转果聚糖合成关键酶基因多年生黑麦草获得及抗旱性的提高
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摘要
多年生黑麦草是重要的冷季型禾草,既可做草坪草,也可做牧草,但其抗旱性较差,限制了在生产中的应用。采用生物技术分离抗旱基因,通过转基因方法培育新品种是提高黑麦草抗旱能力的有效途径。本研究以黑麦草卡特胚性愈伤组织为转化受体材料,通过农杆菌侵染和基因枪转化的方法将从冰草中克隆的果聚糖:果聚糖-1-果糖基转移酶基因(Ac-1-FFT)导入黑麦草中,获得了抗旱性明显增强的转基因植株。主要研究结果如下:
1.将果聚糖:果聚糖-1-果糖基转移酶基因(Ac-1-FFT)导入黑麦草
从冰草中克隆了Ac-1-FFT基因,cDNA序列全长1950 bp。构建了含有Ac-1-FFT基因的植物表达载体Ac-1-FFT-pCambia3301,该载体的Ac-1-FFT基因由玉米泛素基因-1启动子驱动,以bar基因为选择标记。通过农杆菌侵染和基因枪转化的方法将Ac-1-FFT基因导入黑麦草中,共获得122个再生株系,其中利用农杆菌侵染的方法获得89个株系,而利用基因枪的方法获得了33个株系。
2.转基因植株的分子鉴定
对122个再生株系喷洒5‰的Basta溶液,表明有18个株系正常生长,叶片保持绿色,其余植株和对照叶片均枯黄,甚至死亡。通过PCR法和半定量PCR对这18个再生株系在分子水平上进行了鉴定,结果表明18个株系均能扩增到Ac-1-FFT基因的特异片段,而且该基因在黑麦草中得到有效的表达,但不同株系间的表达量不同。
3.转基因植株抗旱性鉴定
对转Ac-1-FFT基因黑麦草进行了干旱胁迫实验。在干旱的条件下,转基因植株表现出了良好的抗旱能力,干旱胁迫6天时,转基因植株叶片仍保持绿色,仅个别叶片开始枯萎,而此时对照植株叶片整体表现枯萎,甚至死亡。复水实验表明对照植株失去恢复能力,而转基因植株恢复较快,虽然个别叶片仍表现枯黄,但植株整体长势良好。
4.转基因植株生理生化指标的测定
检测结果表明转基因黑麦草株系中的可溶性总糖含量和果聚糖含量明显高于对照植株,干旱胁迫6天时其相对含水量和叶绿素含量明显高于对照植株,且下降速度慢,但其电解质渗漏率和丙二醛含量显著低于对照植株,复水后很快复原,而对照植株无法恢复,说明转基因植株中由于干旱处理发生的损伤是可逆的,而对照植株中的损伤是非可逆的。以上结果表明转基因黑麦草中Ac-1-FFT基因的表达及果聚糖合成可能是其耐旱性提高的最重要原因。
Perennial ryegrass (Lolium perenne L.) is an important cool-season grass; it can be turfgrass and forage. But the weak drought tolerance limits its application in production. Isolation of resistance drought-related genes by biological methods and breeding new varieties through transgenic methods is an effective way to improve the drought resistance of ryegrass. In this study, Ac-1-FFT (fructan: fructan 1-fructosyltransferase) cloned from Agropyron cristatum (L.) Gaertn was transformed into embryogenic callus ryegrass (Lolium perenne L. ) Carter by Agrobacterium mediated method and particle bombardment. We obtained significantly enhanced drought resistance of transgenic plants.The main results are as follows:
1. Ac-1-FFT (fructan: fructan 1-fructosyltransferase) was transformed into perennial ryegrass
Ac-1-FFT gene cloned from Agropyron cristatum (L.) Gaertn, the length of cDNA sequence is 1950 bp.We construct a plant expression vector Ac-1-FFT-pCambia3301, it contains Ac-1-FFT gene, the maize ubiquitin-1 gene as promoter and bar gene as selectable marker. Ac-1-FFT transformed into ryegrass through Agrobacterium infection and particle bombardment method.We totally obtained 122 independent lines of regenerated plants, 89 of which was by Agrobacterium infection and 33 of which was by particle bombardment method.
2. Molecular identification of transgenic plants
Transgenic plants were analyzed by 5‰basta spraying, 18 strains showed normal growth and green leaves, the other plants and control palnts’leaves turned yellow, and even death. The 18 strains were identified on the molecular level through PCR, and semi-quantitative PCR. PCR analysis showed that Ac-1-FFT gene’s specific fragment could be amplified, and RT-PCR analysis showed that Ac-1-FFT gene expressed strongly in transgenic Lolium perenne plants whereas didn’t in control plants. The expression was different between the different strains.
3. Drought resistance of transgenic plants
Under drought stress, transgenic plants showed good drought resistance. After 6 days drought stress, the leaves of transgenic plants remained green, few leaves began to wither, in the same time the control plants showde withered, and even death. Rehydration experiments showed that the control plants lost the ability to recover while transgenic plants recovered rapidly. Although few leaves were still yellow, the whole plants were doing well.
4. Physiological and biochemical determination of transgenic plants
The results showed that water soluble carbohydrate and fructan contents in Ac-1-FFT transgenic ryegrass are significantly higher than those in control plants. Relative water content and chlorophyll content are significantly higher, while electrolyte leakage and malondialdehyde (MDA) content are significantly lower in Ac-1-FFT transgenic ryegrass than those in control plants 6 days after drought treatment. Relative water content, chlorophyll content, electrolyte leakage and MDA content restored to its original level in Ac-1-FFT transgenic plants rather than in control plants 7 days after rewatering, suggesting that the damages in Ac-1-FFT transgenic plants are reversible but not in control plants. These results indicate that Ac-1-FFT expression and fructan biosynthesis might play an important role in the improved drought tolerance in Ac-1-FFT transgenic ryegrass.
1.将果聚糖:果聚糖-1-果糖基转移酶基因(Ac-1-FFT)导入黑麦草
从冰草中克隆了Ac-1-FFT基因,cDNA序列全长1950 bp。构建了含有Ac-1-FFT基因的植物表达载体Ac-1-FFT-pCambia3301,该载体的Ac-1-FFT基因由玉米泛素基因-1启动子驱动,以bar基因为选择标记。通过农杆菌侵染和基因枪转化的方法将Ac-1-FFT基因导入黑麦草中,共获得122个再生株系,其中利用农杆菌侵染的方法获得89个株系,而利用基因枪的方法获得了33个株系。
2.转基因植株的分子鉴定
对122个再生株系喷洒5‰的Basta溶液,表明有18个株系正常生长,叶片保持绿色,其余植株和对照叶片均枯黄,甚至死亡。通过PCR法和半定量PCR对这18个再生株系在分子水平上进行了鉴定,结果表明18个株系均能扩增到Ac-1-FFT基因的特异片段,而且该基因在黑麦草中得到有效的表达,但不同株系间的表达量不同。
3.转基因植株抗旱性鉴定
对转Ac-1-FFT基因黑麦草进行了干旱胁迫实验。在干旱的条件下,转基因植株表现出了良好的抗旱能力,干旱胁迫6天时,转基因植株叶片仍保持绿色,仅个别叶片开始枯萎,而此时对照植株叶片整体表现枯萎,甚至死亡。复水实验表明对照植株失去恢复能力,而转基因植株恢复较快,虽然个别叶片仍表现枯黄,但植株整体长势良好。
4.转基因植株生理生化指标的测定
检测结果表明转基因黑麦草株系中的可溶性总糖含量和果聚糖含量明显高于对照植株,干旱胁迫6天时其相对含水量和叶绿素含量明显高于对照植株,且下降速度慢,但其电解质渗漏率和丙二醛含量显著低于对照植株,复水后很快复原,而对照植株无法恢复,说明转基因植株中由于干旱处理发生的损伤是可逆的,而对照植株中的损伤是非可逆的。以上结果表明转基因黑麦草中Ac-1-FFT基因的表达及果聚糖合成可能是其耐旱性提高的最重要原因。
Perennial ryegrass (Lolium perenne L.) is an important cool-season grass; it can be turfgrass and forage. But the weak drought tolerance limits its application in production. Isolation of resistance drought-related genes by biological methods and breeding new varieties through transgenic methods is an effective way to improve the drought resistance of ryegrass. In this study, Ac-1-FFT (fructan: fructan 1-fructosyltransferase) cloned from Agropyron cristatum (L.) Gaertn was transformed into embryogenic callus ryegrass (Lolium perenne L. ) Carter by Agrobacterium mediated method and particle bombardment. We obtained significantly enhanced drought resistance of transgenic plants.The main results are as follows:
1. Ac-1-FFT (fructan: fructan 1-fructosyltransferase) was transformed into perennial ryegrass
Ac-1-FFT gene cloned from Agropyron cristatum (L.) Gaertn, the length of cDNA sequence is 1950 bp.We construct a plant expression vector Ac-1-FFT-pCambia3301, it contains Ac-1-FFT gene, the maize ubiquitin-1 gene as promoter and bar gene as selectable marker. Ac-1-FFT transformed into ryegrass through Agrobacterium infection and particle bombardment method.We totally obtained 122 independent lines of regenerated plants, 89 of which was by Agrobacterium infection and 33 of which was by particle bombardment method.
2. Molecular identification of transgenic plants
Transgenic plants were analyzed by 5‰basta spraying, 18 strains showed normal growth and green leaves, the other plants and control palnts’leaves turned yellow, and even death. The 18 strains were identified on the molecular level through PCR, and semi-quantitative PCR. PCR analysis showed that Ac-1-FFT gene’s specific fragment could be amplified, and RT-PCR analysis showed that Ac-1-FFT gene expressed strongly in transgenic Lolium perenne plants whereas didn’t in control plants. The expression was different between the different strains.
3. Drought resistance of transgenic plants
Under drought stress, transgenic plants showed good drought resistance. After 6 days drought stress, the leaves of transgenic plants remained green, few leaves began to wither, in the same time the control plants showde withered, and even death. Rehydration experiments showed that the control plants lost the ability to recover while transgenic plants recovered rapidly. Although few leaves were still yellow, the whole plants were doing well.
4. Physiological and biochemical determination of transgenic plants
The results showed that water soluble carbohydrate and fructan contents in Ac-1-FFT transgenic ryegrass are significantly higher than those in control plants. Relative water content and chlorophyll content are significantly higher, while electrolyte leakage and malondialdehyde (MDA) content are significantly lower in Ac-1-FFT transgenic ryegrass than those in control plants 6 days after drought treatment. Relative water content, chlorophyll content, electrolyte leakage and MDA content restored to its original level in Ac-1-FFT transgenic plants rather than in control plants 7 days after rewatering, suggesting that the damages in Ac-1-FFT transgenic plants are reversible but not in control plants. These results indicate that Ac-1-FFT expression and fructan biosynthesis might play an important role in the improved drought tolerance in Ac-1-FFT transgenic ryegrass.
引文
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