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Acta Prataculturae Sinica ›› 2013, Vol. 22 ›› Issue (5): 72-83.DOI: 10.11686/cyxb20130509

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

HU Ke1,2,YAN Xue-feng1,2,LI Dan1,TANG Xiao-mei1,2,YANG Hong1,WANG Yan1,DENG Hong-yuan1,MA Xin-rong1   

  1. 1.Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China;
    2.Graduate University of Chinese Academy of Sciences, Beijing 100049, China
  • Online:2013-10-20 Published:2013-10-20

Abstract: Lignin, one of the main components in vascular plants, is mainly present in the walls of secondarily thickened cells. However, it is a limiting factor in a number of agro-industrial processes, such as chemical pulping, forage digestibility, and lignocellulosic-to-bioethanol conversion. Cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) are two key enzymes that catalyse the penultimate and ultimate steps respectively in the biosynthesis of monolignols. Based on the CCR and CAD gene sequences of perennial ryegrass (Lolium perenne) from NCBI, specific primers were designed to add appropriate restriction enzyme sites to amplify and clone the two gene fragments from cDNA of perennial ryegrass. Two RNA interference (RNAi) vectors (p23-iCCR and p23-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 provided novel germplasms for breeding digestible and improved absorption ryegrass.

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