低温锻炼对黄瓜幼苗抗冷效应的影响及转录因子CBF1的克隆与转化
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摘要
黄瓜是重要的蔬菜作物之一,世界各地普遍栽培。黄瓜属于喜温作物,早春季节经常受到低温的危害,日光温室黄瓜冬春季节则易受低温影响。我国劳动人民经长期实践得出了低温锻炼能增强黄瓜抗冷能力的经验,多年来的大量试验也证明了低温锻炼确实能增强植物耐冷能力。但选取多低的温度和多长时间进行锻炼效果最好,目前尚无定论。植物的一切生命活动均受遗传物质的控制,目前已从多种植物中克隆出了耐寒基因,其中转录因子CBF能激活耐寒基因的表达,CBF超表达能提高植物的耐寒能力。本试验选取不同低温对黄瓜幼苗进行锻炼,以探寻最佳锻炼温度及时间。另外从经过低温锻炼的黄瓜幼苗中克隆出黄瓜CBF1基因,并构建表达载体,成功转入抗病但不耐寒黄瓜自交系,转基因植株耐寒能力明显增强。具体结果如下:
1、以耐寒自交系S11和冷敏感自交系S22为试材,以10/4℃、10/6℃、10/8℃三种温度处理,对三叶一心期幼苗进行低温锻炼。结果表明:10/4℃锻炼导致黄瓜幼苗SOD、POD、CAT保护酶活性下降,Vc含量降低,根系活力降低,冷害指数增加,电解质渗透率增加,这说明10/4℃锻炼已对幼苗造成冷伤害;10/6℃和10/8℃锻炼使黄瓜幼苗SOD、POD、CAT保护酶活性大幅提高,Vc含量升高,减少了活性氧的危害,可溶性糖和可溶性蛋白含量增加,丙二醛含量下降,冷害指数显著降低,根系活力上升,这说明10/6℃和10/8℃锻炼能增强幼苗的抗冷能力。综合评价对耐寒自交系S11来说,以10/6℃锻炼3d效果最好,冷敏感自交系以10/6℃锻炼2d效果最好。
2、以耐寒自交系S11为试材,研究低温锻炼对黄瓜幼苗叶片叶肉细胞超微结构的影响。结果表明:正常条件下的黄瓜幼苗,叶绿体结构完整,基粒多,基粒片层排列紧密,整个膜系统完整。冷胁迫导致叶绿体结构变形,基粒片层松散无序,叶绿体膜受到严重损伤,冷胁迫后受伤的膜系统不能立即修复,反而受损伤程度加重。低温锻炼增强了黄瓜幼苗抗冷能力,使幼苗在冷胁迫条件下维持膜系统较完整状态,叶绿体受损轻,基粒片层排列较紧密,冷胁迫后膜系统修复也较快,这初步说明,适宜的低温锻炼使黄瓜幼苗在冷胁迫下维持膜系统,尤其是叶绿体的较完整性,从而减轻了冷伤害。
3、以耐寒能力强的黄瓜‘山农5号’为试材,以拟南芥CBF1基因的保守序列设计引物,通过RT-PCR方法,从低温锻炼的幼苗叶片中克隆出黄瓜CBF1基因,该基因长671bp,推导的氨基酸序列为213个氨基酸,与GenBank中的同源序列比较,本试验克隆的基因与拟南芥的基因在编码区完全相同,仅在起始密码子5’上游存在部分差异,与无包芥、高山离子芥、马蔺等同源性也很高。用SacI和SacII消化pCBF1,获得CBF1基因,用SacI和BamHI对pROK2双酶切,回收大片断,然后进行连接,构建了黄瓜转录因子CBF1基因的正义表达载体,经SacI和HindIII双酶切鉴定,证明了该基因已经成功连入pROK2中,命名为pROK2-CBF1。该载体的构建,为开展转基因研究,以提高植物抗寒、抗旱、抗盐等逆境及基因功能分析,提供了保障。
4、利用花粉管通道途径转化的优势,采用子房微量注射的方式,将重组质粒转化抗病但不耐寒的黄瓜自交系。经Kan初步筛选、PCR检测和Southern杂交检测,确定有两株转基因植株,转化率为0.05%。不同花龄子房注射的结果表明,以花前一天子房注射效果最好,此时注射出现了转基因植株。转基因植株后代出现分离,其分离不符合孟德尔遗传规律,可能是由基因沉默所致。但随世代增加,转基因植株出现的比率明显增加。
5、转基因植株抗冷性明显增强。T1代转基因植株经苗期低温胁迫发现幼苗SOD活性、POD活性比野生型显著提高,脯氨酸含量、可溶性蛋白含量比野生型大幅提高,而冷害指数明显降低,T1代转基因植株耐寒能力显著提高。T2代转基因植株经自然低温检测,发现野生型因低温死亡早。而转基因植株因低温致死时间延迟,T2代转基因植株耐寒能力优于野生型。T3代转基因植株在冬季低温条件下生长势明显优于野生型,转基因植株能安全越冬,而野生型已冻死。转基因植株抗霜雪病和白粉病能力强,具有野生型同等的抗病性,而抗冷能力明显增强,至此我们已得到了即抗病耐寒能力又大幅提高的转基因新种质,目前已得到了T3代株系,为今后开展相关黄瓜育种工作奠定了基础。
Cucumber (Cucumis sativus L.) is an important vegetable and planted all over the world. Cucumber belongs to thermophilic crops and is easily suffered chilling damage in early spring in solar greenhouse as well as in open field. Many papers reported that cold acclimation can enhance cold tolerances of cucumber whereas the proper temperature was in debate. All life activities of plant are controlled by genetic materials. At present, the cold tolerance genes have been cloned in many plants and found that the activator of transcription CBF can activate expression of cold tolerance genes and the cold tolerance of plants can be increased by CBF over-expression. In this research, we chose three different low temperatures to train cucumber seedlings and try to find the optimum low temperature and time. At the same time, we cloned CBF1 gene from cucumber seedlings during cold acclimation, and CBF1 gene sense expression vector was constructed and transferred into cucumber inbred line, the cold tolerance of the transferred plants were increased significantly.
The details were as follows:
1.S11 (cold tolerance and self-bred line) and S22 (cold sensitive and self-bred line) seedlings were treated under three low day/night temperature combinations (10/4℃, 10/6℃and 10/8℃) at three true leaves stage. The results showed that the activities of SOD, POD, CAT and root activity,Vc content decreased significantly, the chilling injury index and electrolyticleakage increased significantly under the treatment of 10/4℃,these indicated that cold damage has arisen in cucumber seedling leaves under this treatment. The activities of SOD, POD, CAT and the content of Vc, soluble protein, soluble sugar increased significantly under the treatments of 10/6℃and 10/8℃, thus reduced the active oxygen damages. At the same time, the MDA contents and chilling injury index decreased significantly, but root activity increased, all these indicated that the cold tolerance of cucumber seedlings can be enhanced by use of cold acclimation under the treatments of 10/6℃and 10/8℃. In general, the best cold acclimation condition for S11 was 10/6℃and lasting for 3d, as for S22 was 10/6℃and lasting for 2d.
2. Using S11 as test material, experiments were conducted to study the effect of cold acclimation on mesophyll cell ultrastructure of cucumber seedlings. The results showed that in normal growth conditions, the grana lamella in cucumber seedlings leaves were arranged regularly with more grana, chloroplast structure and the whole membrane system was complete. The chloroplast structure were changed, the grana lamella become loose and disorder under cold stress, and the chloroplast membrane was injured seriously under cold stress. Furthermore, the damage to membrane system was increased instead of repaired. Cold acclimation enhanced cold tolerances of cucumber seedlings. Compared to cucumber seedling without cold acclimation, the grana lamella in cucumber seedlings with cold acclimation were arranged relative closely, the whole membrane system was more complete, the damage to chloroplast was slighter and the damage can be repaired in a short time under cold stress. All these indicated that cold acclimation with optimum temperature can maintained the integrity of membrane system (especially chloroplast) under cold stress, thus alleviated the cold damage to cucumber seedlings.
3. Using the high cold tolerance cucumber plant as test material. PCR primers were designed based on the conservation sequence of CBF1 gene in arabidopsis thaliana. By means of RT-PCR methods, we had cloned CBF1 gene from cucumber seedlings leaves with cold acclimation, and the gene contained 671bp and 213 amino acids determined by this gene. The gene had 100 percent homology to that of arabidopsis thaliana in coding region, and had minor difference in start codon 5′-upstream region. The gene also has high homology compared to that of chorispora bungeana, olimarabidopsis pumila and Chinese small iris.
The pCBF1 was digested with SacI and SacII, and then CBF1 gene was obtained. After the double digestion to PROk2 with SacI/BamHI, fragments were ligated. Then the sense expression vector of activating transcription factor of cucumber was constructed and ordered pROK2-CBF1. With this expression vector, we can transfer the gene to other plants to increase their resistance to low temperature, drought and salt.
4. The target gene was transferred into cucumber self-bred line (disease resistance and cold sensitive) by pollen-tube pathway via ovary-injecting. Two transgenic plants were found by use of preliminary screening of Kan, PCR and Southern hybridization detection, and the conversion is 0.05%. The optimum time for ovary-injecting was one day before flowering, and can get transgenic plants at this time. The separation law of transformation generation was not as agreement with the Mendelian genetic law, the reason may be gene silencing. The transformants probability of appearance increased with adding of generation.
5. Under cold stress, the activities of SOD, POD and the contents of proline, soluble protein of T1 generation increased significantly compared with those of the wild type. The generation of T2 grew longer time under nature low temperature and generation of T3 had strong growth potential under low temperature in winter compared with the wild type, and can live through the winter, while the wild type all died. The cold tolerance was enhanced and the disease resistance to downy mildew, powdery mildew was kept in transgenic plants. At present, the generation of T3 was obtained.
1、以耐寒自交系S11和冷敏感自交系S22为试材,以10/4℃、10/6℃、10/8℃三种温度处理,对三叶一心期幼苗进行低温锻炼。结果表明:10/4℃锻炼导致黄瓜幼苗SOD、POD、CAT保护酶活性下降,Vc含量降低,根系活力降低,冷害指数增加,电解质渗透率增加,这说明10/4℃锻炼已对幼苗造成冷伤害;10/6℃和10/8℃锻炼使黄瓜幼苗SOD、POD、CAT保护酶活性大幅提高,Vc含量升高,减少了活性氧的危害,可溶性糖和可溶性蛋白含量增加,丙二醛含量下降,冷害指数显著降低,根系活力上升,这说明10/6℃和10/8℃锻炼能增强幼苗的抗冷能力。综合评价对耐寒自交系S11来说,以10/6℃锻炼3d效果最好,冷敏感自交系以10/6℃锻炼2d效果最好。
2、以耐寒自交系S11为试材,研究低温锻炼对黄瓜幼苗叶片叶肉细胞超微结构的影响。结果表明:正常条件下的黄瓜幼苗,叶绿体结构完整,基粒多,基粒片层排列紧密,整个膜系统完整。冷胁迫导致叶绿体结构变形,基粒片层松散无序,叶绿体膜受到严重损伤,冷胁迫后受伤的膜系统不能立即修复,反而受损伤程度加重。低温锻炼增强了黄瓜幼苗抗冷能力,使幼苗在冷胁迫条件下维持膜系统较完整状态,叶绿体受损轻,基粒片层排列较紧密,冷胁迫后膜系统修复也较快,这初步说明,适宜的低温锻炼使黄瓜幼苗在冷胁迫下维持膜系统,尤其是叶绿体的较完整性,从而减轻了冷伤害。
3、以耐寒能力强的黄瓜‘山农5号’为试材,以拟南芥CBF1基因的保守序列设计引物,通过RT-PCR方法,从低温锻炼的幼苗叶片中克隆出黄瓜CBF1基因,该基因长671bp,推导的氨基酸序列为213个氨基酸,与GenBank中的同源序列比较,本试验克隆的基因与拟南芥的基因在编码区完全相同,仅在起始密码子5’上游存在部分差异,与无包芥、高山离子芥、马蔺等同源性也很高。用SacI和SacII消化pCBF1,获得CBF1基因,用SacI和BamHI对pROK2双酶切,回收大片断,然后进行连接,构建了黄瓜转录因子CBF1基因的正义表达载体,经SacI和HindIII双酶切鉴定,证明了该基因已经成功连入pROK2中,命名为pROK2-CBF1。该载体的构建,为开展转基因研究,以提高植物抗寒、抗旱、抗盐等逆境及基因功能分析,提供了保障。
4、利用花粉管通道途径转化的优势,采用子房微量注射的方式,将重组质粒转化抗病但不耐寒的黄瓜自交系。经Kan初步筛选、PCR检测和Southern杂交检测,确定有两株转基因植株,转化率为0.05%。不同花龄子房注射的结果表明,以花前一天子房注射效果最好,此时注射出现了转基因植株。转基因植株后代出现分离,其分离不符合孟德尔遗传规律,可能是由基因沉默所致。但随世代增加,转基因植株出现的比率明显增加。
5、转基因植株抗冷性明显增强。T1代转基因植株经苗期低温胁迫发现幼苗SOD活性、POD活性比野生型显著提高,脯氨酸含量、可溶性蛋白含量比野生型大幅提高,而冷害指数明显降低,T1代转基因植株耐寒能力显著提高。T2代转基因植株经自然低温检测,发现野生型因低温死亡早。而转基因植株因低温致死时间延迟,T2代转基因植株耐寒能力优于野生型。T3代转基因植株在冬季低温条件下生长势明显优于野生型,转基因植株能安全越冬,而野生型已冻死。转基因植株抗霜雪病和白粉病能力强,具有野生型同等的抗病性,而抗冷能力明显增强,至此我们已得到了即抗病耐寒能力又大幅提高的转基因新种质,目前已得到了T3代株系,为今后开展相关黄瓜育种工作奠定了基础。
Cucumber (Cucumis sativus L.) is an important vegetable and planted all over the world. Cucumber belongs to thermophilic crops and is easily suffered chilling damage in early spring in solar greenhouse as well as in open field. Many papers reported that cold acclimation can enhance cold tolerances of cucumber whereas the proper temperature was in debate. All life activities of plant are controlled by genetic materials. At present, the cold tolerance genes have been cloned in many plants and found that the activator of transcription CBF can activate expression of cold tolerance genes and the cold tolerance of plants can be increased by CBF over-expression. In this research, we chose three different low temperatures to train cucumber seedlings and try to find the optimum low temperature and time. At the same time, we cloned CBF1 gene from cucumber seedlings during cold acclimation, and CBF1 gene sense expression vector was constructed and transferred into cucumber inbred line, the cold tolerance of the transferred plants were increased significantly.
The details were as follows:
1.S11 (cold tolerance and self-bred line) and S22 (cold sensitive and self-bred line) seedlings were treated under three low day/night temperature combinations (10/4℃, 10/6℃and 10/8℃) at three true leaves stage. The results showed that the activities of SOD, POD, CAT and root activity,Vc content decreased significantly, the chilling injury index and electrolyticleakage increased significantly under the treatment of 10/4℃,these indicated that cold damage has arisen in cucumber seedling leaves under this treatment. The activities of SOD, POD, CAT and the content of Vc, soluble protein, soluble sugar increased significantly under the treatments of 10/6℃and 10/8℃, thus reduced the active oxygen damages. At the same time, the MDA contents and chilling injury index decreased significantly, but root activity increased, all these indicated that the cold tolerance of cucumber seedlings can be enhanced by use of cold acclimation under the treatments of 10/6℃and 10/8℃. In general, the best cold acclimation condition for S11 was 10/6℃and lasting for 3d, as for S22 was 10/6℃and lasting for 2d.
2. Using S11 as test material, experiments were conducted to study the effect of cold acclimation on mesophyll cell ultrastructure of cucumber seedlings. The results showed that in normal growth conditions, the grana lamella in cucumber seedlings leaves were arranged regularly with more grana, chloroplast structure and the whole membrane system was complete. The chloroplast structure were changed, the grana lamella become loose and disorder under cold stress, and the chloroplast membrane was injured seriously under cold stress. Furthermore, the damage to membrane system was increased instead of repaired. Cold acclimation enhanced cold tolerances of cucumber seedlings. Compared to cucumber seedling without cold acclimation, the grana lamella in cucumber seedlings with cold acclimation were arranged relative closely, the whole membrane system was more complete, the damage to chloroplast was slighter and the damage can be repaired in a short time under cold stress. All these indicated that cold acclimation with optimum temperature can maintained the integrity of membrane system (especially chloroplast) under cold stress, thus alleviated the cold damage to cucumber seedlings.
3. Using the high cold tolerance cucumber plant as test material. PCR primers were designed based on the conservation sequence of CBF1 gene in arabidopsis thaliana. By means of RT-PCR methods, we had cloned CBF1 gene from cucumber seedlings leaves with cold acclimation, and the gene contained 671bp and 213 amino acids determined by this gene. The gene had 100 percent homology to that of arabidopsis thaliana in coding region, and had minor difference in start codon 5′-upstream region. The gene also has high homology compared to that of chorispora bungeana, olimarabidopsis pumila and Chinese small iris.
The pCBF1 was digested with SacI and SacII, and then CBF1 gene was obtained. After the double digestion to PROk2 with SacI/BamHI, fragments were ligated. Then the sense expression vector of activating transcription factor of cucumber was constructed and ordered pROK2-CBF1. With this expression vector, we can transfer the gene to other plants to increase their resistance to low temperature, drought and salt.
4. The target gene was transferred into cucumber self-bred line (disease resistance and cold sensitive) by pollen-tube pathway via ovary-injecting. Two transgenic plants were found by use of preliminary screening of Kan, PCR and Southern hybridization detection, and the conversion is 0.05%. The optimum time for ovary-injecting was one day before flowering, and can get transgenic plants at this time. The separation law of transformation generation was not as agreement with the Mendelian genetic law, the reason may be gene silencing. The transformants probability of appearance increased with adding of generation.
5. Under cold stress, the activities of SOD, POD and the contents of proline, soluble protein of T1 generation increased significantly compared with those of the wild type. The generation of T2 grew longer time under nature low temperature and generation of T3 had strong growth potential under low temperature in winter compared with the wild type, and can live through the winter, while the wild type all died. The cold tolerance was enhanced and the disease resistance to downy mildew, powdery mildew was kept in transgenic plants. At present, the generation of T3 was obtained.
引文
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