杨树木质素生物合成调控的研究
摘要
木质素是一类酚类次生代谢产物,具有重要的生物学功能。然而它却阻碍了人们对资源植物的利用。通过基因工程手段调控木质素生物合成,降低木质素含量或改变组分以培育适合于工农业生产的资源植物具有较大的应用价值和环保效益。本实验主要研究木质素合成途径涉及的四种相关酶——4-香豆酸:CoA连接酶(4CL)、肉桂酰乙醇脱氢酶(CAD)、阿魏酸-5-羟化酶(F5H)和咖啡酰辅酶A-O-甲基转移酶(CCoAOMT)对植物木质素生物合成的调节作用,取得如下进展:
1.通过对生长4年的转基因普通毛白杨Southern检测未发现基因逃逸;Klason木质素含量测定其木质素含量依然保持降低水平;抑制4CL基因表达S/G比值未发生明显变化;切片显微观察发现,转基因植株中存在极少数轻度变形或发育不良的导管,主要表现为导管周径变小,呈扁平状,与正常导管团簇或线形排列,但生长状态良好。再次验证了通过抑制4CL表达改良林木植物的可行性。
2.在杨树中同时抑制表达CCoAOMT基因和过量表达F5H基因,Klason木质素含量测定显示木质素含量有不同程度的下降;硫酸解法分析木质素组分S/G比值有不同程度的升高。这也预示着双或多基因调控木质素生物合成途径可能会更好地修饰木质素特性,从而创造出更适合工农业生产的林木品种。
3.通过RT-PCR方法分离了白杨杂种木质素单体合成途径中的两个关键酶基因F5H和CAD全长cDNA序列,同时通过PCR方法分离了C4H(肉桂酸-4-羟化酶)基因启动子1.1kb序列;分别构建C4H启动子启动正向F5H基因表达(pC4H-F5H)和反义CAD基因表达(pC4H-CAD)的载体。建立了农杆菌介导双基因共转化烟草的转化体系,得到48株PCR检测阳性苗;并用此体系指导转化白杨杂种获得22株PCR检测阳性苗。使用MSA培养基缩短了外植体筛选分化后期的出苗时间。转双基因烟草和白杨杂种阳性苗的筛选与后期检测正在进行中。
Lignins are aromatic polymers that are present mainly in secondarily thickened plant cell walls,which play important roles in plants but represent a major obstacle in chemical pulping,forage digestibility,and processing of plant biomass to biofuels. Thus there is of significantly economical and environmental value on dissecting complex biosysthetic pathways and engineering polygenic agronomic traits in plants. Several genes involved in the lignin biosynthetic pathways,including F5H,4CL,CAD and CCoAOMT,were studied by gene overexpresion and down-regulation in this paper.The main results are as follows:
1.Transgenic Chinese white poplar(Populus) with 4CL-suppressing kept showing the present of transgene after growing in field for 4 years by Southern blotting analysis;The decrement of lignin content was maintained by Klason lignin content evaluation;Plants with 4CL gene down-regualtion have little shift in lignin compositional ratio(S/G ratio).Light micrographs of stem transverse sections revealed that vascular tissue of transgenic plants was in good condition,except that there were a few vessels of slight deformation or dysplasia,showing as smaller circumference,compressed shape,lined with normal vessels or being in clusters. These results imply that it is a good way to suppress the gene of 4CL to improve chemical pulping,and biofuels processing of poplar.
2.The analysis of the Klason lignin content and thioacidolysis methods showed that lignin contents decreased in varying degrees in dual-gene transgenic plants with sense F5H and antisense CCoAOMT,and lignin components S/G ratio increased in varying degrees.This also indicates that dual or multi-gene regulation of lignin biosynthetic pathway might modify lignin properties more efficiently to create more feasible plants for industrial and agricultural production.
3.Two full-length cDNA sequences named F5H and CAD were isolated by RT-PCR,as well as C4H promoter sequence were harvested by PCR with 1.1kb length in hybrid poplar(Populus tremula×Populus alba);the vetor which harbored respectively sense F5H or antisense CAD driven by C4H promoter was constructed, namely pC4H-F5H and pC4H-CAD.We used Agrobacterium to cotransfer dual genes into tobacco and obtained 48 independent kanamycin-resistant transgenic plants based on PCR analysis;with the same cotransfermation system and PCR analysis,22 independent kanamycin-resistant transgenic hybrid poplar plants were harvested.The use of culture medium MSA accelerated the growth of seedling,to reduce the period of tissue culture.Dual-gene transgenic tobacco and hybrid poplar plants are being identified and tested physiologically.
1.通过对生长4年的转基因普通毛白杨Southern检测未发现基因逃逸;Klason木质素含量测定其木质素含量依然保持降低水平;抑制4CL基因表达S/G比值未发生明显变化;切片显微观察发现,转基因植株中存在极少数轻度变形或发育不良的导管,主要表现为导管周径变小,呈扁平状,与正常导管团簇或线形排列,但生长状态良好。再次验证了通过抑制4CL表达改良林木植物的可行性。
2.在杨树中同时抑制表达CCoAOMT基因和过量表达F5H基因,Klason木质素含量测定显示木质素含量有不同程度的下降;硫酸解法分析木质素组分S/G比值有不同程度的升高。这也预示着双或多基因调控木质素生物合成途径可能会更好地修饰木质素特性,从而创造出更适合工农业生产的林木品种。
3.通过RT-PCR方法分离了白杨杂种木质素单体合成途径中的两个关键酶基因F5H和CAD全长cDNA序列,同时通过PCR方法分离了C4H(肉桂酸-4-羟化酶)基因启动子1.1kb序列;分别构建C4H启动子启动正向F5H基因表达(pC4H-F5H)和反义CAD基因表达(pC4H-CAD)的载体。建立了农杆菌介导双基因共转化烟草的转化体系,得到48株PCR检测阳性苗;并用此体系指导转化白杨杂种获得22株PCR检测阳性苗。使用MSA培养基缩短了外植体筛选分化后期的出苗时间。转双基因烟草和白杨杂种阳性苗的筛选与后期检测正在进行中。
Lignins are aromatic polymers that are present mainly in secondarily thickened plant cell walls,which play important roles in plants but represent a major obstacle in chemical pulping,forage digestibility,and processing of plant biomass to biofuels. Thus there is of significantly economical and environmental value on dissecting complex biosysthetic pathways and engineering polygenic agronomic traits in plants. Several genes involved in the lignin biosynthetic pathways,including F5H,4CL,CAD and CCoAOMT,were studied by gene overexpresion and down-regulation in this paper.The main results are as follows:
1.Transgenic Chinese white poplar(Populus) with 4CL-suppressing kept showing the present of transgene after growing in field for 4 years by Southern blotting analysis;The decrement of lignin content was maintained by Klason lignin content evaluation;Plants with 4CL gene down-regualtion have little shift in lignin compositional ratio(S/G ratio).Light micrographs of stem transverse sections revealed that vascular tissue of transgenic plants was in good condition,except that there were a few vessels of slight deformation or dysplasia,showing as smaller circumference,compressed shape,lined with normal vessels or being in clusters. These results imply that it is a good way to suppress the gene of 4CL to improve chemical pulping,and biofuels processing of poplar.
2.The analysis of the Klason lignin content and thioacidolysis methods showed that lignin contents decreased in varying degrees in dual-gene transgenic plants with sense F5H and antisense CCoAOMT,and lignin components S/G ratio increased in varying degrees.This also indicates that dual or multi-gene regulation of lignin biosynthetic pathway might modify lignin properties more efficiently to create more feasible plants for industrial and agricultural production.
3.Two full-length cDNA sequences named F5H and CAD were isolated by RT-PCR,as well as C4H promoter sequence were harvested by PCR with 1.1kb length in hybrid poplar(Populus tremula×Populus alba);the vetor which harbored respectively sense F5H or antisense CAD driven by C4H promoter was constructed, namely pC4H-F5H and pC4H-CAD.We used Agrobacterium to cotransfer dual genes into tobacco and obtained 48 independent kanamycin-resistant transgenic plants based on PCR analysis;with the same cotransfermation system and PCR analysis,22 independent kanamycin-resistant transgenic hybrid poplar plants were harvested.The use of culture medium MSA accelerated the growth of seedling,to reduce the period of tissue culture.Dual-gene transgenic tobacco and hybrid poplar plants are being identified and tested physiologically.
引文
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