农杆菌介导SAMS基因的玉米自交系遗传转化与验证
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摘要
干旱是影响玉米生产的第一限制因素,通过导入抗旱基因已成为增强玉米抗旱性的重要途径。本研究通过构建pBI121-SAMS表达载体,以玉米自交系茎尖和合子为受体,采用农杆菌介导法将抗旱基因SAMS导入玉米中,用PCR和RT-PCR技术对转化玉米进行检测,并以18%PEG-6000模拟水分胁迫对T1代转基因玉米和非转基因玉米进行抗旱性分析。主要研究结果如下:
1.采用PCR同源扩增,从晋麦47中扩增获得抗旱相关基因SAMS,构建以pBI121为基础的35S启动子调控的植物表达载体pBI121-SAMS。
2.通过研究掖478和郑58三叶期玉米叶片对不同卡那霉素浓度的敏感性,确定卡那霉素叶片涂抹筛选的最佳浓度为20000mg/L,观察时间以10天为宜。
3.农杆菌介导法转化玉米茎尖,共转化玉米茎尖518个,获得转化苗453株。经PCR初步检测,获得T0代阳性植株16株,转化率为3.53%,自交结实14株,经RT-PCR检测,T1代转基因玉米有9个株系为稳定遗传阳性株系。说明SAMS基因已整合到玉米基因组中,并进行正常表达。
4.农杆菌介导法转化玉米合子,共转化掖478和郑58玉米合子各50个,掖478自交系自交结实的有32株,郑58自交系自交结实的有34株,各穗结实较少。经卡那霉素筛选和PCR初步检测,掖478获得T1代阳性植株3株,郑58获得T1代阳性植株4株。说明SAMS基因已整合到玉米基因组中。
5.通过测定叶片相对含水量、叶绿素含量、脯氨酸含量、SOD活性、电导率和MDA含量等重要抗旱生理指标,统计分析显示,在同一水分胁迫时间下,转基因玉米的叶片相对含水量、叶绿素含量、脯氨酸含量和SOD活性均高于非转基因玉米,而转基因玉米叶片的电导率、MDA含量均低于非转基因玉米。在60h水分胁迫处理下,转基因玉米叶片相对含水量、叶绿素含量、脯氨酸含量和SOD活性比非转基因玉米上升8.16%、20.02%、32.21%、22.77%,而转基因玉米叶片的电导率、MDA含量比非转基因玉米下降14.38%、29.41%。研究表明,通过导入SAMS基因,可以提高玉米的抗旱性。
Drought is the first limiting factor in maize production, So drought-resistant gene was transferred into the maize, which has become an important measure. This study constructed expression vector pBI121-SAMS, And with the seedling shoot tips and zygote of maize inbred line as explants, the SAMS gene was transferred into the maize by using Agrobacterium-mediated in plant transformation, transgenic maize were identified by PCR and RT-PCR, T1 transgenic maize and non-modified maize were treated by using 18% PEG-6000 to simulate drought stress conditions. The results are as follows:
1. The study obtained SAMS gene from Jinmai47 by PCR amplification, and constructed plant expression vector pBI121-SAMS that was regulated by 35S Promoter.
2. Using kanamycin of different concentration to Ye 478 and Zheng58 in three leaf stage, The results show that the Optimal concentration is 20000mg/L,and time is about 10 days 3. 453 transgenic plants were obtained after having transformed 518 shoot tips. 16 T0 positive transgenic plants were screened by PCR, The frequency of transformation reached
3.53%, 14 of them get seeds finally. 9 of T1 transgenic maize were transcription normally confirmed by RT-PCR. These indicated that SAMS genes had been integrated to corn genomes and normal expression
4. Ye478 and Zheng58 maize zygotes were transformed for 50 respectively by using Agrobacterium-mediated in plant transformation. 32 plants were got from Ye478 inbred line, and 34 from Zheng58 inbred lines, but less per ear decreased. 3 of T1 positive plants from Ye478 and 4 of T1 positive plants from Zheng58 were got by kanamycin and PCR. It proved that the SAMS gene has been integrated to the genome of maize
5. The study found that Leaf relative water content、Chlorophyll content、Proline content and SOD activity of transgenic maize were more than CK plants while Conductance and MDA content were less under the same water stress time. When the time reached 60h, Leaf relative water content、Chlorophyll content、Proline content and SOD activity of transgenic maize were 8.16%、20.02%、32.21%、22.77% higher than the physiological indexes of CK, but Conductance and MDA content were 14.38%、29.41% lower than them were in CK. We can conclude that genetically modified maize has improved the drought-resistance by introducing of the foreign SAMS genes.
1.采用PCR同源扩增,从晋麦47中扩增获得抗旱相关基因SAMS,构建以pBI121为基础的35S启动子调控的植物表达载体pBI121-SAMS。
2.通过研究掖478和郑58三叶期玉米叶片对不同卡那霉素浓度的敏感性,确定卡那霉素叶片涂抹筛选的最佳浓度为20000mg/L,观察时间以10天为宜。
3.农杆菌介导法转化玉米茎尖,共转化玉米茎尖518个,获得转化苗453株。经PCR初步检测,获得T0代阳性植株16株,转化率为3.53%,自交结实14株,经RT-PCR检测,T1代转基因玉米有9个株系为稳定遗传阳性株系。说明SAMS基因已整合到玉米基因组中,并进行正常表达。
4.农杆菌介导法转化玉米合子,共转化掖478和郑58玉米合子各50个,掖478自交系自交结实的有32株,郑58自交系自交结实的有34株,各穗结实较少。经卡那霉素筛选和PCR初步检测,掖478获得T1代阳性植株3株,郑58获得T1代阳性植株4株。说明SAMS基因已整合到玉米基因组中。
5.通过测定叶片相对含水量、叶绿素含量、脯氨酸含量、SOD活性、电导率和MDA含量等重要抗旱生理指标,统计分析显示,在同一水分胁迫时间下,转基因玉米的叶片相对含水量、叶绿素含量、脯氨酸含量和SOD活性均高于非转基因玉米,而转基因玉米叶片的电导率、MDA含量均低于非转基因玉米。在60h水分胁迫处理下,转基因玉米叶片相对含水量、叶绿素含量、脯氨酸含量和SOD活性比非转基因玉米上升8.16%、20.02%、32.21%、22.77%,而转基因玉米叶片的电导率、MDA含量比非转基因玉米下降14.38%、29.41%。研究表明,通过导入SAMS基因,可以提高玉米的抗旱性。
Drought is the first limiting factor in maize production, So drought-resistant gene was transferred into the maize, which has become an important measure. This study constructed expression vector pBI121-SAMS, And with the seedling shoot tips and zygote of maize inbred line as explants, the SAMS gene was transferred into the maize by using Agrobacterium-mediated in plant transformation, transgenic maize were identified by PCR and RT-PCR, T1 transgenic maize and non-modified maize were treated by using 18% PEG-6000 to simulate drought stress conditions. The results are as follows:
1. The study obtained SAMS gene from Jinmai47 by PCR amplification, and constructed plant expression vector pBI121-SAMS that was regulated by 35S Promoter.
2. Using kanamycin of different concentration to Ye 478 and Zheng58 in three leaf stage, The results show that the Optimal concentration is 20000mg/L,and time is about 10 days 3. 453 transgenic plants were obtained after having transformed 518 shoot tips. 16 T0 positive transgenic plants were screened by PCR, The frequency of transformation reached
3.53%, 14 of them get seeds finally. 9 of T1 transgenic maize were transcription normally confirmed by RT-PCR. These indicated that SAMS genes had been integrated to corn genomes and normal expression
4. Ye478 and Zheng58 maize zygotes were transformed for 50 respectively by using Agrobacterium-mediated in plant transformation. 32 plants were got from Ye478 inbred line, and 34 from Zheng58 inbred lines, but less per ear decreased. 3 of T1 positive plants from Ye478 and 4 of T1 positive plants from Zheng58 were got by kanamycin and PCR. It proved that the SAMS gene has been integrated to the genome of maize
5. The study found that Leaf relative water content、Chlorophyll content、Proline content and SOD activity of transgenic maize were more than CK plants while Conductance and MDA content were less under the same water stress time. When the time reached 60h, Leaf relative water content、Chlorophyll content、Proline content and SOD activity of transgenic maize were 8.16%、20.02%、32.21%、22.77% higher than the physiological indexes of CK, but Conductance and MDA content were 14.38%、29.41% lower than them were in CK. We can conclude that genetically modified maize has improved the drought-resistance by introducing of the foreign SAMS genes.
引文
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Sharp R E,Poroyko V,Hejlek L G,SpollenPOLLEN W G,Springer G K,Bohnert H J,Nguyen H T. 2004.Root growth main tenance during water deficits physiology to functional genomies.Joural of Experim ental Botany,55(407):2343~2351
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