百脉根（Lotus corniculatus）是研究茎叶中积累缩合单宁（Proanthocyanidins, PAs）的理想牧草，而PAs是豆科牧草的重要品质性状，申请人以235份百脉根种质为材料，通过定性、定量测定PAs含量及其合成途径相关基因的表达量，筛选出了调控缩合单宁合成的关键转录因子LcMYB5，且酵母单杂交证明LcMYB5能与无色花色素还原酶LcLAR1启动子结合；拟在此基础上，通过ChIP-qPCR、EMSA技术鉴定LcMYB5结合不同百脉根种质LAR启动子的顺式元件，运用瞬时转录激活方法分析在不同细胞系中LcMYB5调控LAR表达的活性，采用定点突变LcMYB5结构域的方法，鉴定影响调控LAR表达活性的关键位点，以期揭示百脉根LcMYB5通过调控PAs特异合成途径关键酶基因LAR表达提高叶片中积累缩合单宁的的分子机制，为苜蓿等豆科牧草品质改良与分子育种提供理论与技术支持。

The moderate quantities of proanthocyanidins (PAs) protects ruminants against pasture bloat, reduces the accumulation of methane and ammonium inurine, which could decrease the concentration of potent greenhouse gases methane and nitrous oxide in pastures. Thus, understanding the regulation mechanisms of PAs biosynthetic pathway and obtaining legumes with moderate quantities of PAs are one of the priorities of forage breeders worldwide. Two hundred and thirty-five accessions of Lotus corniculatus around the world were collected. Detection of the contents of PAs in leaves using DMACA staining and vanillin hydrochloric acid method showed that 150, 50, 35 Lotus corniculatus contained higher level, lower level PAs and null PAs, respectively. Meanwhile, Using the qRT-PCR method, The the relative expression level of transcription factor LcMYB5 and gene LAR were positively correlated with the PA content. Therefore, understanding the transcriptional regulation mechanism of LcMYB5 contributes to develop legumes with moderate quantities of PAs. In this proposal, the objectives are: (1) Isolation and identification the LcMYB5 binding DNA sequence of LAR gene based on Chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) and electrophoresis mobility shift assay (EMSA); (2) Understanding LcMYB5 protein how to regulate the transcriptional activity of LAR genes through protoplast trans-activation assay and functional analysis of transgenic plants; (3) identification the amino acid residues that affect the level of transcriptional activation of LcMYB5. This could open an excellent opportunity to develop new cultivars which show balanced PAs concentration of forage plants in the future.