苎麻CCoAOMT基因干扰表达载体构建及其遗传转化
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摘要
咖啡酰辅酶A氧甲基转移酶(CCoAOMT)是植物木质素合成过程的一种关键酶。运用已克隆的苎麻CCoAOMT基因cDNA,构建成该基因干扰表达载体,分别开展了模式烟草、亚麻和苎麻的遗传转化。以期通过基因表达的干扰作用,降低苎麻和亚麻中CCoAOMT的活性,进而降低其木质素含量,提高纤维品质。
以pFGC5941为载体,将苎麻CCoAOMT(BnCCoAOMT)基因cDNA核心区段624 bp序列以相反方向插入其CHS内含子两侧的克隆位点,构建了35S启动子控制的BnCCoAOMT干扰表达重组质粒(pFGC5941-BnCCoAOMTi),该重组分子在植物中的表达,能拼接成624 bp dsRNA,作为CCoAOMT的干扰分子。为验证CCoAOMT干扰表达抑制木质素含量的可行性,通过农杆菌介导叶盘转化法,先行转化了模式植物烟草WS38,获得了转基因烟草。BnCCoAOMT基因在烟草中的干扰表达,对烟草生长产生了一定影响,转基因植株比对照植株生长较缓慢,其中一株植株表现为茎秆卷曲。叶柄切片的染色分析表明,干扰表达的转基因烟草细胞木质素含量下降。化学分析也证实转基因烟草木质素下降的趋势。
鉴于亚麻对pFGC5941载体的选择标记(Bar)的草胺膦(PPT)有较高耐受性,通过载体改造,以潮霉素抗性基因(Hyg)取代草胺膦抗性的选择标记基因,构建具有潮霉素抗性标记的改建干扰表达载体(pFH-BnCCoAOMT)。同样采用根癌农杆菌的共培转化法,对亚麻茎段进行了共培遗传转化。通过潮霉素筛选及分化培养,获得了转BnCCoAOMTi的转基因亚麻。BnCCoAOMT基因干扰表达的转基因亚麻,表型无明显变化,长势和形态与对照基本相同。茎秆切片的染色分析证实,转基因亚麻木质素含量一定程度下降。
在上述研究的基础上,开展了苎麻BnCCoAOMTi的转基因研究。目前已获得抗PPT的苎麻愈伤组织。
研究结果表明,CCoAOMT基因的干扰表达可以有效抑制转基因植物CCoAOMT活性,降低木质素含量,为利用苎麻CCoAOMT基因开展木质素合成的基因工程调控,培育低木质素或木质素组分改良的苎麻新种质积累了经验。
Caffeoyl-CoA 3-O-methyltransferase(CCoAOMT) plays a key role in lignin biosynthesis in plant.In our previous research,the cDNA sequence of ramie CCoAOMT has been cloned and studied.In this paper,the gene cDNA is cloned into a plant expression interference vector,pFGC5941,as an RNAi recombinant.The recombinant is transformed into Tobacco(Nicotiana tabacurn) "WS38",Flax(Linum usitutissimum L.) and Ramie (Boehmeria nivea) via Agrobacteriurn turnefaciens mediated transformation.It is expected the expression of this interference gene to reduce the activity of CCoAOMT in ramie and flax so as to reduce lignin content in cortex of the crops and improve the fiber quality.
The Ramie CCoAOMT cDNA conservative sequence of 624 bp was inserted into pFGC5941 two clone sites border to CHS intron in a reverse direction.Thus the plasmid pFGC5941-BnCCoAOMTi is constructed the gene is controlled by 35S promoter and it can form a 624 bp dsRNA after splicing.It will be the interference molecule to CCoAOMT in the transgenics.For testing the interference fuction of the recombinant the molecule is first transformed into the Tobacco(Nicotiana tabacum) "WS38",a model variety for lignin research,by Agrobacteriurn turnefaciens mediated transformation.Several transgenics obtained and the transformed plantlets show slower growth and one of them shows a willowy stem.In both staining of petiole slice and chemical analysis,an obviously decline of lignin content is detected.
Whereas flax shows a higher PPT resistant which is the selection marker(Bar~r) for pFGC5941 transformation.We reconstruct the vector with hygromycin as selection marker (Hyg~r) to replace the PPT resistance(Bar~r).A modified recombinant interference vector (pFH-BnCCoAOMT) was constructed and used to transform flax hypocotyls.The regenerated transgenic flax seedlings were obtained after hygromycin screening.The phenotype of the transgenic are roughly the same as the control on the characters of growing form and configuration while the biopsy of stem straining confirmed that the transgenic flax content less lignin than non-transgenic partner.
Based on the study above,we tried the ramie transformation with BnCCoAOMT RNAi.Some PPT tolerance ramie calluses are obtained.But it is difficult to make it differentiation so we did not get transgenic seedling yet.
But our researches give an amazing result that the RNAi of BnCCoAOMT can effectively inhibit the CCoAOMT activity in transgenic tobacco and flax and show a reduced lignin contents.Much experience have been extracted from this study to benefit our research on Ramie gene engineering to reduce its lignin contents or improve components of the lignin monopoly for a better fiber quality.
以pFGC5941为载体,将苎麻CCoAOMT(BnCCoAOMT)基因cDNA核心区段624 bp序列以相反方向插入其CHS内含子两侧的克隆位点,构建了35S启动子控制的BnCCoAOMT干扰表达重组质粒(pFGC5941-BnCCoAOMTi),该重组分子在植物中的表达,能拼接成624 bp dsRNA,作为CCoAOMT的干扰分子。为验证CCoAOMT干扰表达抑制木质素含量的可行性,通过农杆菌介导叶盘转化法,先行转化了模式植物烟草WS38,获得了转基因烟草。BnCCoAOMT基因在烟草中的干扰表达,对烟草生长产生了一定影响,转基因植株比对照植株生长较缓慢,其中一株植株表现为茎秆卷曲。叶柄切片的染色分析表明,干扰表达的转基因烟草细胞木质素含量下降。化学分析也证实转基因烟草木质素下降的趋势。
鉴于亚麻对pFGC5941载体的选择标记(Bar)的草胺膦(PPT)有较高耐受性,通过载体改造,以潮霉素抗性基因(Hyg)取代草胺膦抗性的选择标记基因,构建具有潮霉素抗性标记的改建干扰表达载体(pFH-BnCCoAOMT)。同样采用根癌农杆菌的共培转化法,对亚麻茎段进行了共培遗传转化。通过潮霉素筛选及分化培养,获得了转BnCCoAOMTi的转基因亚麻。BnCCoAOMT基因干扰表达的转基因亚麻,表型无明显变化,长势和形态与对照基本相同。茎秆切片的染色分析证实,转基因亚麻木质素含量一定程度下降。
在上述研究的基础上,开展了苎麻BnCCoAOMTi的转基因研究。目前已获得抗PPT的苎麻愈伤组织。
研究结果表明,CCoAOMT基因的干扰表达可以有效抑制转基因植物CCoAOMT活性,降低木质素含量,为利用苎麻CCoAOMT基因开展木质素合成的基因工程调控,培育低木质素或木质素组分改良的苎麻新种质积累了经验。
Caffeoyl-CoA 3-O-methyltransferase(CCoAOMT) plays a key role in lignin biosynthesis in plant.In our previous research,the cDNA sequence of ramie CCoAOMT has been cloned and studied.In this paper,the gene cDNA is cloned into a plant expression interference vector,pFGC5941,as an RNAi recombinant.The recombinant is transformed into Tobacco(Nicotiana tabacurn) "WS38",Flax(Linum usitutissimum L.) and Ramie (Boehmeria nivea) via Agrobacteriurn turnefaciens mediated transformation.It is expected the expression of this interference gene to reduce the activity of CCoAOMT in ramie and flax so as to reduce lignin content in cortex of the crops and improve the fiber quality.
The Ramie CCoAOMT cDNA conservative sequence of 624 bp was inserted into pFGC5941 two clone sites border to CHS intron in a reverse direction.Thus the plasmid pFGC5941-BnCCoAOMTi is constructed the gene is controlled by 35S promoter and it can form a 624 bp dsRNA after splicing.It will be the interference molecule to CCoAOMT in the transgenics.For testing the interference fuction of the recombinant the molecule is first transformed into the Tobacco(Nicotiana tabacum) "WS38",a model variety for lignin research,by Agrobacteriurn turnefaciens mediated transformation.Several transgenics obtained and the transformed plantlets show slower growth and one of them shows a willowy stem.In both staining of petiole slice and chemical analysis,an obviously decline of lignin content is detected.
Whereas flax shows a higher PPT resistant which is the selection marker(Bar~r) for pFGC5941 transformation.We reconstruct the vector with hygromycin as selection marker (Hyg~r) to replace the PPT resistance(Bar~r).A modified recombinant interference vector (pFH-BnCCoAOMT) was constructed and used to transform flax hypocotyls.The regenerated transgenic flax seedlings were obtained after hygromycin screening.The phenotype of the transgenic are roughly the same as the control on the characters of growing form and configuration while the biopsy of stem straining confirmed that the transgenic flax content less lignin than non-transgenic partner.
Based on the study above,we tried the ramie transformation with BnCCoAOMT RNAi.Some PPT tolerance ramie calluses are obtained.But it is difficult to make it differentiation so we did not get transgenic seedling yet.
But our researches give an amazing result that the RNAi of BnCCoAOMT can effectively inhibit the CCoAOMT activity in transgenic tobacco and flax and show a reduced lignin contents.Much experience have been extracted from this study to benefit our research on Ramie gene engineering to reduce its lignin contents or improve components of the lignin monopoly for a better fiber quality.
引文
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