农杆菌介导的acdS基因转化烟草和中苜3号的研究
摘要
ACC脱氨酶基因可以将乙烯合成的前体物质ACC(1-氨基环丙烷-1羧酸)降解为α-丁酮酸和氨,从而降低植物体内乙烯的生成量,达到增强植株的抗逆性的目的。本研究以普通烟草品种NC89和紫花苜蓿品种“中苜3号”作为基因转化受体,利用农杆菌介导法将连接到CaMV35S-2和rolD两种启动子下游、来自于根际促生菌-假单胞杆菌UW4的acdS基因转入其外植体。在叶盘法转化苜蓿的过程中,优化了紫花苜蓿高频再生组织培养体系,得到了转基因烟草和紫花苜蓿植株。对转基因植株进行检测,以期获得耐逆性更好的烟草和苜蓿材料。经过分子生物学鉴定表明,表达元件已经整合到转基因植株的基因组中,并且能转录为mRNA,在转录水平上没有沉默。
主要结果如下:目的基因acdS的转化及对转基因烟草的初步鉴定。对转基因植株进行PCR、RT-PCR检测和耐盐性分析,结果表明,目的基因acdS已经整合到烟草基因组中,在转录水平上得到表达,且rolD启动子调控的含有acdS基因的转基因烟草有更强的耐盐性;建立和优化了“中苜3号”体胚发生体系。选用培养5~7 d的无菌苗子叶为外植体,通过不同处理结果的比较表明,UM +KT 1.5mgL-1的培养基是胚状体分化诱导效果较好的培养基;农杆菌介导的acdS基因转化“中苜3号”遗传转化体系的建立与优化。通过对试验现象的观察和数据的统计分析表明,将培养5~7d的苜蓿无菌苗的子叶先预培养2 d,经OD600≈0.5左右的农杆菌菌液浸染10min,共培养3 d,然后转接到含500mgL-1的头孢霉素的UM培养基上培养一周左右。最后转入含有50mgL-1的卡那霉素和300mgL-1的头孢霉素的UM培养基上进行筛选培养的转化体系效果最佳;目的基因acdS的转化及对转基因苜蓿的初步鉴定。将UM+2,4-D 2mgL-1+KT 0.25mgL-1+Kan 50mgL-1+Cef 300mgL-1培养基所诱导出的抗性愈伤组织转接到UM+KT 1.5mgL-1 +Kan 50mgL-1+Cef 100mgL-1培养基上进行胚状体的诱导,然后在1/2MS培养基上使其生根成苗。最后,对转基因植株进行针对目的基因acdS的PCR检测,结果部分植株呈阳性。对PCR检测呈阳性的植株进行Southern检测、RT-PCR检测和cDNA测序鉴定,结果表明,目的基因acdS已经整合到苜蓿基因组中,且在转录水平上得到正确的表达。
ACC deaminase can catalyzes metabolism of ACC to ammonoa anα-ketobutyrate.The gene transformation acceptors selected in this experiment are tobacco NC89 and the Alfalfa cultivar Medicago sativa L.“Zhongmu NO.3. The acdS gene, which had been cloned from Pseudomonas putida UW4 and ligated to CaMV35S-2 and rolD promoter, was transformed into tobacco NC89 by Agrobacterium -mediated. In the process of transferring alfalfa by leaf disks, we optimized high -frequency regeneration system. Based on that, gene acdS, was also transformed into Medicago sativa L.“Zhongmu NO.3”by Agrobacterium-mediated. The transgenic alfalfa plants were gained and the expression elements could be integrated into the genome of alfalfa by identification.
The main results were as following: Transformation of acdS and identification of transgenic tobacco. By PCR, RT-PCR and salt-tolerance analysis, the results indicated that acdS gene had already inserted into the genome in the plants and transcripted to mRNA. The transgenic tobacco palnts of the gene acdS under the transcriptional control of the rolD promoter had stronger salt-tolerance than the other; Select the cotyledon as the explant, conduct the tissue culture systematic research and seek a high differentiation rate explant and the basic culture medium that suitable for this variety. It was indicated that UM +KT 1.5mgL-1 medium had a better effect on embryo induction;Establishment and optimization of genetic transformation system by Agrobacterium-mediated.The optimized genetic transformation procedure was: The edges of cotyledons from aseptic plants which had been cultured for 5~7 days were cut, pre-culture for 2 days then moved to a suspension of Agrobacterium cells. Cell density was adjusted to fall 0.5 at OD600. After 10min inoculation and co-culture 3 days, the explants were transferred to UM containing 500 mgL-1 cefotaxime for a week.Then they were transferred to UM containing 300mgL-1 cefotaxime and 50mgL-1 kanamycin for induction of Kan- resistance callus; Transformation of acdS and identification of transgenic plants.The acdS gene was transformed into Medicago sativa L.“Zhongmu NO.3”according to transformation procedure showed above. Kan-resistance callus which derived from explants were transferred to UM containing 1.5mgL-1 kinetin, 100 mgL-1 cefotaxime and 50mgL-1 Kanamycin for embryo induction and development. Embryos derived from callus were transferred to 1/2MS for the formation of roots. Finally, molecular identification of regeneration plants was carried out; the results of PCR and southern Blot identification of acdS gene showed that some alfalfa plants had inserted into the genome.In addition, the results of RT-PCR and cDNA sequencing showed that the gene of acdS had transcribed into mRNA.
主要结果如下:目的基因acdS的转化及对转基因烟草的初步鉴定。对转基因植株进行PCR、RT-PCR检测和耐盐性分析,结果表明,目的基因acdS已经整合到烟草基因组中,在转录水平上得到表达,且rolD启动子调控的含有acdS基因的转基因烟草有更强的耐盐性;建立和优化了“中苜3号”体胚发生体系。选用培养5~7 d的无菌苗子叶为外植体,通过不同处理结果的比较表明,UM +KT 1.5mgL-1的培养基是胚状体分化诱导效果较好的培养基;农杆菌介导的acdS基因转化“中苜3号”遗传转化体系的建立与优化。通过对试验现象的观察和数据的统计分析表明,将培养5~7d的苜蓿无菌苗的子叶先预培养2 d,经OD600≈0.5左右的农杆菌菌液浸染10min,共培养3 d,然后转接到含500mgL-1的头孢霉素的UM培养基上培养一周左右。最后转入含有50mgL-1的卡那霉素和300mgL-1的头孢霉素的UM培养基上进行筛选培养的转化体系效果最佳;目的基因acdS的转化及对转基因苜蓿的初步鉴定。将UM+2,4-D 2mgL-1+KT 0.25mgL-1+Kan 50mgL-1+Cef 300mgL-1培养基所诱导出的抗性愈伤组织转接到UM+KT 1.5mgL-1 +Kan 50mgL-1+Cef 100mgL-1培养基上进行胚状体的诱导,然后在1/2MS培养基上使其生根成苗。最后,对转基因植株进行针对目的基因acdS的PCR检测,结果部分植株呈阳性。对PCR检测呈阳性的植株进行Southern检测、RT-PCR检测和cDNA测序鉴定,结果表明,目的基因acdS已经整合到苜蓿基因组中,且在转录水平上得到正确的表达。
ACC deaminase can catalyzes metabolism of ACC to ammonoa anα-ketobutyrate.The gene transformation acceptors selected in this experiment are tobacco NC89 and the Alfalfa cultivar Medicago sativa L.“Zhongmu NO.3. The acdS gene, which had been cloned from Pseudomonas putida UW4 and ligated to CaMV35S-2 and rolD promoter, was transformed into tobacco NC89 by Agrobacterium -mediated. In the process of transferring alfalfa by leaf disks, we optimized high -frequency regeneration system. Based on that, gene acdS, was also transformed into Medicago sativa L.“Zhongmu NO.3”by Agrobacterium-mediated. The transgenic alfalfa plants were gained and the expression elements could be integrated into the genome of alfalfa by identification.
The main results were as following: Transformation of acdS and identification of transgenic tobacco. By PCR, RT-PCR and salt-tolerance analysis, the results indicated that acdS gene had already inserted into the genome in the plants and transcripted to mRNA. The transgenic tobacco palnts of the gene acdS under the transcriptional control of the rolD promoter had stronger salt-tolerance than the other; Select the cotyledon as the explant, conduct the tissue culture systematic research and seek a high differentiation rate explant and the basic culture medium that suitable for this variety. It was indicated that UM +KT 1.5mgL-1 medium had a better effect on embryo induction;Establishment and optimization of genetic transformation system by Agrobacterium-mediated.The optimized genetic transformation procedure was: The edges of cotyledons from aseptic plants which had been cultured for 5~7 days were cut, pre-culture for 2 days then moved to a suspension of Agrobacterium cells. Cell density was adjusted to fall 0.5 at OD600. After 10min inoculation and co-culture 3 days, the explants were transferred to UM containing 500 mgL-1 cefotaxime for a week.Then they were transferred to UM containing 300mgL-1 cefotaxime and 50mgL-1 kanamycin for induction of Kan- resistance callus; Transformation of acdS and identification of transgenic plants.The acdS gene was transformed into Medicago sativa L.“Zhongmu NO.3”according to transformation procedure showed above. Kan-resistance callus which derived from explants were transferred to UM containing 1.5mgL-1 kinetin, 100 mgL-1 cefotaxime and 50mgL-1 Kanamycin for embryo induction and development. Embryos derived from callus were transferred to 1/2MS for the formation of roots. Finally, molecular identification of regeneration plants was carried out; the results of PCR and southern Blot identification of acdS gene showed that some alfalfa plants had inserted into the genome.In addition, the results of RT-PCR and cDNA sequencing showed that the gene of acdS had transcribed into mRNA.
引文
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