Four methods were used to prepare four crude elicitors containing different ingredients from mycelia of fungus F 5: (a) Polysaccharides A containing lipids and proteins; (b) Micromolecular polysaccharides B without li...Four methods were used to prepare four crude elicitors containing different ingredients from mycelia of fungus F 5: (a) Polysaccharides A containing lipids and proteins; (b) Micromolecular polysaccharides B without lipids and proteins; (c) Macromolecular polysaccharides F without lipids and proteins; (d) Polysaccharides H containing proteins but no lipids. The crude preparation B had the highest ability to induce taxol biosynthesis among other preparations did. The crude preparation B was through Sephadex G15 column to separate two compositions: BⅠ whose molecular weight was over 1500 D and BⅡ whose molecular weight between 700～ 1500 D. The BⅡ activity to induce taxol biosynthesis was 2.3 times higher than that of the crude preparation B, while BⅠ hadn’t the activity to induce taxol biosynthesis. By analysis of PAL activity induced by elicitors, we conclude that the changes of PAL activity could be regarded as a useful physiological indicator to select the effective elicitor.展开更多
【目的】克隆有壳美洲南瓜种皮PAL基因(CP-PAL),研究PAL基因在有壳和裸仁美洲南瓜种皮发育过程中的表达特性,为揭示美洲南瓜种皮发育机理及木质素积累在南瓜种皮发育中的作用等方面提供理论依据。【方法】利用RT-PCR,结合RACE技术克隆CP...【目的】克隆有壳美洲南瓜种皮PAL基因(CP-PAL),研究PAL基因在有壳和裸仁美洲南瓜种皮发育过程中的表达特性,为揭示美洲南瓜种皮发育机理及木质素积累在南瓜种皮发育中的作用等方面提供理论依据。【方法】利用RT-PCR,结合RACE技术克隆CP-PAL的全长序列并进行生物信息学分析;利用实时荧光定量PCR技术,采用2-△△Ct方法对种皮发育过程中PAL基因的表达进行分析。【结果】CP-PAL序列全长为1 720 bp,含有一个1 359bp的ORF,114 bp 5′端非翻译区、236 bp 3′端非翻译区及11 bp polyA结构,可编码452个氨基酸,分子量为48.86 kD,等电点为6.55,原子总数为6 885个,分子式为C2158H3449N607O657S14。通过BLASTX比对表明CP-PAL核苷酸序列及其氨基酸序列与黄瓜PAL核苷酸及其氨基酸序列的相似性最高。CP-PAL包含PAL-HAL、PLN02457及phe_am_lyase 3个结构域及酶活性中心序列(GTITASGDLVPLSYIA),属于Lyase_I_Like超家族。CP-PAL不具有导肽及信号肽,为非跨膜蛋白,可能定位于细胞质及内质网上,属可溶性蛋白。CP-PAL蛋白含有4个酪蛋白激酶Ⅱ识别位点、6个蛋白激酶C识别位点、12个豆蔻酰化位点及2个糖基化位点。此外,分析可知CP-PAL有18个丝氨酸磷酸化位点、6个苏氨酸磷酸化位点及5个酪氨酸磷酸化位点。无规则卷曲是CP-PAL蛋白二级结构中最大量的结构元件,α-螺旋和延伸链分散于整个蛋白质中,且N-末端以无规则卷曲形式存在,C-末端以延伸链形式存在。CP-PAL氨基酸序列同挑选的其他14种植物的PAL氨基酸序列进行多重序列比较,发现功能区域的氨基酸序列较为保守,N-端的差异最大。系统进化树分析表明CP-PAL和黄瓜PAL蛋白的亲缘关系最近。CP-PAL蛋白三级结构以α-螺旋为主要结构元件,β-转角和无规则卷曲较少。实时荧光定量PCR分析表明PAL基因在有壳和裸仁美洲南瓜种皮发育中呈现反向对应的变化趋势:有壳美洲南瓜种展开更多
A fragment of PAL (phenylalanine ammonia_lyase) gene was amplified by RT_PCR from poplar (Populus×euramericana cv. “74/76”) developing second xylem mRNA. It was cloned into pGEM-T Easy vector and identified by ...A fragment of PAL (phenylalanine ammonia_lyase) gene was amplified by RT_PCR from poplar (Populus×euramericana cv. “74/76”) developing second xylem mRNA. It was cloned into pGEM-T Easy vector and identified by restriction enzyme, PCR amplification and sequencing. The sequence of the amplified DNA fragment was 565 base pairs. Alignment with the P. kitakamiensis PAL cDNA sequence retrieved from EMBL nucleotide acid database (accession number D30656) showed that the first 400 base pairs in both sequences were almost identical. Therefore the fragment was part of PAL gene. And both of sense and anti-sense expressional vectors were constructed.展开更多
文摘Four methods were used to prepare four crude elicitors containing different ingredients from mycelia of fungus F 5: (a) Polysaccharides A containing lipids and proteins; (b) Micromolecular polysaccharides B without lipids and proteins; (c) Macromolecular polysaccharides F without lipids and proteins; (d) Polysaccharides H containing proteins but no lipids. The crude preparation B had the highest ability to induce taxol biosynthesis among other preparations did. The crude preparation B was through Sephadex G15 column to separate two compositions: BⅠ whose molecular weight was over 1500 D and BⅡ whose molecular weight between 700～ 1500 D. The BⅡ activity to induce taxol biosynthesis was 2.3 times higher than that of the crude preparation B, while BⅠ hadn’t the activity to induce taxol biosynthesis. By analysis of PAL activity induced by elicitors, we conclude that the changes of PAL activity could be regarded as a useful physiological indicator to select the effective elicitor.
文摘【目的】克隆有壳美洲南瓜种皮PAL基因(CP-PAL),研究PAL基因在有壳和裸仁美洲南瓜种皮发育过程中的表达特性,为揭示美洲南瓜种皮发育机理及木质素积累在南瓜种皮发育中的作用等方面提供理论依据。【方法】利用RT-PCR,结合RACE技术克隆CP-PAL的全长序列并进行生物信息学分析;利用实时荧光定量PCR技术,采用2-△△Ct方法对种皮发育过程中PAL基因的表达进行分析。【结果】CP-PAL序列全长为1 720 bp,含有一个1 359bp的ORF,114 bp 5′端非翻译区、236 bp 3′端非翻译区及11 bp polyA结构,可编码452个氨基酸,分子量为48.86 kD,等电点为6.55,原子总数为6 885个,分子式为C2158H3449N607O657S14。通过BLASTX比对表明CP-PAL核苷酸序列及其氨基酸序列与黄瓜PAL核苷酸及其氨基酸序列的相似性最高。CP-PAL包含PAL-HAL、PLN02457及phe_am_lyase 3个结构域及酶活性中心序列(GTITASGDLVPLSYIA),属于Lyase_I_Like超家族。CP-PAL不具有导肽及信号肽,为非跨膜蛋白,可能定位于细胞质及内质网上,属可溶性蛋白。CP-PAL蛋白含有4个酪蛋白激酶Ⅱ识别位点、6个蛋白激酶C识别位点、12个豆蔻酰化位点及2个糖基化位点。此外,分析可知CP-PAL有18个丝氨酸磷酸化位点、6个苏氨酸磷酸化位点及5个酪氨酸磷酸化位点。无规则卷曲是CP-PAL蛋白二级结构中最大量的结构元件,α-螺旋和延伸链分散于整个蛋白质中,且N-末端以无规则卷曲形式存在,C-末端以延伸链形式存在。CP-PAL氨基酸序列同挑选的其他14种植物的PAL氨基酸序列进行多重序列比较,发现功能区域的氨基酸序列较为保守,N-端的差异最大。系统进化树分析表明CP-PAL和黄瓜PAL蛋白的亲缘关系最近。CP-PAL蛋白三级结构以α-螺旋为主要结构元件,β-转角和无规则卷曲较少。实时荧光定量PCR分析表明PAL基因在有壳和裸仁美洲南瓜种皮发育中呈现反向对应的变化趋势:有壳美洲南瓜种
文摘A fragment of PAL (phenylalanine ammonia_lyase) gene was amplified by RT_PCR from poplar (Populus×euramericana cv. “74/76”) developing second xylem mRNA. It was cloned into pGEM-T Easy vector and identified by restriction enzyme, PCR amplification and sequencing. The sequence of the amplified DNA fragment was 565 base pairs. Alignment with the P. kitakamiensis PAL cDNA sequence retrieved from EMBL nucleotide acid database (accession number D30656) showed that the first 400 base pairs in both sequences were almost identical. Therefore the fragment was part of PAL gene. And both of sense and anti-sense expressional vectors were constructed.
