胀果甘草细胞培养及查尔酮合成酶基因的cDNA克隆
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摘要
甘草是中国常用的中药,是重要的药用植物。除药用价值外,甘草在食品、饮料、卷烟、化妆品等工业中也广泛使用。近年来,由于人们破坏性地对野生甘草采挖,使野生植物几乎灭绝。故开展甘草组织培养工作,尤其是大规模悬浮培养具有重要意义。
黄酮类化合物是甘草中一大类生物活性成分,它们具有抗溃疡、抗菌、抗炎、抗氧化、抗HIV、抗肿瘤等作用。查尔酮合成酶是所有黄酮类化合物合成的关键酶。它催化3分子的丙二酰辅酶A与1分子对羟基苯丙烯酰辅酶A缩合成柚皮素查尔酮,以此为支架其他黄酮类化合物得以衍生出来。查尔酮合成酶活性的积累与黄酮类化合物的积累是紧密相关的。
本实验切取胀果甘草无菌苗的子叶、胚轴、根段3种外植体进行了愈伤组织诱导和继代培养,建立了甘草悬浮细胞培养体系,测定了悬浮培养的细胞生长曲线；并且研究了接种量、条件培养基和外源激素对悬浮细胞的影响。结果表明子叶来源的愈伤组织最适合悬浮培养；悬浮培养的最佳接种量为35g/L；条件培养基的最佳用量为总培养体积的1/3；最适合胀果甘草悬浮培养的的培养基是附加NAA 1.0mg/L+KT 0.5mg/L激素组合的MS培养基。
利用RT-PCR方法,从胀果甘草愈伤组织中克隆了查尔酮合酶基因的cDNA片断,并进行了序列分析。测序结果表明,我们克隆所得到的cDNA片段包含一个开放阅读框架,由1170bp核苷酸组成,编码一个由389个氨基酸残基组成的多肽。与紫花苜蓿、大豆、豌豆等几种豆科植物CHS基因的核苷酸同源率在80%以上,氨基酸同源率在90%以上。已将得到的序列提交GenBank/EMBL/DDBJ注册,序列号为EU706287。
Liquorice is a famous medicinal plant. Besides the massive use in Chinese medicine, it is widely used in food, beverage, cigarette, cosmetic and other industries. And tissue culture are considered to be hopeful way to alleviate the crisis of natural resources.
Flavonoids form a class of secondary metabolites which are abundant in liquorice, they have stronger bio-activity. Pharmacological investigation concluded that they had antioxidant, antibacterial, anti-ulcerous, anti-inflammatory, antitumer, anty-HIV and many other activities. Chalcone Synthase (CHS) is a key enzyme in the biosynthesis of all classes of flavonoids, CHS can catalyze the condensation of 3 moleculars of malonyl-CoA and one molecular of hydroxycinnamoyl-CoA ester to form a naringenin-chalcone intermediate. Further isomerization and substitution of this central intermediate leads to the synthesis of other flavonoids. And variation of the expression of CHS might change the content of flavonoids in liquorice.
Different explants of plantlet of Glycyrrhiza inflate were cultured on the MS media containing different plant hormones to induce callus and Glycyrrhiza inflate suspension cell culture was established when the induced callus were transferred to liquid medium. The cultures were incubated at 25±1℃in the dark with continuing agitation (120rpm). In basis of this, the growth curve of suspension cell of Glycyrrhiza inflate was measured. In this study, we also have investigated the effects of the initial callus amount, conditioned medium (CM) and hormone on the growth of suspension cell. The results indicated that the callus induced from cotyledon was the best initial callus for cell suspension culture among the three different explants; the optimal initial callus amount for cell suspension culture is 35g/L; the optimal use amount of condition medium is one third of total volume and the optimal medium was MS+NAA 1.0 mg/L+KT 0.5mg/L.
By using RT-PCR method, a cDNA fragment of 1170bp length was successful obtained, it contained a complete opening reading frame and encoded a protein of 389 amino acids. The CHS gene cloned from Glycyrrhiza inflate has more than 80% sequence homology at the nucleotide level and more than 90% similarity at the amino acid level compared with Medicago sativa, Glycine max, Pisum sativum etc. The G. inflate CHS sequence was submitted to the GenBank and the accession number as EU706287.
黄酮类化合物是甘草中一大类生物活性成分,它们具有抗溃疡、抗菌、抗炎、抗氧化、抗HIV、抗肿瘤等作用。查尔酮合成酶是所有黄酮类化合物合成的关键酶。它催化3分子的丙二酰辅酶A与1分子对羟基苯丙烯酰辅酶A缩合成柚皮素查尔酮,以此为支架其他黄酮类化合物得以衍生出来。查尔酮合成酶活性的积累与黄酮类化合物的积累是紧密相关的。
本实验切取胀果甘草无菌苗的子叶、胚轴、根段3种外植体进行了愈伤组织诱导和继代培养,建立了甘草悬浮细胞培养体系,测定了悬浮培养的细胞生长曲线；并且研究了接种量、条件培养基和外源激素对悬浮细胞的影响。结果表明子叶来源的愈伤组织最适合悬浮培养；悬浮培养的最佳接种量为35g/L；条件培养基的最佳用量为总培养体积的1/3；最适合胀果甘草悬浮培养的的培养基是附加NAA 1.0mg/L+KT 0.5mg/L激素组合的MS培养基。
利用RT-PCR方法,从胀果甘草愈伤组织中克隆了查尔酮合酶基因的cDNA片断,并进行了序列分析。测序结果表明,我们克隆所得到的cDNA片段包含一个开放阅读框架,由1170bp核苷酸组成,编码一个由389个氨基酸残基组成的多肽。与紫花苜蓿、大豆、豌豆等几种豆科植物CHS基因的核苷酸同源率在80%以上,氨基酸同源率在90%以上。已将得到的序列提交GenBank/EMBL/DDBJ注册,序列号为EU706287。
Liquorice is a famous medicinal plant. Besides the massive use in Chinese medicine, it is widely used in food, beverage, cigarette, cosmetic and other industries. And tissue culture are considered to be hopeful way to alleviate the crisis of natural resources.
Flavonoids form a class of secondary metabolites which are abundant in liquorice, they have stronger bio-activity. Pharmacological investigation concluded that they had antioxidant, antibacterial, anti-ulcerous, anti-inflammatory, antitumer, anty-HIV and many other activities. Chalcone Synthase (CHS) is a key enzyme in the biosynthesis of all classes of flavonoids, CHS can catalyze the condensation of 3 moleculars of malonyl-CoA and one molecular of hydroxycinnamoyl-CoA ester to form a naringenin-chalcone intermediate. Further isomerization and substitution of this central intermediate leads to the synthesis of other flavonoids. And variation of the expression of CHS might change the content of flavonoids in liquorice.
Different explants of plantlet of Glycyrrhiza inflate were cultured on the MS media containing different plant hormones to induce callus and Glycyrrhiza inflate suspension cell culture was established when the induced callus were transferred to liquid medium. The cultures were incubated at 25±1℃in the dark with continuing agitation (120rpm). In basis of this, the growth curve of suspension cell of Glycyrrhiza inflate was measured. In this study, we also have investigated the effects of the initial callus amount, conditioned medium (CM) and hormone on the growth of suspension cell. The results indicated that the callus induced from cotyledon was the best initial callus for cell suspension culture among the three different explants; the optimal initial callus amount for cell suspension culture is 35g/L; the optimal use amount of condition medium is one third of total volume and the optimal medium was MS+NAA 1.0 mg/L+KT 0.5mg/L.
By using RT-PCR method, a cDNA fragment of 1170bp length was successful obtained, it contained a complete opening reading frame and encoded a protein of 389 amino acids. The CHS gene cloned from Glycyrrhiza inflate has more than 80% sequence homology at the nucleotide level and more than 90% similarity at the amino acid level compared with Medicago sativa, Glycine max, Pisum sativum etc. The G. inflate CHS sequence was submitted to the GenBank and the accession number as EU706287.
引文
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