沉默CCR和CAD基因培育低木质素含量转基因多年生黑麦草  

Genetic improvement of perennial ryegrass with low lignin content by silencing genes of CCRand CAD

作　　者：胡可[1,2] 严雪锋 栗丹[1] 唐晓梅[1,2] 杨宏[1] 王艳[1] 邓洪渊[1] 马欣荣[1] 

机构地区：[1]中国科学院成都生物研究所,四川成都610041 [2]中国科学院研究生院,北京100049

出　　处：《草业学报》2013年第5期72-83,共12页Acta Prataculturae Sinica

基　　金：国家863计划项目(2009AA10Z108;2008AA10Z409);国家自然科学基金项目(30170589)资助

摘　　要：木质素作为维管植物的重要成分之一,主要存在于细胞的次生壁中。然而,木质素却是许多工农业加工过程的限制因素,例如在化学制浆、牧草消化以及木质纤维转化为生物酒精等过程中。肉桂酰辅酶A还原酶(CCR)和肉桂醇脱氢酶(CAD)是催化木质素单体生物合成最后两步的关键酶。本研究根据NCBI中黑麦草CCR和CAD基因序列设计特异引物并添加相应酶切位点,从野生型多年生黑麦草cDNA分离克隆CCR和CAD基因片段,分别构建了含正反方向目的片段的植物表达干扰载体p23-iCCR和p23-iCAD。通过根癌农杆菌EHA105介导转入多年生黑麦草胚性愈伤组织,经过巴龙霉素筛选和PCR检测获得导入了干扰CCR和CAD基因片段的转基因株系i-CCR和i-CAD。常规方法测定相对木质素含量,结果显示,与对照相比,有9株i-CCR植株和11株i-CAD植株木质素含量显著降低,分别平均降低了34.67%,33.86%,且生长正常。本研究表明通过干扰CCR和CAD基因表达,可以获得低木质素含量的多年生黑麦草,为进一步培育易消化吸收的黑麦草提供了良好的种质资源。thickened ce gnln, lls. H one of the main components in vascular plants, is mainly present in the walls of secondarily owever, it is a ing factor in a number of agro-industrial processes, such as chemical pul ping, forage digestibility, and lignocellulosic-to-bioethanol conversion. Cinnamoyl CoA reductase （CCR） and cinnamyl alcohol dehydrogenase （CAD） are two key enzymes respectively in ryegrass （Loli sites to amplif Y the biosynthesis of monolignols. m perenne） from NCBI, specific and clone the two gene fragmen （RNAi） vectors （p23-iCCR and at catalyse the penultimate and ultimate steps Based on the CCR and CAD gene sequences of perennial pr ts imers were designed to add appropriate restriction enzyme from cDNA of perennial ryegrass. Two RNA interference 3-iCAD） were constructed using inverse repeat fragments. The constructs were introduced into embryogenic calli of perennial ryegrass by Agrobacterium-mediated transformation with EHA105. They were screened using paromomycin and detected by PCR, and independent i-CCR and i-CAD transgenic plants were obtained. Analysis of relative lignin content was conducted by conventional methods. The lignin content was distinctly reduced compared with that of wild controls in 9 plants of i-CCR and 11 plants of i-CAD transgenic plants. The lignin content of these i-CCR and i-CAD plants was reduced by 34. 67% and 33.86 % respectively. The transgenic plants had normal morphology and grew well. This study showed that low-lignin-content perennial ryegrass could be obtained by silencing CCR and CAD gene expression and has pro- vided novel germplasms for breeding digestible and improved absorption ryegrass.

关 键 词：木质素 肉桂酰辅酶A还原酶 肉桂醇脱氢酶 多年生黑麦草 RNAi 遗传转化 

分 类 号：S543.6[农业科学—作物学]