优质蓝莓转LEA基因及耐寒性分析研究
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摘要
随着人们生活质量和生活水平的提高,人们的保健意识逐渐增强,许多外国水果备受人们的青睐,其中蓝莓更是深受欢迎。由于蓝莓鲜果价格昂贵、加工工艺不断完善,以及人们逐渐认识到蓝莓对身体健康有益,世界各地蓝莓的种植面积逐年增加。近年来,我国的蓝莓产业也逐渐受到了重视。在我国北方地区,由于冬季低温干燥,蓝莓产业的发展受到了一定限制。因此,为我国北方地区培育营养价值高、对低温耐受能力强的蓝莓新品种已迫在眉睫。本研究对我国北方地区主要蓝莓栽培品种进行了营养价值分析。同时,分别以蓝莓的茎段和叶片为外植体,建立了高效组培再生体系。通过根癌农杆菌介导法将来源于柽柳cDNA文库中的LEA基因转化至营养价值相对较高的蓝莓品种——北陆基因组中,为蓝莓品种选育开辟新的途径。具体研究结果如下：
1.对供试的7个蓝莓品种分别进行了氨基酸含量、总糖含量、蛋白质含量、维生素含量及矿质元素含量的测定。结果表明,北陆各测试指标表现突出,除Vb2和钙含量位居第二外,其他测试指标均列于首位；
2.以不同蓝莓品种的茎段和叶片为外植体,选择基本培养基类型,同时确定植物生长调节物质的种类和适宜浓度。结果表明,在附加玉米素(Zt)的改良WPM培养基中,各品种分化率均较高,但Zt的适宜浓度在各基因型中有所差异；
3.筛选出头孢噻肟钠为蓝莓遗传转化体系的抑菌抗生素,使用浓度为250 mg·L-1；蓝莓遗传转化的选择类抗生素为卡那霉素,其使用浓度为30 mg·L-1；应用农杆菌介导法对蓝莓进行遗传转化,将抗逆LEA基因转化至北陆基因组中,并确定了最优北陆遗传转化体系为：8-10周龄不定芽的中部叶片于25±1℃条件下暗培养48h,然后将其浸泡在工程菌液(OD600=0.5)中于28℃侵染60min。侵染之后于共培养培养基中暗培养4 d,转移到延迟选择培养基上,于25±1℃条件下暗培养48 h后将叶片转移到抗性芽筛选培养基上进行选择培养,此阶段需先暗培养7 d后再转至光下培养,并每隔5-7d脱菌一次；
4.利用农杆菌介导法对蓝莓进行遗传转化初步获得卡那霉素抗性植株13个,经增大选择压,排除5个假阳性株系。对剩余8个阳性株系分别进行了PCR、Southern杂交和RT-PCR检测。结果表明,LEA基因已经整合到北陆基因组中,但不同转化子拷贝数不同。经一步法RT-PCR检测2个株系为转录水平沉默,其他转化子能够正常转录；
5.对8个转基因株系进行低温胁迫,分别测定了胁迫前后丙二醛(MDA)含量、相对电导率(REL)、超氧化物歧化酶(SOD)活性和过氧化物酶(POD)活性。结果表明,在未发生转录水平沉默的6个转基因株系中,胁迫后的MDA含量和REL均低于对照,SOD活性和POD活性在胁迫后均高于对照,且均存在显著差异。说明转入的LEA基因能够正常表达,并提高了转基因北陆的耐寒能力。
With improving quality and standards of people's lives, people gradually increase health awareness. Many people favor the subject of foreign fruit, in which blueberry is much more welcome. As the blueberry fruit is expensive, processing technology constantly improving, and the fruit benefit to the physical health, planted area of blueberry is increased year by year around the world. In recent years, Chinese blueberry industry is gradually being taken seriously. In North China, due to the winter cold and dry, blueberry industriy has been limited. Therefore, in northern China, cultivation new variety of blueberry with high nutritional value and low temperature tolerance ability is imminent. In this study, the nutritional value of mainly blueberry cultivars in northern China was analysed. Meanwhile, the stems and leaves were used as explants to establish an efficient system for tissue regeneration, respectively. By Agrobacterium-mediated method, the LEA gene from cDNA library of Tamarix androssowii was introduced into Northland genome, which is a new way to genetic transformation to blueberry. The results are as follows:
1. The content of amino acid, total sugar, protein, vitamin and mineral element were carried out in the tested seven blueberry varieties. The results showed that the test targets of Northland were outstanding, except Vb2 and calcium content in second place, the other test indicators were all listed in the first place;
2. In different varieties of blueberry, the stems and leaves were used as explants, selected the type of basic medium, while identifying the types and the appropriate concentration of plant growth regulators. The results indicated that the modified WPM medium containing zeatin (Zt) was benefitted to differentiation, but the suitable concentration of Zt is different in different genotypes;
3. Cefotaxime sodium is the optimized antibacterial antibiotics, it's effective concentration is 250 mg-L-1; Kanamycin is the optimized antibiotics for the transformant selection, it's effective concentration is 30 mg·L-1. The resistance LEA gene was transformed into blueberry by Agrobacterium-mediated method. The optimal genetic transformation system for Northland as follows:Leaves from 8- to 10-week-old adventitious shoot were used as explants for pre-culturing for 48 h in the dark at 25±1℃. The pre-cultured explants were then infected with A. tumefaciens diluted by liquid MWPMZ to OD600=0.5 for 1 h at 28℃. The infected explants were co-cultivated on solid MWPMZ in the dark for 4 days. Then the explants were transferred to MWPMZ+250 mg l-1 cefotaxime for 48 h in the dark at 25℃. The explants were then transferred to selection medium (solid MWPMZ+30 mg l-1 Km+250 mg l-1 cefotaxime) for 7 d in the dark at 25℃, followed by subculturing on fresh selection medium at 5-7 d intervals under a 16 h photoperiod with a photosynthetic photon flux density of 50μmol m-2 s-1.
4.13 kanamycin resistant shoots were initially gained using Agrobacterium-mediated genetic transformation of blueberry. Five false-positive clones were removed by increasing the selection pressure. The remaining eight positive clones were carried out PCR, Southern blotting and RT-PCR detection. The results involved that, LEA gene was integrated into the genome of Northland, but different transformants with different copy numbers. The results of the one-step RT-PCR indicated that two lines were transcription silence, while the other transgenic lines were normal transcription;
5. The 8 transgenic lines were tested for cold tolerance by measuring the activities of peroxidase (POD) and superoxide dismutase (SOD), malondialdehyde (MDA) content and relative electrolyte leakage (REL). The results showed that the MDA content and REL were lower than the control, SOD activity and POD activity was higher than the control after stress in the six transgenic lines which normal transcription, and there were significant differences. The results above illuminate that the exogenous LEA genes in Northland could normal expression, and increased transgenic Northland cold tolerance ability.
1.对供试的7个蓝莓品种分别进行了氨基酸含量、总糖含量、蛋白质含量、维生素含量及矿质元素含量的测定。结果表明,北陆各测试指标表现突出,除Vb2和钙含量位居第二外,其他测试指标均列于首位；
2.以不同蓝莓品种的茎段和叶片为外植体,选择基本培养基类型,同时确定植物生长调节物质的种类和适宜浓度。结果表明,在附加玉米素(Zt)的改良WPM培养基中,各品种分化率均较高,但Zt的适宜浓度在各基因型中有所差异；
3.筛选出头孢噻肟钠为蓝莓遗传转化体系的抑菌抗生素,使用浓度为250 mg·L-1；蓝莓遗传转化的选择类抗生素为卡那霉素,其使用浓度为30 mg·L-1；应用农杆菌介导法对蓝莓进行遗传转化,将抗逆LEA基因转化至北陆基因组中,并确定了最优北陆遗传转化体系为：8-10周龄不定芽的中部叶片于25±1℃条件下暗培养48h,然后将其浸泡在工程菌液(OD600=0.5)中于28℃侵染60min。侵染之后于共培养培养基中暗培养4 d,转移到延迟选择培养基上,于25±1℃条件下暗培养48 h后将叶片转移到抗性芽筛选培养基上进行选择培养,此阶段需先暗培养7 d后再转至光下培养,并每隔5-7d脱菌一次；
4.利用农杆菌介导法对蓝莓进行遗传转化初步获得卡那霉素抗性植株13个,经增大选择压,排除5个假阳性株系。对剩余8个阳性株系分别进行了PCR、Southern杂交和RT-PCR检测。结果表明,LEA基因已经整合到北陆基因组中,但不同转化子拷贝数不同。经一步法RT-PCR检测2个株系为转录水平沉默,其他转化子能够正常转录；
5.对8个转基因株系进行低温胁迫,分别测定了胁迫前后丙二醛(MDA)含量、相对电导率(REL)、超氧化物歧化酶(SOD)活性和过氧化物酶(POD)活性。结果表明,在未发生转录水平沉默的6个转基因株系中,胁迫后的MDA含量和REL均低于对照,SOD活性和POD活性在胁迫后均高于对照,且均存在显著差异。说明转入的LEA基因能够正常表达,并提高了转基因北陆的耐寒能力。
With improving quality and standards of people's lives, people gradually increase health awareness. Many people favor the subject of foreign fruit, in which blueberry is much more welcome. As the blueberry fruit is expensive, processing technology constantly improving, and the fruit benefit to the physical health, planted area of blueberry is increased year by year around the world. In recent years, Chinese blueberry industry is gradually being taken seriously. In North China, due to the winter cold and dry, blueberry industriy has been limited. Therefore, in northern China, cultivation new variety of blueberry with high nutritional value and low temperature tolerance ability is imminent. In this study, the nutritional value of mainly blueberry cultivars in northern China was analysed. Meanwhile, the stems and leaves were used as explants to establish an efficient system for tissue regeneration, respectively. By Agrobacterium-mediated method, the LEA gene from cDNA library of Tamarix androssowii was introduced into Northland genome, which is a new way to genetic transformation to blueberry. The results are as follows:
1. The content of amino acid, total sugar, protein, vitamin and mineral element were carried out in the tested seven blueberry varieties. The results showed that the test targets of Northland were outstanding, except Vb2 and calcium content in second place, the other test indicators were all listed in the first place;
2. In different varieties of blueberry, the stems and leaves were used as explants, selected the type of basic medium, while identifying the types and the appropriate concentration of plant growth regulators. The results indicated that the modified WPM medium containing zeatin (Zt) was benefitted to differentiation, but the suitable concentration of Zt is different in different genotypes;
3. Cefotaxime sodium is the optimized antibacterial antibiotics, it's effective concentration is 250 mg-L-1; Kanamycin is the optimized antibiotics for the transformant selection, it's effective concentration is 30 mg·L-1. The resistance LEA gene was transformed into blueberry by Agrobacterium-mediated method. The optimal genetic transformation system for Northland as follows:Leaves from 8- to 10-week-old adventitious shoot were used as explants for pre-culturing for 48 h in the dark at 25±1℃. The pre-cultured explants were then infected with A. tumefaciens diluted by liquid MWPMZ to OD600=0.5 for 1 h at 28℃. The infected explants were co-cultivated on solid MWPMZ in the dark for 4 days. Then the explants were transferred to MWPMZ+250 mg l-1 cefotaxime for 48 h in the dark at 25℃. The explants were then transferred to selection medium (solid MWPMZ+30 mg l-1 Km+250 mg l-1 cefotaxime) for 7 d in the dark at 25℃, followed by subculturing on fresh selection medium at 5-7 d intervals under a 16 h photoperiod with a photosynthetic photon flux density of 50μmol m-2 s-1.
4.13 kanamycin resistant shoots were initially gained using Agrobacterium-mediated genetic transformation of blueberry. Five false-positive clones were removed by increasing the selection pressure. The remaining eight positive clones were carried out PCR, Southern blotting and RT-PCR detection. The results involved that, LEA gene was integrated into the genome of Northland, but different transformants with different copy numbers. The results of the one-step RT-PCR indicated that two lines were transcription silence, while the other transgenic lines were normal transcription;
5. The 8 transgenic lines were tested for cold tolerance by measuring the activities of peroxidase (POD) and superoxide dismutase (SOD), malondialdehyde (MDA) content and relative electrolyte leakage (REL). The results showed that the MDA content and REL were lower than the control, SOD activity and POD activity was higher than the control after stress in the six transgenic lines which normal transcription, and there were significant differences. The results above illuminate that the exogenous LEA genes in Northland could normal expression, and increased transgenic Northland cold tolerance ability.
引文
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