烟草西柏三烯一醇合成酶基因（CYC）过表达研究
发布时间：2018-12-15 06:30
【摘要】：烟草叶面化学成分与烟株抗性和烟叶品质密切相关。为调控烟草叶面化学的含量和组分,本文克隆了烟草西柏三烯一醇合成酶基因(Cembratrien-ol Synthetase,CYC),分别构建了组成型植物过表达载体(p K7WG2-CYC)和腺毛特异表达载体(p CAMBIA1391-CYP-CYC)。采用Ti质粒介导法转化烟草,经PCR、Southern-blot、qPCR、Northern-blot检测,获得了CYC基因组成型过表达和腺毛特异过表达烟草转基因株系,并对转基因因株系进行了形态学、叶面化学成分分析。为阐明CYC基因过表达对烟草叶面成分及烟株抗性和品质的影响奠定了基础。主要研究结果如下:1.CYC基因克隆及分析:根据NCBI中烟草序列设计引物,以c DNA为模板进行PCR扩增,获得烟草Nicotiana tabacum K326中的西柏三烯一醇合成的关键基因CYC的ORF序列。序列比对发现,所获得的核苷酸序列与林烟草中的西柏三烯一醇合成酶m RNA的序列同源性高达99%。2.CYC转化烟草研究。CYC基因在烟草中组成型表达:构建了植物表达载体p K7WG2-CYC;采用农杆菌介导方法转化烟草K326,获得了转基因阳性植株68株。对转基因植株进行PCR鉴定,结果显示CYC基因成功整合至烟草基因组DNA。qPCR结果表明,68个再生植株中共有51个表达量高于对照野生型K326。其中,CYC的表达量最高可达对照的40倍以上。3.CYC基因在烟草腺毛特异过表达:将CYC基因亚克隆至腺毛特异表达载体p CAMBIA1391-CYP中,构建了CYC的腺毛特异表达载体p CAMBIA1391-CYP-CYC。DNA的PCR结果显示7株再生植株中CYC基因都成功插入烟草基因组DNA,取六株进行Southern-blot检测,结果显示,共有四株检测到CYC基因序列成功插入基因组DNA,其中三株插入位点可能是一样的。qPCR结果证明,6个再生植株中,共有5个表达量高于对照野生型K326。6个再生植株除株系9外的CYC基因的表达量明显高于未转基因的对照植株,最高达到74.8倍。Northern-blot检测结果显示,6个再生株系株系5、6、7、8、9、10均出现目的条带,表明CYC基因在转基因植株中实现了转录。4.转基因植株分析:对CYC组成型转基因植株和CYC腺毛特异过表达转基因植株进行了形态学分析。大部分组成型转基因株系与对照无明显差异,部分株系表型变化很大,出现了茎秆弯曲、植株矮小等现象。CYC腺毛特异表达转基因株系的表型无明显变化。采用GC/MS对转基因植株叶面化学成分进行分析,结果发现叶面分泌物中西柏三烯一醇含量在转基因株系C-14、C-20、C-38、C-53中均明显高于对照,其中,株系C-20最高,可达对照的五倍左右,而株系C-7和C-40中西柏三烯一醇含量低于对照;叶面分泌物中西柏三烯二醇含量在转基因株系C-14,C-53和C-38中均明显高于对照,其中,C-14接近对照的三倍。表明CYC的过表达有利于提高烟草叶面分泌物中西柏三烯一醇、西柏三烯二醇的含量,而对糖酯类和烷烃类化合物的积累无明显影响。
[Abstract]:The chemical composition of tobacco leaf surface is closely related to the resistance of tobacco plant and the quality of tobacco leaf. In order to control the content and composition of tobacco leaf surface chemistry, the sibtriene-synthase gene (Cembratrien-ol Synthetase,CYC) was cloned. Constitutive plant overexpression vectors (p K7WG2-CYC) and glandular hair specific expression vectors (p CAMBIA1391-CYP-CYC) were constructed. Transgenic tobacco lines were transformed by Ti plasmid mediated transformation. After PCR,Southern-blot,qPCR,Northern-blot detection, CYC genome-forming overexpression and glandular hair specific overexpression were obtained, and the transgenic lines were morphologically studied. Chemical composition analysis of leaf surface. The results laid a foundation for elucidating the effect of CYC gene overexpression on leaf composition, resistance and quality of tobacco plants. The main results are as follows: 1.CYC gene was cloned and analyzed. According to the tobacco sequence in NCBI, primers were designed and PCR amplified by using c DNA as template. The ORF sequence of the key gene CYC in tobacco Nicotiana tabacum K326 was obtained. Sequence alignment found, The nucleotide sequence obtained was highly homologous to that of sibertrienol synthase m RNA in forest tobacco. The CYC gene was expressed constitutively in tobacco. A plant expression vector, pK7WG2-CYC, was constructed. 68 transgenic plants were obtained by Agrobacterium tumefaciens mediated transformation of tobacco K326. The results of PCR identification of transgenic plants showed that CYC gene was successfully integrated into tobacco genome DNA.qPCR. 51 of 68 regenerated plants had higher expression than wild-type K326. The expression of CYC was 40 times higher than that of the control. The 3.CYC gene was overexpressed in tobacco glandular hair. The CYC gene was subcloned into the glandular hair specific expression vector p CAMBIA1391-CYP. The PCR results showed that the CYC gene of 7 regenerated plants was successfully inserted into the genomic DNA, of tobacco for Southern-blot detection. A total of four CYC gene sequences were successfully inserted into genomic DNA,. QPCR results showed that three of the six regenerated plants had the same insertion site. The expression of CYC gene in 5 regenerated plants with wild type K326.6 was significantly higher than that of the control, except for line 9, and the highest expression was 74.8 times. The results of Northern-blot analysis showed that the expression of CYC gene in 5 regenerated plants was 74.8 times higher than that in the control. The target bands were found in all the 6 regenerative lines, indicating that the CYC gene was transcribed in transgenic plants. Analysis of transgenic plants: morphological analysis of CYC constitutive transgenic plants and CYC glandular hair specific overexpression transgenic plants was carried out. There was no significant difference between most of the transgenic lines and the control. The phenotypic changes of some lines were very great, such as stem bending and plant dwarfing. The phenotypes of CYC glandular hair specifically expressed transgenic lines had no obvious change. The chemical composition of the leaf surface of transgenic plants was analyzed by GC/MS. The results showed that the contents of berbertrienol in leaf surface secretion were significantly higher than those of the control in the transgenic line C-14, C-20, C-38, C-53, and the results showed that the chemical composition of the leaves of the transgenic plants was significantly higher than that of the control. The content of C-20 was the highest, about five times of that of the control, while the contents of C-7 and C-40 were lower than those of the control. The content of ezeparaxadiol in leaf secretion was significantly higher than that of the control in the transgenic lines C-14, C-53 and C-38, and C-14 was nearly three times of that of the control. The results showed that the overexpression of CYC was beneficial to the increase of the contents of estetrienol and sibertrienediol in tobacco leaf secretion, but had no significant effect on the accumulation of carbohydrates and alkanes.
【学位授予单位】：河南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S572
本文编号：2380141
[Abstract]:The chemical composition of tobacco leaf surface is closely related to the resistance of tobacco plant and the quality of tobacco leaf. In order to control the content and composition of tobacco leaf surface chemistry, the sibtriene-synthase gene (Cembratrien-ol Synthetase,CYC) was cloned. Constitutive plant overexpression vectors (p K7WG2-CYC) and glandular hair specific expression vectors (p CAMBIA1391-CYP-CYC) were constructed. Transgenic tobacco lines were transformed by Ti plasmid mediated transformation. After PCR,Southern-blot,qPCR,Northern-blot detection, CYC genome-forming overexpression and glandular hair specific overexpression were obtained, and the transgenic lines were morphologically studied. Chemical composition analysis of leaf surface. The results laid a foundation for elucidating the effect of CYC gene overexpression on leaf composition, resistance and quality of tobacco plants. The main results are as follows: 1.CYC gene was cloned and analyzed. According to the tobacco sequence in NCBI, primers were designed and PCR amplified by using c DNA as template. The ORF sequence of the key gene CYC in tobacco Nicotiana tabacum K326 was obtained. Sequence alignment found, The nucleotide sequence obtained was highly homologous to that of sibertrienol synthase m RNA in forest tobacco. The CYC gene was expressed constitutively in tobacco. A plant expression vector, pK7WG2-CYC, was constructed. 68 transgenic plants were obtained by Agrobacterium tumefaciens mediated transformation of tobacco K326. The results of PCR identification of transgenic plants showed that CYC gene was successfully integrated into tobacco genome DNA.qPCR. 51 of 68 regenerated plants had higher expression than wild-type K326. The expression of CYC was 40 times higher than that of the control. The 3.CYC gene was overexpressed in tobacco glandular hair. The CYC gene was subcloned into the glandular hair specific expression vector p CAMBIA1391-CYP. The PCR results showed that the CYC gene of 7 regenerated plants was successfully inserted into the genomic DNA, of tobacco for Southern-blot detection. A total of four CYC gene sequences were successfully inserted into genomic DNA,. QPCR results showed that three of the six regenerated plants had the same insertion site. The expression of CYC gene in 5 regenerated plants with wild type K326.6 was significantly higher than that of the control, except for line 9, and the highest expression was 74.8 times. The results of Northern-blot analysis showed that the expression of CYC gene in 5 regenerated plants was 74.8 times higher than that in the control. The target bands were found in all the 6 regenerative lines, indicating that the CYC gene was transcribed in transgenic plants. Analysis of transgenic plants: morphological analysis of CYC constitutive transgenic plants and CYC glandular hair specific overexpression transgenic plants was carried out. There was no significant difference between most of the transgenic lines and the control. The phenotypic changes of some lines were very great, such as stem bending and plant dwarfing. The phenotypes of CYC glandular hair specifically expressed transgenic lines had no obvious change. The chemical composition of the leaf surface of transgenic plants was analyzed by GC/MS. The results showed that the contents of berbertrienol in leaf surface secretion were significantly higher than those of the control in the transgenic line C-14, C-20, C-38, C-53, and the results showed that the chemical composition of the leaves of the transgenic plants was significantly higher than that of the control. The content of C-20 was the highest, about five times of that of the control, while the contents of C-7 and C-40 were lower than those of the control. The content of ezeparaxadiol in leaf secretion was significantly higher than that of the control in the transgenic lines C-14, C-53 and C-38, and C-14 was nearly three times of that of the control. The results showed that the overexpression of CYC was beneficial to the increase of the contents of estetrienol and sibertrienediol in tobacco leaf secretion, but had no significant effect on the accumulation of carbohydrates and alkanes.
【学位授予单位】：河南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S572
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