朝鲜碱茅P5CS基因的克隆及其转化紫花苜蓿的研究
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摘要
本研究利用RT-PCR的方法在朝鲜碱茅（Puccinellia chinampoensis）中分离到一个P5CS的同源新基因，命名为：PuP5CS（Genebank：HQ637435），并通过农杆菌介导法将其转入公农2号紫花苜蓿中，以期改良其耐盐性，结果如下：
表达分析的结果显示，PuP5CS基因在朝鲜碱茅的根部和叶片均有表达，但是根部的表达量较低，叶片中的表达量较高。在根部，PuP5CS基因受盐胁迫、碱胁迫和盐碱复合胁迫诱导的表达量比受干旱胁迫诱导的表达量要高，在叶片中4种胁迫处理均可以诱导PuP5CS基因显著表达，但变化规律不同。
对PuP5CS基因的亚细胞定位的结果显示，其主要表达在细胞膜和细胞核中。
在对转基因烟草进行耐盐性和抗寒性检测中，显示正义转基因烟草在受到高盐胁迫和低温胁迫时，能够明显提高其脯氨酸积累，其叶绿素含量、可溶性糖含量的增长幅度也显著高于对照。而反义转基因烟草在受到非生物胁迫的过程中，其脯氨酸积累、可溶性糖含量等都低于对照。表明PuP5CS基因不仅影响了烟草体内脯氨酸的合成过程，而且还参与了体内可溶性糖、丙二醛、叶绿素a和叶绿素b的代谢调控，从增加渗透调节物质，缓解膜质氧化反应，增强光合作用等几个方面在整体上提高了烟草的抗逆性。
建立了用于公农2号（Medicago sativa L.cv.Gongnong No.2）紫花苜蓿的组织培养高频再生体系。选择子叶作为外植体材料，UM培养基+2mg/L2,4-D+0.25mg/L KT为愈伤诱导培养基，UM培养基+1.2mg/L KT作为分化培养基，1/2MS培养基为生根培养基。共培养时间为3d，转移至含500mg/L头孢霉素的愈伤诱导培养基上进行培养，20d后转至分化培养基上，其中含1.2mg/L KT、500mg/L头孢霉素、2mg/L草铵膦。约20d~40d后，分化出小植株，转入1/2MS+1mg/L草铵膦+500mg/L Cef培养基上，生根后移栽至蛭石：土壤为1:1的混合土壤中，得到了草铵膦阳性植株。经PCR和RT-PCR鉴定，PuP5CS基因已整合至再生植株的基因组上。
In this study, a new P5CS homologous genes was separated by RT-PCR method in Puccinelliachinampoensis, named: PuP5CS (from Genebank: HQ637435). And introduced it into Medicago sativaL.cv.Gongnong No.2by Agrobacterium-mediated genetic transformation, in order to gain the newbreeding of alfalfa.The main result were as following:
Semi-quantitative RT-PCR results show that in the roots and leaves, the gene PuP5CS is expressed,but the expression level in the root of the PuP5CS gene is lower than that in the leaves. In the leaves,PuP5CS gene was expressed higher under salt stress, alkaline stress and salinization combined stress,compares to the drought stress. In the leaves, the four kinds of stress can induce PuP5CS geneexpression with different pattern.
The subcellular localization result shows that PuP5CS genes are mainly expressed in the cellmembrane and nucleus.
When testing the salt tolerance and cold resistance of transgenic tobacco, the overexprssiontransgenic tobacco plants under low high salt stress and temperature stress can significantly improve itsproline accumulation, chlorophyll content and soluble sugar content compares to the control, whilethese in antisense transgenic tobacco plants under abiotic stress is relatively lower than the control.According to the above, it can be concluded that PuP5CS gene affects not only the tobacco prolinesynthesis process, but also participates in the soluble sugar, MDA, chlorophyll a and chlorophyll b.From the increased osmotic adjustment substances alleviate membranous oxidation, enhancedphotosynthesis and other aspects, PuP5CS gene increases the overall stress resistance of tobacco.
An efficient regeneration method of Medicago sativa L. cv. Gongnong No.2has been estanblished.The cotyledon was selected as explant for induction. Select the UM medium+2,4-D2mg/L+KT0.25mg/L as the callus induction medium，UM medium containing1.2mg/L KT for regeneration and1/2MS medium as the rooting medium. After co-cultured for3days, the cotyledon was then transferred onthe UM medium containing2,4-D, KT20days, then transferred on the embruogenesis inductionmedium, which contains1.2mg/L KT,2mg/L glufosinate,500mg/L cefotaxime. After20-40days,shoots grew up from the surface of the calli, then transferred on the1/2MS medium which contains1mg/L glufosinate,500mg/L cefotaxime, after rooting transplant the shoots to vermiculite: soil1:1mixture soil. By using PCR and RT-PCR validation, the result shows that the PuP5CS gene has beenintegrated into the genome of the regenerated plants.
表达分析的结果显示，PuP5CS基因在朝鲜碱茅的根部和叶片均有表达，但是根部的表达量较低，叶片中的表达量较高。在根部，PuP5CS基因受盐胁迫、碱胁迫和盐碱复合胁迫诱导的表达量比受干旱胁迫诱导的表达量要高，在叶片中4种胁迫处理均可以诱导PuP5CS基因显著表达，但变化规律不同。
对PuP5CS基因的亚细胞定位的结果显示，其主要表达在细胞膜和细胞核中。
在对转基因烟草进行耐盐性和抗寒性检测中，显示正义转基因烟草在受到高盐胁迫和低温胁迫时，能够明显提高其脯氨酸积累，其叶绿素含量、可溶性糖含量的增长幅度也显著高于对照。而反义转基因烟草在受到非生物胁迫的过程中，其脯氨酸积累、可溶性糖含量等都低于对照。表明PuP5CS基因不仅影响了烟草体内脯氨酸的合成过程，而且还参与了体内可溶性糖、丙二醛、叶绿素a和叶绿素b的代谢调控，从增加渗透调节物质，缓解膜质氧化反应，增强光合作用等几个方面在整体上提高了烟草的抗逆性。
建立了用于公农2号（Medicago sativa L.cv.Gongnong No.2）紫花苜蓿的组织培养高频再生体系。选择子叶作为外植体材料，UM培养基+2mg/L2,4-D+0.25mg/L KT为愈伤诱导培养基，UM培养基+1.2mg/L KT作为分化培养基，1/2MS培养基为生根培养基。共培养时间为3d，转移至含500mg/L头孢霉素的愈伤诱导培养基上进行培养，20d后转至分化培养基上，其中含1.2mg/L KT、500mg/L头孢霉素、2mg/L草铵膦。约20d~40d后，分化出小植株，转入1/2MS+1mg/L草铵膦+500mg/L Cef培养基上，生根后移栽至蛭石：土壤为1:1的混合土壤中，得到了草铵膦阳性植株。经PCR和RT-PCR鉴定，PuP5CS基因已整合至再生植株的基因组上。
In this study, a new P5CS homologous genes was separated by RT-PCR method in Puccinelliachinampoensis, named: PuP5CS (from Genebank: HQ637435). And introduced it into Medicago sativaL.cv.Gongnong No.2by Agrobacterium-mediated genetic transformation, in order to gain the newbreeding of alfalfa.The main result were as following:
Semi-quantitative RT-PCR results show that in the roots and leaves, the gene PuP5CS is expressed,but the expression level in the root of the PuP5CS gene is lower than that in the leaves. In the leaves,PuP5CS gene was expressed higher under salt stress, alkaline stress and salinization combined stress,compares to the drought stress. In the leaves, the four kinds of stress can induce PuP5CS geneexpression with different pattern.
The subcellular localization result shows that PuP5CS genes are mainly expressed in the cellmembrane and nucleus.
When testing the salt tolerance and cold resistance of transgenic tobacco, the overexprssiontransgenic tobacco plants under low high salt stress and temperature stress can significantly improve itsproline accumulation, chlorophyll content and soluble sugar content compares to the control, whilethese in antisense transgenic tobacco plants under abiotic stress is relatively lower than the control.According to the above, it can be concluded that PuP5CS gene affects not only the tobacco prolinesynthesis process, but also participates in the soluble sugar, MDA, chlorophyll a and chlorophyll b.From the increased osmotic adjustment substances alleviate membranous oxidation, enhancedphotosynthesis and other aspects, PuP5CS gene increases the overall stress resistance of tobacco.
An efficient regeneration method of Medicago sativa L. cv. Gongnong No.2has been estanblished.The cotyledon was selected as explant for induction. Select the UM medium+2,4-D2mg/L+KT0.25mg/L as the callus induction medium，UM medium containing1.2mg/L KT for regeneration and1/2MS medium as the rooting medium. After co-cultured for3days, the cotyledon was then transferred onthe UM medium containing2,4-D, KT20days, then transferred on the embruogenesis inductionmedium, which contains1.2mg/L KT,2mg/L glufosinate,500mg/L cefotaxime. After20-40days,shoots grew up from the surface of the calli, then transferred on the1/2MS medium which contains1mg/L glufosinate,500mg/L cefotaxime, after rooting transplant the shoots to vermiculite: soil1:1mixture soil. By using PCR and RT-PCR validation, the result shows that the PuP5CS gene has beenintegrated into the genome of the regenerated plants.
引文
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