MdSPDS1基因的功能分析及转化毛白杨的初步研究
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摘要
目前,土壤盐渍化严重影响农林业生产和生态环境,如何使大片的盐碱地以及丰富的咸水资源被充分利用,成为当前研究的热点问题。而多胺(polyamines,PAs)对提高植物抗逆性起着重要的作用,为此,本研究开展了对多胺合成途径中的相关基因苹果亚精胺合成酶(apple spermidine synthase genes1,MdSPDS1)的基因功能分析,并首次将其转入毛白杨(Populus tomentosa Carr.)中,从而提高转基因毛白杨的耐盐能力。本研究工作结果如下:
(1)农杆菌介导法将MdSPDS1基因转入普通烟草(Nicotiana tabacum)中,采用实时荧光定量PCR方法和高效液相色谱法(HPLC)分别检测了植株中MdSPDS1基因的相对表达情况,以及三种主要多胺——腐胺、亚精胺、精胺(Put、Spd、Spm)的含量,结果发现转MdSPDS1基因的烟草中Spd含量是非转基因的2-3倍。
(2)在初步探讨MdSPDS1基因功能中,耐盐性分析时发现MdSPDS1表达量高的转基因烟草D14可以在400mM的NaCl胁迫下生存,在进一步的多胺含量变化趋势测定中,结果表明Spd的含量和(Spd+Spm)/Put比值的增加对提高植物的耐盐性起到重要的作用。同时在转基因烟草抗病性分析中,D14的抗烟草花叶病毒(TMV)能力强于为转基因植株WT,WT植株感病后,Put含量比初始增加了5倍多,而D14中Spd含量增加4倍左右,暗示Spd的增加可能是促进植物抗烟草花叶病毒能力提高的原因。
(3)初步建立了毛白杨遗传转化体系,对卡那霉素抗性芽进行了PCR和Southern检测,结果表明,成功获得了转MdSPDS1基因的毛白杨植株。并测定了转基因毛白杨中MdSPDS1基因相对表达量和多胺的含量,其中T7的Spd含量是未转化植株的1.65倍。转基因毛白杨耐盐性初步分析中发现转MdSPDS1基因的毛白杨可以在200mM的盐胁迫下生存。
The problem of the salty soil has represented some of the most significant constraints to agricultural and forestry productivity. It has become an important topic and hotspot on the researching of the future development in agriculture and forestry that how to make use of the large quantity of salty soil and salty water resources. Polyamines play an important role in plants to enhance environmental stress tolerance. So, functional analysis of MdSPDS1 gene which is encoding Polyamines biosynthesis has been carrying out. And transgenic Populus tomentosa Carr. with MdSPDS1 was abtained. The major results and conclusions are described as follows.
(1) MdSPDS1 was transferred to Nicotiana tabacum by Agrobacterium–mediated, and the expression of MdSPDS1 gene was analyzed by Real time Quantitative PCR. The contents of three main polyamines -- Put、Spd、Spm were detected by HPLC. The result showed that the Spd levels in transgenic plants were 2-3 fold when compared to untransgenic plants.
(2) It was found that transgenic plant D14 which has high expression of MdSPDS1 could live under 400mM NaCl stress. Under this stress, contents of polyamines were detected to learn the changes of three main polyamines. The results illustrated that the increase of Spd content and (Spd+Spm)/Put played an important role in enhancing tolerance to salt stress. On the other hand, it was found that the ability of resistance to TMV in D14 was higher than WT, and PAs levels altered by TMV infected. Put levels in WT were enhanced more than 5-folds when compared to healthy control; while Spd levels in D14 were enhanced 4-folds. The result showed that increase of Spd was one of the reseaons of enhancing plant resistance to TMV.
(3) The primary transformation system was established in Populus tomentosa. The integration of the MdSPDS1 gene into plant genomes of transgenic Populus tomentosa was confirmed by PCR and Southern blot analysis. The expressions of MdSPDS1 gene and polyamines levels were also detected. The result showed that the Spd levels in transgenic plants were 1.65-folds when compared to untransgenic plants. It was also found that transgenic Populus tomentosa could live under 200mM NaCl stress.
(1)农杆菌介导法将MdSPDS1基因转入普通烟草(Nicotiana tabacum)中,采用实时荧光定量PCR方法和高效液相色谱法(HPLC)分别检测了植株中MdSPDS1基因的相对表达情况,以及三种主要多胺——腐胺、亚精胺、精胺(Put、Spd、Spm)的含量,结果发现转MdSPDS1基因的烟草中Spd含量是非转基因的2-3倍。
(2)在初步探讨MdSPDS1基因功能中,耐盐性分析时发现MdSPDS1表达量高的转基因烟草D14可以在400mM的NaCl胁迫下生存,在进一步的多胺含量变化趋势测定中,结果表明Spd的含量和(Spd+Spm)/Put比值的增加对提高植物的耐盐性起到重要的作用。同时在转基因烟草抗病性分析中,D14的抗烟草花叶病毒(TMV)能力强于为转基因植株WT,WT植株感病后,Put含量比初始增加了5倍多,而D14中Spd含量增加4倍左右,暗示Spd的增加可能是促进植物抗烟草花叶病毒能力提高的原因。
(3)初步建立了毛白杨遗传转化体系,对卡那霉素抗性芽进行了PCR和Southern检测,结果表明,成功获得了转MdSPDS1基因的毛白杨植株。并测定了转基因毛白杨中MdSPDS1基因相对表达量和多胺的含量,其中T7的Spd含量是未转化植株的1.65倍。转基因毛白杨耐盐性初步分析中发现转MdSPDS1基因的毛白杨可以在200mM的盐胁迫下生存。
The problem of the salty soil has represented some of the most significant constraints to agricultural and forestry productivity. It has become an important topic and hotspot on the researching of the future development in agriculture and forestry that how to make use of the large quantity of salty soil and salty water resources. Polyamines play an important role in plants to enhance environmental stress tolerance. So, functional analysis of MdSPDS1 gene which is encoding Polyamines biosynthesis has been carrying out. And transgenic Populus tomentosa Carr. with MdSPDS1 was abtained. The major results and conclusions are described as follows.
(1) MdSPDS1 was transferred to Nicotiana tabacum by Agrobacterium–mediated, and the expression of MdSPDS1 gene was analyzed by Real time Quantitative PCR. The contents of three main polyamines -- Put、Spd、Spm were detected by HPLC. The result showed that the Spd levels in transgenic plants were 2-3 fold when compared to untransgenic plants.
(2) It was found that transgenic plant D14 which has high expression of MdSPDS1 could live under 400mM NaCl stress. Under this stress, contents of polyamines were detected to learn the changes of three main polyamines. The results illustrated that the increase of Spd content and (Spd+Spm)/Put played an important role in enhancing tolerance to salt stress. On the other hand, it was found that the ability of resistance to TMV in D14 was higher than WT, and PAs levels altered by TMV infected. Put levels in WT were enhanced more than 5-folds when compared to healthy control; while Spd levels in D14 were enhanced 4-folds. The result showed that increase of Spd was one of the reseaons of enhancing plant resistance to TMV.
(3) The primary transformation system was established in Populus tomentosa. The integration of the MdSPDS1 gene into plant genomes of transgenic Populus tomentosa was confirmed by PCR and Southern blot analysis. The expressions of MdSPDS1 gene and polyamines levels were also detected. The result showed that the Spd levels in transgenic plants were 1.65-folds when compared to untransgenic plants. It was also found that transgenic Populus tomentosa could live under 200mM NaCl stress.
引文
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