域。
2.成功构建RNAi干扰载体pBI121-NaCBF1-RNAi和NaCBF1基因过表达载体pBI121-NaCBFl,并将后者通过叶盘法进行烟草转化研究。通过预培养、侵染、筛选、生根等步骤得到大量转化幼苗。常规PCR检测获得了含有NaCBF1基因的转基因烟草阳性植株,初步证明该表达载体已导入烟草叶片基因组中；半定量RT-PCR检测结果表明,NaCBF1基因在转基因烟草(如OE1、OE2和OE3株系)中的表达量与未转化的对照相比,明显增加。
3.为检测NaCBF1基因过表达对转基因烟草耐寒性影响,本研究对野生型烟草(WT)和T2代过表达NaCBF1转基因烟草(OE1、OE2和OE3),在不同温度胁迫(25℃、15℃、4℃、0℃和-2℃)下处理0h、3h、6h、12h和24h,测定其叶绿素荧光参数,及其逆境响应相关生理指标。结果表明,随着胁迫温度的降低,与野生型烟草相比,过表达NaCBF1转基因烟草的叶绿素荧光参数除NPQ值增大外,Fv/Fm、ΦPSⅡ、ETR, qP、和Fv/Fo值都减小,降幅缓慢,表明NaCBF1过表达的转基因烟草叶片的光系统受损较小,PSH反应中心丌放度增大,光合电子传递活性增强,对逆境胁迫具有更强的抗性。不同温度胁迫下,转基因烟草的MDA和RC相对较低,抗氧化酶系统(SOD、POD、CAT)均保持较高的活性,同时渗透保护物质脯氨酸、可溶性糖含量也明显担高,表明转基因烟草电解质外渗量相对较小,膜脂过氧化程度相对较低,活性氧的清除能力提高,有利于维护细胞膜结构和功能,从而缓解温度胁迫的伤害,可见NaCBF1在烟草中过表达可增加转基因烟草的耐寒性。
4.相关性分析表明,过表达NaCBF1转基因烟草的POD活性与各叶绿素荧光参数呈显著或极显著的正相关,表明其在低温或冷冻胁迫下仍能保持较高光合能力,可能与其POD活性的提高有关
CRT-binding factor/Dehydration responsive element binding (CBF/DREB1) is a small family of transcription factors which plays an important role in response to cold. It controls expressions of genes involved in cold-resistance of plant. In our study, a Neosinocalamus affinis CBF1gene (NaCBF1) was cloned by RACE. Then, a NaCBF1overexpression vector and an RNAi expression vector were generated. Furthermore, the NaCBFl overexpression vector (pBI-NaCBF1) was transformed into tobacco by A. tumefaciens-mediated transformation, which lays a foundation for studying the mechanism of cold-resistance of bamboo. Main results are as follows:
1. According to several known CBF1cDNA sequences, a pair of degenerate primer was designed. Then, a conserved sequence of CBF1gene was cloned from Neosinocalamus affinis. With the cloned fragment as the template, a full-length CBF1gene sequence from Neosinocalamus affinis was obtained through5'RACE and3'RACE, and designated as NaCBFl (Genbank:JN896707). The results of bioinformatics analysis indicated that the length of NaCBFl was663bp, encoding221amino acids with the conserved amino acid blocks PKRRAGRTKFKETRHP and LNFADS.
2. In order to analyze the function of NaCBF1, an RNAi plant expression vector (pBI-NaCBF1-CBFl) and an overexpression vector (pB1-NaCBFl) of NaCBF1gene were generated. Then, the pB1-NaCBFl vector was transformed into tobacco by A. tumefaciens-mediated transformation. Several transformation plants obtained were further confirmed by PCR and RT-PCR testing. The results showed that the expression of NaCBF1gene in OE1, OE2and OE3lines of transgenic tobaccos were higher than that of untransformed wild type tobaccos.
3. In order to detect the effects of NaCBF1overexpression on cold-resistance of transformed tobacco, wild-type and transgenic tobaccos were treated by the different temperature treatments (25℃,15℃,4℃,0℃and-2℃) with different times (0h,3h,6h,12h and24h). The results showed that under chilling or cold stress, the levels of maximal photochemical efficiency of PS II (Fv/Fm), actual photochemical efficiency of system Ⅱ (ΦPS Ⅱ), electron transport rate(ETR), photochemical quenching (qP) and potential activity of PS Ⅱ (Fv/F0) in the overexpressing NaCBFl transgenic tobacco were significantly decreased compared to wild type tobacco. However, the level of non-photochemical quenching (NPQ) in transgenic tobacco was higher than wild type tobacco under chilling stress or cold stress, which indicated that the cold resistance of transgenic tobacco with overexpressing NaCBF1was improved compared with wild type tobacco. The results also showed that, under the conditions of chilling-stress or cold-stress, the contents of relative conductivity(RC) and MDA in transgenic tobacco plants were lower than that of wild-type tobacco plants, which resulted in the decrease of electrolyte leakage and the extent of the membrane lipid perioxidation. Furthermore, in all stresses, the transgenic tobacco plants with overexpression of NaCBFl always sustained higher activities in SOD, POD and CAT, which resulted in more increase of scavenging active oxygen and less injury. Under chilling stress or cold stress, in transgenic tobacco plants with overexpression of NaCBFl gene, more prolines, soluble sugars and soluble protein were accumulated compared with wild type tobacco plants, which were favorable to maintain the function of cell membrane and improve cold resistance in transgenic tobacco.
4. Correlation analysis showed in the transgenic tobacco plants with overexpression of NaCBF1gene, POD activity was significantly correlated with chlorophyll fluorescence parameters. This result indicated that under the condition of chilling or cold stress, higher photosynthetic capacity in the transgenic tobacco plants with overexpression of NaCBF1gene was correlated with increased POD activity.
1.以慈竹叶片为材料,根据已报道的CBF1(CRT-Binding factor1)基因的cDNA序列设计简并引物,通过TD-PCR技术,得到1条新的CBF1基因保守区片段。以这条保守区片段为模板,结合5'RACE和3'RACE得到一条新的CBF1基因全长序列,命名为NaCBF1,并提交Genbank注册,注册号为JN896707.生物信息学分析结果表明,该序列编码区(coding sequence, CDS)全长为663bp,编码221个氨基酸,其序列中存在PKRRAGRTKFKETRHP和LNFADS两大CBF1氨基酸保守域。
2.成功构建RNAi干扰载体pBI121-NaCBF1-RNAi和NaCBF1基因过表达载体pBI121-NaCBFl,并将后者通过叶盘法进行烟草转化研究。通过预培养、侵染、筛选、生根等步骤得到大量转化幼苗。常规PCR检测获得了含有NaCBF1基因的转基因烟草阳性植株,初步证明该表达载体已导入烟草叶片基因组中；半定量RT-PCR检测结果表明,NaCBF1基因在转基因烟草(如OE1、OE2和OE3株系)中的表达量与未转化的对照相比,明显增加。
3.为检测NaCBF1基因过表达对转基因烟草耐寒性影响,本研究对野生型烟草(WT)和T2代过表达NaCBF1转基因烟草(OE1、OE2和OE3),在不同温度胁迫(25℃、15℃、4℃、0℃和-2℃)下处理0h、3h、6h、12h和24h,测定其叶绿素荧光参数,及其逆境响应相关生理指标。结果表明,随着胁迫温度的降低,与野生型烟草相比,过表达NaCBF1转基因烟草的叶绿素荧光参数除NPQ值增大外,Fv/Fm、ΦPSⅡ、ETR, qP、和Fv/Fo值都减小,降幅缓慢,表明NaCBF1过表达的转基因烟草叶片的光系统受损较小,PSH反应中心丌放度增大,光合电子传递活性增强,对逆境胁迫具有更强的抗性。不同温度胁迫下,转基因烟草的MDA和RC相对较低,抗氧化酶系统(SOD、POD、CAT)均保持较高的活性,同时渗透保护物质脯氨酸、可溶性糖含量也明显担高,表明转基因烟草电解质外渗量相对较小,膜脂过氧化程度相对较低,活性氧的清除能力提高,有利于维护细胞膜结构和功能,从而缓解温度胁迫的伤害,可见NaCBF1在烟草中过表达可增加转基因烟草的耐寒性。
4.相关性分析表明,过表达NaCBF1转基因烟草的POD活性与各叶绿素荧光参数呈显著或极显著的正相关,表明其在低温或冷冻胁迫下仍能保持较高光合能力,可能与其POD活性的提高有关
CRT-binding factor/Dehydration responsive element binding (CBF/DREB1) is a small family of transcription factors which plays an important role in response to cold. It controls expressions of genes involved in cold-resistance of plant. In our study, a Neosinocalamus affinis CBF1gene (NaCBF1) was cloned by RACE. Then, a NaCBF1overexpression vector and an RNAi expression vector were generated. Furthermore, the NaCBFl overexpression vector (pBI-NaCBF1) was transformed into tobacco by A. tumefaciens-mediated transformation, which lays a foundation for studying the mechanism of cold-resistance of bamboo. Main results are as follows:
1. According to several known CBF1cDNA sequences, a pair of degenerate primer was designed. Then, a conserved sequence of CBF1gene was cloned from Neosinocalamus affinis. With the cloned fragment as the template, a full-length CBF1gene sequence from Neosinocalamus affinis was obtained through5'RACE and3'RACE, and designated as NaCBFl (Genbank:JN896707). The results of bioinformatics analysis indicated that the length of NaCBFl was663bp, encoding221amino acids with the conserved amino acid blocks PKRRAGRTKFKETRHP and LNFADS.
2. In order to analyze the function of NaCBF1, an RNAi plant expression vector (pBI-NaCBF1-CBFl) and an overexpression vector (pB1-NaCBFl) of NaCBF1gene were generated. Then, the pB1-NaCBFl vector was transformed into tobacco by A. tumefaciens-mediated transformation. Several transformation plants obtained were further confirmed by PCR and RT-PCR testing. The results showed that the expression of NaCBF1gene in OE1, OE2and OE3lines of transgenic tobaccos were higher than that of untransformed wild type tobaccos.
3. In order to detect the effects of NaCBF1overexpression on cold-resistance of transformed tobacco, wild-type and transgenic tobaccos were treated by the different temperature treatments (25℃,15℃,4℃,0℃and-2℃) with different times (0h,3h,6h,12h and24h). The results showed that under chilling or cold stress, the levels of maximal photochemical efficiency of PS II (Fv/Fm), actual photochemical efficiency of system Ⅱ (ΦPS Ⅱ), electron transport rate(ETR), photochemical quenching (qP) and potential activity of PS Ⅱ (Fv/F0) in the overexpressing NaCBFl transgenic tobacco were significantly decreased compared to wild type tobacco. However, the level of non-photochemical quenching (NPQ) in transgenic tobacco was higher than wild type tobacco under chilling stress or cold stress, which indicated that the cold resistance of transgenic tobacco with overexpressing NaCBF1was improved compared with wild type tobacco. The results also showed that, under the conditions of chilling-stress or cold-stress, the contents of relative conductivity(RC) and MDA in transgenic tobacco plants were lower than that of wild-type tobacco plants, which resulted in the decrease of electrolyte leakage and the extent of the membrane lipid perioxidation. Furthermore, in all stresses, the transgenic tobacco plants with overexpression of NaCBFl always sustained higher activities in SOD, POD and CAT, which resulted in more increase of scavenging active oxygen and less injury. Under chilling stress or cold stress, in transgenic tobacco plants with overexpression of NaCBFl gene, more prolines, soluble sugars and soluble protein were accumulated compared with wild type tobacco plants, which were favorable to maintain the function of cell membrane and improve cold resistance in transgenic tobacco.
4. Correlation analysis showed in the transgenic tobacco plants with overexpression of NaCBF1gene, POD activity was significantly correlated with chlorophyll fluorescence parameters. This result indicated that under the condition of chilling or cold stress, higher photosynthetic capacity in the transgenic tobacco plants with overexpression of NaCBF1gene was correlated with increased POD activity.
引文
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