大白菜DREB类转录因子cDNA的克隆及植物表达载体的构建
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摘要
干旱、盐碱、低温等非生物逆境胁迫对植物的生长发育有重要影响,是农作物减产的直接原因。提高植物的抗逆性可以减轻胁迫伤害、提高产量、节约淡水资源,对农业增产、生态环境改良、实现可持续发展具有重要意义。DREB转录因子能特异性地与DRE元件(脱水应答元件)结合,激活许多逆境诱导基因的表达,它在植物对干旱、高盐及低温胁迫的分子反应中起着重要的调控作用。逆境胁迫下DREB转录因子能够调节功能基因的表达,普遍提高植物对逆境的抵抗和忍耐能力,因而能够综合改良植物的抗逆性状,是迄今为止最理想的植物抗逆工程基因。
本研究以逆境处理的大白菜为试验材料,运用RT-PCR技术,克隆大白菜DREB转录因子基因cDNA序列,对逆境胁迫下的基因诱导表达模式进行了研究,并成功构建了植物表达载体,结果如下:
1.根据已发表的甘蓝型油菜DREB转录因子基因序列设计引物DR2-1和DR2-2,进行RT-PCR扩增,得到两个长645bp编码214个氨基酸的基因,分别命名为BcDREB1(GenBank登录号为:EU924266)和BcDREB2(GenBank登录号为:GQ122331)。同源性分析表明,两个基因与甘蓝型油菜DREB2-2基因、芥菜DREB1B基因的氨基酸序列同源性均在90%以上。
2.利用SMART和Protparam分析表明,BcDREB1推断的氨基酸相对分子量为23.90KD、等电点为4.88;BcDREB2推断的氨基酸相对分子量为23.85KD,等电点5.21,它们都包含一个典型的AP2结构域。
3.半定量RT-PCR分析表明,低温、干旱、高盐均能诱导大白菜DREB基因表达。在低温胁迫时1h开始表达,3h到达最高;在高盐胁迫时1h开始表达,12h到达最高;在干旱胁迫时3h开始表达,12h到达最高。
4.设计含有XbaI和BamHI酶切位点的正义特异性引物DR-F和DR-R,用BamHI和XbaI分别酶切含有BcDREB1基因的质粒和pCAMBIA2301载体,用T4 DNA Ligase连接,构建成大白菜BcDREB1基因的正义植物表达载体pCAM-BcDREB1。
5.设计含有BamHI和XbaI酶切位点的反义特异性引物DR1-F和DR1-R,用BamHI和XbaI分别酶切含有BcDREB1、BcDREB2基因的质粒和pCAMBIA2301载体,分别用T4 DNA Ligase连接,构建成大白菜BcDREB1、BcDREB2基因的反义植物表达载体anti-pCAM-BcDREB1和anti-pCAM- BcDREB2。
6.利用冻融法将植物表达载体pCAM-BcDREB1、anti-pCAM-BcDREB1和anti-pCAM-BcDREB2转化到农杆菌EHA105中,经特异性引物对转化菌液进行PCR扩增,证实表达载体已转入农杆菌。
Drought, salinity, low-temperature and other abiotic adversity stress, impacting on plant growth development significantly, are the direct cause of reduction in crop production. Increasing the stress resistance of plants is important in agricultural production, improvement of ecological environment and sustainable development, because it can reduce the stress injury, increase crop production and save fresh water resources. The DREB transcription factor can specifically combine with the DRE (dehydration responsion element) and activate expression of many stress-induced genes , resulting in regulating and controlling the molecules response to drought, hyperhaline and low-temperature stress. The DREB transcription factor, regulating the expression of functional genes under the stress and increasing the capacity of resistance and endurance to adversity of plant, is the best adversity resistance gene in plant by far.
In this study, Chinese cabbage dealt with the adversity was selected as experiment material. Using RT-PCR technology, we obtained the cDNA sequences of Chinese cabbage DREB transcription factor’s. Under adversity stress, stress-induced expression patterns of DREB transcription factor in Chinese cabbage was studied and plant expression vector was constructed successfully. The results are as follows:
1. According to published gene sequence of Brassica napus DREB transcription factor, primers DR2-1 and DR2-2 were designed and two genes with 645bp in length were amplified by RT-PCR. Each gene contained a complete sequence encoding 214 amino acids, named BcDREB1 (GenBank accession number: EU924266) and BcDREB2 (GenBank accession number: GQ122331) respectively. Homological analysis showed that over 90% amino acids in the two sequences were similar to Brassica napus DREB2-2 gene and Brassica juncea Coss DREB1B gene.
2. SMART and Protparam analysis indicated that the deduced relative molecular weight of the amino acid was 23.90KD for BcDREB1 and 23.85KD for BcDREB2, and the isoelectric point was 4.88 BcDREB1 and 5.21 for BcDREB2. Furthermore, a typical AP2 structure domain was found in both of gene structures.
3. Using semi-quantitative RT-PCR. analysis of expression modes showed that BcDREB gene expression induced by low temperature, drought and hyperhalin. The gene started to express after 1h and reached the highest level after 3h under the low-temperature stress, started to express after 1h and reached the highest level after 12h under the drought stress, and started to express after 3h and reached highest level after 12h under the high-salt stress.
4. The sensed specific primer DR-F and DR-R,containing XbaI and BamHI restriction sites, were designed.Plasmid containing BcDREB1 gene and pCAMBIA2301 vector was digested with BamHI and XbaI,respectively. The T4 DNA Ligase was used to be ligated, sensed plant expression vector pCAM-BcDREB1 of BcDREB1 gene was built in Chinese cabbage.
5. The antisensed specific primer DR-F and DR-R were designed which contained BamHI and XbaI restriction sites. Plasmid containing gene BcDREB1, BcDREB2 and pCAMBIA2301 vector were digested with BamHI and XbaI, respectively, connected using T4 DNA Ligase, antisense plant expression vector anti-pCAM-BcDREB1 and anti-pCAM-BcDREB2 of BcDREB1, BcDREB2 gene were built in Chinese cabbage.
6. The plant expression vectors pCAM-BcDREB1, anti-pCAM-BcDREB1 and anti-pCAM-BcDREB2 were transformed into Agrobacterium EHA105 using freeze-thaw method. The PCR amplification specific primers was further carried out in transformed bacteria, expression vectors were confirmed to transfer into the Agrobacterium.
本研究以逆境处理的大白菜为试验材料,运用RT-PCR技术,克隆大白菜DREB转录因子基因cDNA序列,对逆境胁迫下的基因诱导表达模式进行了研究,并成功构建了植物表达载体,结果如下:
1.根据已发表的甘蓝型油菜DREB转录因子基因序列设计引物DR2-1和DR2-2,进行RT-PCR扩增,得到两个长645bp编码214个氨基酸的基因,分别命名为BcDREB1(GenBank登录号为:EU924266)和BcDREB2(GenBank登录号为:GQ122331)。同源性分析表明,两个基因与甘蓝型油菜DREB2-2基因、芥菜DREB1B基因的氨基酸序列同源性均在90%以上。
2.利用SMART和Protparam分析表明,BcDREB1推断的氨基酸相对分子量为23.90KD、等电点为4.88;BcDREB2推断的氨基酸相对分子量为23.85KD,等电点5.21,它们都包含一个典型的AP2结构域。
3.半定量RT-PCR分析表明,低温、干旱、高盐均能诱导大白菜DREB基因表达。在低温胁迫时1h开始表达,3h到达最高;在高盐胁迫时1h开始表达,12h到达最高;在干旱胁迫时3h开始表达,12h到达最高。
4.设计含有XbaI和BamHI酶切位点的正义特异性引物DR-F和DR-R,用BamHI和XbaI分别酶切含有BcDREB1基因的质粒和pCAMBIA2301载体,用T4 DNA Ligase连接,构建成大白菜BcDREB1基因的正义植物表达载体pCAM-BcDREB1。
5.设计含有BamHI和XbaI酶切位点的反义特异性引物DR1-F和DR1-R,用BamHI和XbaI分别酶切含有BcDREB1、BcDREB2基因的质粒和pCAMBIA2301载体,分别用T4 DNA Ligase连接,构建成大白菜BcDREB1、BcDREB2基因的反义植物表达载体anti-pCAM-BcDREB1和anti-pCAM- BcDREB2。
6.利用冻融法将植物表达载体pCAM-BcDREB1、anti-pCAM-BcDREB1和anti-pCAM-BcDREB2转化到农杆菌EHA105中,经特异性引物对转化菌液进行PCR扩增,证实表达载体已转入农杆菌。
Drought, salinity, low-temperature and other abiotic adversity stress, impacting on plant growth development significantly, are the direct cause of reduction in crop production. Increasing the stress resistance of plants is important in agricultural production, improvement of ecological environment and sustainable development, because it can reduce the stress injury, increase crop production and save fresh water resources. The DREB transcription factor can specifically combine with the DRE (dehydration responsion element) and activate expression of many stress-induced genes , resulting in regulating and controlling the molecules response to drought, hyperhaline and low-temperature stress. The DREB transcription factor, regulating the expression of functional genes under the stress and increasing the capacity of resistance and endurance to adversity of plant, is the best adversity resistance gene in plant by far.
In this study, Chinese cabbage dealt with the adversity was selected as experiment material. Using RT-PCR technology, we obtained the cDNA sequences of Chinese cabbage DREB transcription factor’s. Under adversity stress, stress-induced expression patterns of DREB transcription factor in Chinese cabbage was studied and plant expression vector was constructed successfully. The results are as follows:
1. According to published gene sequence of Brassica napus DREB transcription factor, primers DR2-1 and DR2-2 were designed and two genes with 645bp in length were amplified by RT-PCR. Each gene contained a complete sequence encoding 214 amino acids, named BcDREB1 (GenBank accession number: EU924266) and BcDREB2 (GenBank accession number: GQ122331) respectively. Homological analysis showed that over 90% amino acids in the two sequences were similar to Brassica napus DREB2-2 gene and Brassica juncea Coss DREB1B gene.
2. SMART and Protparam analysis indicated that the deduced relative molecular weight of the amino acid was 23.90KD for BcDREB1 and 23.85KD for BcDREB2, and the isoelectric point was 4.88 BcDREB1 and 5.21 for BcDREB2. Furthermore, a typical AP2 structure domain was found in both of gene structures.
3. Using semi-quantitative RT-PCR. analysis of expression modes showed that BcDREB gene expression induced by low temperature, drought and hyperhalin. The gene started to express after 1h and reached the highest level after 3h under the low-temperature stress, started to express after 1h and reached the highest level after 12h under the drought stress, and started to express after 3h and reached highest level after 12h under the high-salt stress.
4. The sensed specific primer DR-F and DR-R,containing XbaI and BamHI restriction sites, were designed.Plasmid containing BcDREB1 gene and pCAMBIA2301 vector was digested with BamHI and XbaI,respectively. The T4 DNA Ligase was used to be ligated, sensed plant expression vector pCAM-BcDREB1 of BcDREB1 gene was built in Chinese cabbage.
5. The antisensed specific primer DR-F and DR-R were designed which contained BamHI and XbaI restriction sites. Plasmid containing gene BcDREB1, BcDREB2 and pCAMBIA2301 vector were digested with BamHI and XbaI, respectively, connected using T4 DNA Ligase, antisense plant expression vector anti-pCAM-BcDREB1 and anti-pCAM-BcDREB2 of BcDREB1, BcDREB2 gene were built in Chinese cabbage.
6. The plant expression vectors pCAM-BcDREB1, anti-pCAM-BcDREB1 and anti-pCAM-BcDREB2 were transformed into Agrobacterium EHA105 using freeze-thaw method. The PCR amplification specific primers was further carried out in transformed bacteria, expression vectors were confirmed to transfer into the Agrobacterium.
引文
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