毛竹木质素合成相关基因的克隆与表达
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摘要
我国是世界上竹类资源最丰富的国家,毛竹是竹类资源中最主要且应用最广泛的造林、造纸竹种,因具有速生、纤维素含量高等特点,成为优良的纸浆原材料。由于其独特的生物学特性,传统育种手段的应用受到严重制约。竹材的固有缺陷制约了竹板材加工的高速发展,竹纤维原料匮乏制约了竹浆造纸业的规模化发展。如何定向改良现有的毛竹材性,培育满足我国日益增长的工业化利用所需的多功能、多用途、高性能、高附加值的专用竹材新品种,已成为当前竹类植物遗传育种的首要任务。因此,利用生物技术等手段改变木质素含量或改变其制浆性能,创造出符合制浆造纸原料特性要求和适用于板材加工的竹材新品种,对于竹林培育和竹材工业化利用的技术进步具有重要意义。
PAL是木质素生物合成途径的核心酶之一,位于苯丙烷代谢途径的入口;CCoAOMT和CAD是两种位于木质素特异合成途径下游的酶类,对于木质素合成调控也具有重要作用。本文主要在毛竹木质素生物合成酶相关基因PAL、CCoAOMT和CAD的克隆、表达鉴定以及其烟草转化等方面展开研究工作,具体研究内容如下:
(1)本研究利用改良的CTAB法和Trizol法提取毛竹的DNA和RNA,质量较好,纯度高,可以用作进一步的分子生物学研究。
(2)本研究利用Genome walking法克隆得到毛竹PAL基因的DNA序列,长2 736 bp,基因含有一个内含子和一个预测的启动子序列。推测的毛竹PAL基因DNA序列编码区编码712个氨基酸,分子量约为77 KD。与己知的禾本科植物绿竹、水稻的PAL基因氨基酸序列相似性分别达到95%、94%。
(3)本研究利用RT-PCR和RACE法获得了毛竹PAL基因全长cDNA(命名为PAL1),全长共2 678bp,开放阅读框架(ORF)2 142bp,编码713个氨基酸,分子量约为77 KD,与禾本科植物水稻、绿竹的PAL基因有90%以上的相似性。组织特异性表达表明,PAL1在毛竹根、茎、叶和幼苗中都有表达,但表达量存在较明显差异,根中表达量最高。
(4)分别获得了长度为787 bp和1 019 bp的CCoAOMT和CAD基因片段。编码区分别编码197、282个氨基酸,与水稻、玉米、梯牧草等植物的CCoAOMT、CAD基因均具有90%以上的相似性,GenBank登录号分别为EF549579、EF549577。
(5)构建了毛竹PAL1基因的原核表达载体pET16b-PAL1,在大肠杆菌BL21中诱导表达后纯化出目的蛋白。以L-苯丙氨酸为底物,测定Km值为(0.421±0.023 )×10-3mol/L;最适反应温度是50℃;最适反应pH值为8.8。
(6)构建了毛竹PAL1、CCoAOMT和CAD基因正反义植物表达载体pBI121-PAL1、pBI121-anti PAL1、pBI121-anti CCoAOMT和pBI121-anti CAD,转化农杆菌后通过叶盘法侵染烟草,经过愈伤组织继代培养得到植株幼苗。经PCR初步检测,得到转基因植株,为进一步的研究奠定了基础。
China is one of the most abundant bamboo resource countries in the world. Because of its fast-growing and high cellulose content and its utility in afforestation and paper pulping, Phyllostachys edulis is the most important and widely used bamboo species in China. But the application of P. edulis was restricted by its biological characteristics and the traditional breeding methods. The development of high-speed bamboo process was restricted by the inherent shortcomings of P. edulis and the application in paper pulping industry development was also restricted by the shortages of fiber. So, the most important is how to breed new varieties to meet the growing multi-purpose industrialization of P. edulis at present. Therefore, it is of great significance to use molecular biological technologies to improve lignin content of P. edulis.
PAL is one of the key enzymes in the early stages of the phenylpropanoid pathway and has significant effects on the lignin content of plant. CCoAOMT and CAD are another two enzymes in the lignin synthesization pathway. Both of them also play an important role on the regulation of lignin content in plant. In this study, PAL, CCoAOMT, CAD genes in P. edulis were cloned and the expressions were also studied. The results were summarized as follows:
(1) The DNA and RNA were isolated respectively from P. edulis leaves by modified CTAB and Trizol methods. The result showed that the quality was good enough for further research.
(2) A full length of 2 736 bp DNA fragment encoded PAL of P. edulis was obtained by genome walking, which contained an intron and a forecasted promoter sequences. The PAL fragment encoded 712 amino acids with a molecular weight of 77KD. The deduced amino acid sequence shared 95% homology with the PAL of Bambusa oldhamii and 94% with the PAL of Oryza sativa.
(3) A full length of PAL cDNA named PAL1 was obtained by RT-PCR and RACE. The size of the cDNA fragment was 2 678 bp, which included a 2 142 bp open reading frame. It shared 90% homology with PAL of Oryza sativa and Bambusa oldhami. Tissue specific expression test revealed that there were significant differences in roots, stems, leaves and seedlings.The expression of PAL in root was the highest.
(4) A 787 bp DNA fragment of CCoAOMT and a 1019 bp DNA fragment of CAD were cloned from the genomic DNA of P. edulis which encoded 197 amino acids and 282 amino acids respectively. The CCoAOMT and CAD DNA fragment in P. edulis shared 90% homology with that of Oryza sativa and Phleum pratens. The deduced amino acids sequence of CCoAOMT and CAD in P. edulis shared 85% homology with that of Oryza sativa and Phleum pratens. GenBank accession numbers were EF549579 and EF549577 respectively.
(5) The PAL1 gene was cloned into a prokaryotic expression vector pET-16b and then it was used to transform E.coli. BL21. The Km, the optimum temperature and the optimum pH of purified recombinant protein was (0.421±0.023)×10-3 mol/L, 50℃and 8.8 respectively.
(6) The pBI121-PAL1、pBI121-anti PAL1、pBI121-anti CCoAOMT and pBI121-anti CAD plant expression vectors were constructed. The vectors were introduced into tabacoo plant by agrobacterium-mediated transformation. PCR analyses confirmed the integration of the target gene into the genome of the transformed tobacco plant.
PAL是木质素生物合成途径的核心酶之一,位于苯丙烷代谢途径的入口;CCoAOMT和CAD是两种位于木质素特异合成途径下游的酶类,对于木质素合成调控也具有重要作用。本文主要在毛竹木质素生物合成酶相关基因PAL、CCoAOMT和CAD的克隆、表达鉴定以及其烟草转化等方面展开研究工作,具体研究内容如下:
(1)本研究利用改良的CTAB法和Trizol法提取毛竹的DNA和RNA,质量较好,纯度高,可以用作进一步的分子生物学研究。
(2)本研究利用Genome walking法克隆得到毛竹PAL基因的DNA序列,长2 736 bp,基因含有一个内含子和一个预测的启动子序列。推测的毛竹PAL基因DNA序列编码区编码712个氨基酸,分子量约为77 KD。与己知的禾本科植物绿竹、水稻的PAL基因氨基酸序列相似性分别达到95%、94%。
(3)本研究利用RT-PCR和RACE法获得了毛竹PAL基因全长cDNA(命名为PAL1),全长共2 678bp,开放阅读框架(ORF)2 142bp,编码713个氨基酸,分子量约为77 KD,与禾本科植物水稻、绿竹的PAL基因有90%以上的相似性。组织特异性表达表明,PAL1在毛竹根、茎、叶和幼苗中都有表达,但表达量存在较明显差异,根中表达量最高。
(4)分别获得了长度为787 bp和1 019 bp的CCoAOMT和CAD基因片段。编码区分别编码197、282个氨基酸,与水稻、玉米、梯牧草等植物的CCoAOMT、CAD基因均具有90%以上的相似性,GenBank登录号分别为EF549579、EF549577。
(5)构建了毛竹PAL1基因的原核表达载体pET16b-PAL1,在大肠杆菌BL21中诱导表达后纯化出目的蛋白。以L-苯丙氨酸为底物,测定Km值为(0.421±0.023 )×10-3mol/L;最适反应温度是50℃;最适反应pH值为8.8。
(6)构建了毛竹PAL1、CCoAOMT和CAD基因正反义植物表达载体pBI121-PAL1、pBI121-anti PAL1、pBI121-anti CCoAOMT和pBI121-anti CAD,转化农杆菌后通过叶盘法侵染烟草,经过愈伤组织继代培养得到植株幼苗。经PCR初步检测,得到转基因植株,为进一步的研究奠定了基础。
China is one of the most abundant bamboo resource countries in the world. Because of its fast-growing and high cellulose content and its utility in afforestation and paper pulping, Phyllostachys edulis is the most important and widely used bamboo species in China. But the application of P. edulis was restricted by its biological characteristics and the traditional breeding methods. The development of high-speed bamboo process was restricted by the inherent shortcomings of P. edulis and the application in paper pulping industry development was also restricted by the shortages of fiber. So, the most important is how to breed new varieties to meet the growing multi-purpose industrialization of P. edulis at present. Therefore, it is of great significance to use molecular biological technologies to improve lignin content of P. edulis.
PAL is one of the key enzymes in the early stages of the phenylpropanoid pathway and has significant effects on the lignin content of plant. CCoAOMT and CAD are another two enzymes in the lignin synthesization pathway. Both of them also play an important role on the regulation of lignin content in plant. In this study, PAL, CCoAOMT, CAD genes in P. edulis were cloned and the expressions were also studied. The results were summarized as follows:
(1) The DNA and RNA were isolated respectively from P. edulis leaves by modified CTAB and Trizol methods. The result showed that the quality was good enough for further research.
(2) A full length of 2 736 bp DNA fragment encoded PAL of P. edulis was obtained by genome walking, which contained an intron and a forecasted promoter sequences. The PAL fragment encoded 712 amino acids with a molecular weight of 77KD. The deduced amino acid sequence shared 95% homology with the PAL of Bambusa oldhamii and 94% with the PAL of Oryza sativa.
(3) A full length of PAL cDNA named PAL1 was obtained by RT-PCR and RACE. The size of the cDNA fragment was 2 678 bp, which included a 2 142 bp open reading frame. It shared 90% homology with PAL of Oryza sativa and Bambusa oldhami. Tissue specific expression test revealed that there were significant differences in roots, stems, leaves and seedlings.The expression of PAL in root was the highest.
(4) A 787 bp DNA fragment of CCoAOMT and a 1019 bp DNA fragment of CAD were cloned from the genomic DNA of P. edulis which encoded 197 amino acids and 282 amino acids respectively. The CCoAOMT and CAD DNA fragment in P. edulis shared 90% homology with that of Oryza sativa and Phleum pratens. The deduced amino acids sequence of CCoAOMT and CAD in P. edulis shared 85% homology with that of Oryza sativa and Phleum pratens. GenBank accession numbers were EF549579 and EF549577 respectively.
(5) The PAL1 gene was cloned into a prokaryotic expression vector pET-16b and then it was used to transform E.coli. BL21. The Km, the optimum temperature and the optimum pH of purified recombinant protein was (0.421±0.023)×10-3 mol/L, 50℃and 8.8 respectively.
(6) The pBI121-PAL1、pBI121-anti PAL1、pBI121-anti CCoAOMT and pBI121-anti CAD plant expression vectors were constructed. The vectors were introduced into tabacoo plant by agrobacterium-mediated transformation. PCR analyses confirmed the integration of the target gene into the genome of the transformed tobacco plant.
引文
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