华南象草Pp4CL基因的克隆及其转基因烟草木质素含量分析

为探究华南象草(Pennisetum purpureumcv.Huanan)木质素合成关键酶基因的调控机制,通过同源克隆得到华南象草4-香豆酸:CoA连接酶基因(Pp4CL)的cDNA序列,长度为1 943bp,其中编码区序列1 662bp。Pp4CL蛋白由553个氨基酸组成,分子量为59.57kD,等电点为5.2,属于疏水性蛋白。该蛋白含有AMP结合结构域,属于AFD ClassⅠ超家族。在系统进化分析中,Pp4CL与At4CL1、Os4CL1遗传距离最近,聚为一支。Pp4CL氨基酸序列具有SSGTGLPKGV和GEICIRG等2个保守基序,是典型的植物4CL。构建原核表达载体pGEX-4CL,得到约88kD的Pp4CL-GST融合蛋白,为Pp4CL酶活性测定及Western免疫印迹分析奠定了基础。同时构建植物表达载体pBA-4CL,并通过叶盘法对烟草进行了遗传转化,得到3个转基因阳性株系(OX-9、OX-7、OX-4),它们中叶柄木质素总含量分别比非转基因植株(对照)提高了10.0%、16.2%和94.6%,茎秆基部节木质素总含量分别比对照提高了0.9%、4.0%和13.5%。研究结果表明,Pp4CL蛋白与木质素合成有关,过表达Pp4CL基因能够显著提高植株木质素含量。该研究结果为华南象草木质素改良工作打下了基础,同时也为深入开展牧草分子育种提供了依据。

Isolation of 4-coumarate CoA Ligase Gene from Pennisetum purpureum cv.Huanan and Lignin Content Analysis of Transgenic Tobacco Plants

Huanan elephant grass (Pennisetum purpureum cv.Huanan) is widely used as forage in South China.It is also a highly yielding energy crop for ethanol production.However,the lignin in lignocellulose inhibits the activity of bioconversion.In order to explore the lignin regulation network,we isolated 4-coumarate CoA ligase (4CL) from P.purpureum cv.Huanan.A 1 943 bp cDNA sequence of Pp4CL gene was cloned by homology-based RT-PCR.Pp4CL has an open reading frame of 1 662 bp and encodes a 553 amino acid protein with a predicted molecular mass of 59.57 kD and a isoelectric point of 5.2.Pp4CL protein is a hydrophobin protein,and belongs to AFD class I superfamily as it contains an AMP binding domain.In phylogenetic tree,Pp4CL was clustered with At4CL1 and Os4CL1.Two conserved motifs,SSGTGLPKGV and GEICIRG,were found in Pp4CL,indicating that it was a typical plant 4CL.The pGEX-4CL prokaryotic expression vector was constructed and GST-tagged Pp4CL fusion protein with the molecular mass of 88 kD was obtained.This result laid foundation for the western blot and enzymology analysis.The overexpression vector named pBA-4CL was constructed,and three positive transgenic tobacco lines were obtained by leaf disc method.In petiole,acetyl bromide lignin content was greater by approximately 10% in OX-9,16.2% in OX-7,94.6% in OX-4 compared with WT plants.And in base stem,the three transgenic plants had 0.9%,4.0% and 13.5% more acetyl bromide lignin content than CK plant,respectively.In conclusion,overexpression of Pp4CL in tobacco leaded to increasing of lignin content,thus,Pp4CL was related to lignin biosynthesis.This research provides scientific basis for lignin improvement in elephant grass and molecular breeding in forage grass.