枸杞类胡萝卜素合成酶基因分离
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摘要
类胡萝卜素是自然界广泛存在的一大类色素,具有许多重要的生物学功能,在人类营养中,类胡萝卜素扮演着更重要的角色。目前,人们对增加类胡萝卜素含量提高农作物的营养价值、改善外观品质以及改变观赏植物的花色有着极大的兴趣。
RNA分离提取是cDNA文库构建重要操作。RNA操作包括RNA纯化,RNA反转录PCR等。本实验针对植物根部和茎部干扰成分的存在,得到其相应适宜的提取方法;并且通过调整试剂及操作步骤从而达到理想效果。为下一步cDNA文库构建奠定了良好的基础。
以中华枸杞根、茎及叶片为材料,对比分析TRNzol提取法、异硫氰酸胍法、CTAB提取法和SDS-酚提取法提取总RNA,得到了针对枸杞根、茎及叶片等器官完整的总RNA提取方法。其中克服根部RNA难以提取的困难,得到了质量非常高的枸杞根部总RNA。同时协助进行了cDNA文库的构建,得到了滴度为2.78×10~5 pfu/ml,重组效率为88.1%的枸杞叶片cDNA文库。进行了cDNA保存和扩增。
用Digoxigenin(DIG)标记龙胆草PDS探针、ZDS探针,利用梯度稀释方法检测探针效率,并得到了理想的效果,探针效率达到了0.1pg/μl。并对枸杞叶片cDNA文库进行筛选,通过梯度稀释得到了比较好的文库筛选浓度;通过调整杂交条件和筛选洗脱严谨程度获得了2个ZDS cDNA阳性克隆,得到了枸杞ZDS的同源基因。本研究为利用基因工程手段来调控类胡萝卜素的生物合成奠定了基础。
Carotenoid is a kind of pigment which is wild existent in the nature. Carotenoids are a group of phytochemicals that have strong antioxidant properties and therefore protect biological structures, so it plays an important role in the nutrition of human. At present, people have a great interest on the effect of carotenoid in improving the crop's nutrition, improving appearance character and changing the design and color of ornamental.
RNA extraction plays an important role in the construction of cDNA library. The operation of RNA includes purifying RNA, amplifying via RT-PCR and so on. In the experiment, aimed at the existence of disturb composition, getting the suitable extraction method. This makes a well groundwork for the building of cDNA library.
Using the root, stem and leaf of lycium barbarm as the stuff, with contrast of TRIZOL Reagent Method, CTAB-acidic Phenolic Method, isothiocyanate method and SDS- phenol Method, the ideal method for extraction of total RNA from the root, stem and leaf of lycium barbarm was established. In the experiment, overcome the hardness that the RNA form the root is very hard to be extracted, and getted good quality RNA. At the same time, assisted building the lycium barbarm cDNA library and the capacity of the library was 2.78×10~5pfu, and the recombination rate reached to 88.1%.
Heterologous probe of PDS、ZDS originate from Getina lutea were labeled with digoxingenin-11-dUTP (DIG), were further used to screen the cDNA libraries of leaf of lycium barbarm. Two ZDS positive clones were obtained.This study will lay foundations for regulating carotenoid biosynthesis via genetic engineening.
RNA分离提取是cDNA文库构建重要操作。RNA操作包括RNA纯化,RNA反转录PCR等。本实验针对植物根部和茎部干扰成分的存在,得到其相应适宜的提取方法;并且通过调整试剂及操作步骤从而达到理想效果。为下一步cDNA文库构建奠定了良好的基础。
以中华枸杞根、茎及叶片为材料,对比分析TRNzol提取法、异硫氰酸胍法、CTAB提取法和SDS-酚提取法提取总RNA,得到了针对枸杞根、茎及叶片等器官完整的总RNA提取方法。其中克服根部RNA难以提取的困难,得到了质量非常高的枸杞根部总RNA。同时协助进行了cDNA文库的构建,得到了滴度为2.78×10~5 pfu/ml,重组效率为88.1%的枸杞叶片cDNA文库。进行了cDNA保存和扩增。
用Digoxigenin(DIG)标记龙胆草PDS探针、ZDS探针,利用梯度稀释方法检测探针效率,并得到了理想的效果,探针效率达到了0.1pg/μl。并对枸杞叶片cDNA文库进行筛选,通过梯度稀释得到了比较好的文库筛选浓度;通过调整杂交条件和筛选洗脱严谨程度获得了2个ZDS cDNA阳性克隆,得到了枸杞ZDS的同源基因。本研究为利用基因工程手段来调控类胡萝卜素的生物合成奠定了基础。
Carotenoid is a kind of pigment which is wild existent in the nature. Carotenoids are a group of phytochemicals that have strong antioxidant properties and therefore protect biological structures, so it plays an important role in the nutrition of human. At present, people have a great interest on the effect of carotenoid in improving the crop's nutrition, improving appearance character and changing the design and color of ornamental.
RNA extraction plays an important role in the construction of cDNA library. The operation of RNA includes purifying RNA, amplifying via RT-PCR and so on. In the experiment, aimed at the existence of disturb composition, getting the suitable extraction method. This makes a well groundwork for the building of cDNA library.
Using the root, stem and leaf of lycium barbarm as the stuff, with contrast of TRIZOL Reagent Method, CTAB-acidic Phenolic Method, isothiocyanate method and SDS- phenol Method, the ideal method for extraction of total RNA from the root, stem and leaf of lycium barbarm was established. In the experiment, overcome the hardness that the RNA form the root is very hard to be extracted, and getted good quality RNA. At the same time, assisted building the lycium barbarm cDNA library and the capacity of the library was 2.78×10~5pfu, and the recombination rate reached to 88.1%.
Heterologous probe of PDS、ZDS originate from Getina lutea were labeled with digoxingenin-11-dUTP (DIG), were further used to screen the cDNA libraries of leaf of lycium barbarm. Two ZDS positive clones were obtained.This study will lay foundations for regulating carotenoid biosynthesis via genetic engineening.
引文
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[3]Yamamoto H Y. Xanthophyll cycles Method Enzymol [M]. 1985,110:303~312
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[5]徐昌杰,张上隆,植物类胡萝卜素的生物合成及其调控,植物生理学通讯. 2000,2 36(1):64~70
[6]von Lintig J,Welsch R ,Bonkm et al. Light-dependent regulation of carotenoid biosynthesis occurs at the level of phytoene synthase expression and ismediated by phytochrome in Sinapis alba and Arabidopsis thaliana seedlings. Plant J ,1997,12:625~634
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[11] Bouvier.F.,Keller.Y.,Xanthophy 11 biosynthesis :molecular and functional characterization of carotenoid hydroxylases from pepper fruits Biochim. Biophs. 1998 1391(3):320~828 [12 ] Fraser PD,TruesdalemR,Bird CR et al. Carotenoid biosynthesis duringtomato fruit development. Plant Physiol,1994,105:379~387
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