煙草西柏三烯一醇合成酶基因（CYC）過表達研究
發(fā)布時間：2018-12-15 06:30
【摘要】：煙草葉面化學成分與煙株抗性和煙葉品質(zhì)密切相關(guān)。為調(diào)控煙草葉面化學的含量和組分,本文克隆了煙草西柏三烯一醇合成酶基因(Cembratrien-ol Synthetase,CYC),分別構(gòu)建了組成型植物過表達載體(p K7WG2-CYC)和腺毛特異表達載體(p CAMBIA1391-CYP-CYC)。采用Ti質(zhì)粒介導法轉(zhuǎn)化煙草,經(jīng)PCR、Southern-blot、qPCR、Northern-blot檢測,獲得了CYC基因組成型過表達和腺毛特異過表達煙草轉(zhuǎn)基因株系,并對轉(zhuǎn)基因因株系進行了形態(tài)學、葉面化學成分分析。為闡明CYC基因過表達對煙草葉面成分及煙株抗性和品質(zhì)的影響奠定了基礎(chǔ)。主要研究結(jié)果如下:1.CYC基因克隆及分析:根據(jù)NCBI中煙草序列設(shè)計引物,以c DNA為模板進行PCR擴增,獲得煙草Nicotiana tabacum K326中的西柏三烯一醇合成的關(guān)鍵基因CYC的ORF序列。序列比對發(fā)現(xiàn),所獲得的核苷酸序列與林煙草中的西柏三烯一醇合成酶m RNA的序列同源性高達99%。2.CYC轉(zhuǎn)化煙草研究。CYC基因在煙草中組成型表達:構(gòu)建了植物表達載體p K7WG2-CYC;采用農(nóng)桿菌介導方法轉(zhuǎn)化煙草K326,獲得了轉(zhuǎn)基因陽性植株68株。對轉(zhuǎn)基因植株進行PCR鑒定,結(jié)果顯示CYC基因成功整合至煙草基因組DNA。qPCR結(jié)果表明,68個再生植株中共有51個表達量高于對照野生型K326。其中,CYC的表達量最高可達對照的40倍以上。3.CYC基因在煙草腺毛特異過表達:將CYC基因亞克隆至腺毛特異表達載體p CAMBIA1391-CYP中,構(gòu)建了CYC的腺毛特異表達載體p CAMBIA1391-CYP-CYC。DNA的PCR結(jié)果顯示7株再生植株中CYC基因都成功插入煙草基因組DNA,取六株進行Southern-blot檢測,結(jié)果顯示,共有四株檢測到CYC基因序列成功插入基因組DNA,其中三株插入位點可能是一樣的。qPCR結(jié)果證明,6個再生植株中,共有5個表達量高于對照野生型K326。6個再生植株除株系9外的CYC基因的表達量明顯高于未轉(zhuǎn)基因的對照植株,最高達到74.8倍。Northern-blot檢測結(jié)果顯示,6個再生株系株系5、6、7、8、9、10均出現(xiàn)目的條帶,表明CYC基因在轉(zhuǎn)基因植株中實現(xiàn)了轉(zhuǎn)錄。4.轉(zhuǎn)基因植株分析:對CYC組成型轉(zhuǎn)基因植株和CYC腺毛特異過表達轉(zhuǎn)基因植株進行了形態(tài)學分析。大部分組成型轉(zhuǎn)基因株系與對照無明顯差異,部分株系表型變化很大,出現(xiàn)了莖稈彎曲、植株矮小等現(xiàn)象。CYC腺毛特異表達轉(zhuǎn)基因株系的表型無明顯變化。采用GC/MS對轉(zhuǎn)基因植株葉面化學成分進行分析,結(jié)果發(fā)現(xiàn)葉面分泌物中西柏三烯一醇含量在轉(zhuǎn)基因株系C-14、C-20、C-38、C-53中均明顯高于對照,其中,株系C-20最高,可達對照的五倍左右,而株系C-7和C-40中西柏三烯一醇含量低于對照;葉面分泌物中西柏三烯二醇含量在轉(zhuǎn)基因株系C-14,C-53和C-38中均明顯高于對照,其中,C-14接近對照的三倍。表明CYC的過表達有利于提高煙草葉面分泌物中西柏三烯一醇、西柏三烯二醇的含量,而對糖酯類和烷烴類化合物的積累無明顯影響。
[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.
【學位授予單位】：河南農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S572
本文編號：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.
【學位授予單位】：河南農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S572
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