Cloning and Expression Analysis of a Leucoanthocyanidin Reductase (LAR) Gene from Onobrychis viciifolia cv.Gansu

doi:10.11686/cyxb2014452

Abstract

Abstract: A leucoanthocyanidin reductase (LAR) gene was cloned using RT-PCR from Onobrychis viciifolia (sainfoin) cv. Gansu, and the level of LAR gene expression was analyzed in different plant organs. The LAR gene sequence from O. viciifolia cv. Gansu shared a high level of similarity (98.34% homology) with an O. viciifolia LAR gene in GenBank (accession No: HM152980). However, the coding sequence of amino acids is obviously different from those in other genera of the Fabaceae. The open reading frame (ORF) comprised 1089 bp, encoding 362 amino acid residues. The sequence of the LAR gene from the present study was submitted to GenBank with the accession number KP013623. LAR expression differed between plant organs of O. viciifolia cv. Gansu. LAR expression was highest in leaves, followed by flowers and fruit, and was lowest in stem tissues, which corresponded to concentration of condensed tannins in different organs of O. viciifolia cv. Gansu. It is inferred that LAR gene expression is in some way involved in condensed tannin accumulation in O. viciifolia cv.Gansu. These results provide fundamental information and a method for exploring the regulation mechanism of condensed tannin accumulation.

CHEN Chun-Yan, MA Hui-Ling, DONG Wen-Ke. Cloning and Expression Analysis of a Leucoanthocyanidin Reductase (LAR) Gene from Onobrychis viciifolia cv.Gansu[J]. Acta Prataculturae Sinica, 2015, 24(6): 177-187.

References

[1] Chen B S. Research of yield and nutrition in sainfoin. Grassland of China, 1983, (2): 36-38.
[2] Chen B S. Sainfoin is an excellent leguminous forage and a plant for water and soil conservation. Science of Soil and Water Conservation, 1984, 29(8): 14-18.
[3] Chen B S. Sainfoin[M]. Lanzhou: Gansu Science and Technology Press, 1992: 18.
[4] Howarth R E, Sarkar S K, Fesser A C, et al . Some properties of soluble proteins from alfalfa ( Medicago sativa ) herbage and their possible relation to ruminant bloat. Journal of Agricultural and Food Chemistry, 1977, 25(1): 175-179.
[5] Wu Z L, Niu J L, Zhang Y P, et al . Tannin dynamics study for sainfoin. Chinese Journal of Grassland, 1993, (5): 25-27.
[6] McMahon L R, McAllister T A, Berg B, et al . A review of the effects of forage condensed tannins on ruminal fermentation and bloat in grazing cattle. Canadian Journal of Plant Science, 2000, 80: 469-485.
[7] Wang Y P, Yang Z M, Zhang B. Identification of protein related to biosynthesis of condensed Tannin in Onobrychis tanaitica spreng. Acta Agrestia Sinica, 2000, 8(2): 126-131.
[8] Zhao W J, Zhang D, Ma L J, et al . Biosynthetic pathway, functional genes and metabolic engineering of proanthocyanidins. Plant Physiology Communications, 2009, 45(5): 509-519.
[9] Xie D Y, Sharma S B, Paiva N L, et al . Role of anthocyanidin reductase, encoded by BANYULS in plant flavonoid biosynthesis. Science, 2003, 299: 396-399.
[10] Bogs J, Downey M O, Harvey J S, et al . Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin reductase and anthocyanidin reductase in developing grape berries and grapevine leaves. Plant Physiology, 2005, 139(2): 652-663.
[11] Yuan L, Wang L, Han Z, et al . Molecular cloning and characterization of PtrLAR3, a gene encoding leucoanthocyanidin reductase from Populus trichocarpa , and its constitutive expression enhances fungal resistance in transgenic plants. Journal of Experiment Botany, 2012, 63(7): 2513-2524.
[12] Paolocci F, Robbins M P, Madeo L, et al . Ectopic expression of a basic helix-loop-helix gene transactivates parallel pathways of proanthocyanidin biosyn thesis: structure, expression analysis, and genetic control of leucoanthocyanidin 4-reductase and anthocyanidin reductase genes in Lotus corniculatus. Plant Physiology, 2007, 143(1): 504-516.
[13] Wang Y, Zhang Q L, Luo Z R. Isolation and expression of gene encoding leucoanthocyanidin reductase from Diospyros kaki during fruit development. Biologia Plantarum, 2010, 54(4): 707-710.
[14] Pang Y, Wenger J P, Saathoff K, et al . A WD40 repeat protein from Medicago truncatula is necessary for tissue-specific anthocyanin and proanthocyanidin biosynthesis but not for trichome development. Plant Physiology, 2009,151(3): 1114-1129.
[15] Sambrook J, Russell D W. Molecular Cloning: A Laboratory Manual[M]. Vol.2. Beijing: Beijing Sciences Press, 1995: 343-392.
[16] Shi B S, Zhuo L H. Modification and comparation of total extraction methods from Prunus cerasifera leaves. Molecular Plant Breeding, 2006, 4(5): 721-725.
[17] Terrill T H, Rowan A M, Douglas G B. et al . Determination of extractable and bound condensed tannin concentrations in forage plants, prote in concentrate meals and cereal grains. Journal of the Science of Food and Agriculture, 1992, 58(3): 321-329.
[18] Li H, Wang X L. The difficulties in the isloation of RNA from plant tissues and their resolving strategies. Bio Technology Information, 1999, (1): 36-39.
[19] Gasic K, Hernandez A, Korban S S. RNA extraction from different apple tissues rich in polyphenols and polysaccharides for cDNA library construction. Plant Molecular Biology Reporter, 2004, 22(4): 437-438.
[20] Rodrigues S M, Soares V L F, de Oliveira T M, et al . Isolation and purification of RNA from tissues rich in polyphenols, polysaccharides, and pigments of annatto ( Bixa Orellana L.). Molecular Biotechnology, 2007, 37(3): 220-224.
[21] Ainsworth C. Isolation of RNA from floral tissue of Rumex acetosa (sorrel). Plant Molecular Biology Reporter, 1994, 12(3): 198-203.
[22] Tanner G J, Francki K T, Abrahams S, et al . Proanthocyanidin biosynthesis in plants:purification of legume leucoanthocyanidin reductase and molecular cloning of its cDNA. Journal of Biology Chemistry, 2003, 278: 31647-31656.
[23] Ma J, Wang B, Dai Y, et al . Cloning and expression analysis of leucoanthocyanidin reductase gene in Fagopyrum dibotrys . Acta Pharmaceutica Sinica, 2012, 47(7): 953-961.
[24] Thill J, Regos I, Farag M A, et al . Polyphenol metabolism provides a screening tool for beneficial effects of Onobrychis viciifolia (sainfoin). Phytochemistry, 2012, 82: 67-80.
[25] Shen B Y, Liu Y H, Zhang J L, et al . Analysis of cloning and sequence characteristics of cDNA ending the GBSS I gene from tubers of Sloanum tuberosum . Acta Prataculturae Sinica, 2010, 19(5): 1-8.
[26] Li L Q, Huang Y B, Wang X Y. Cloning, sequence analysis and prokaryotic expression of the WRKY6 gene of potato. Acta Prataculturae Sinica, 2011, 20(2): 177-183.
[27] Lv H Y, Li Y X, Kong F J, et al . Recent progress in studies on Na + /H + antiporter in plants. Chinese Bulletin of Botany, 2003, 20(3): 363-369.
[28] Wang L, Jiang Y, Yuan L, et al .Isolation and characterization of cDNAs encoding leucoanthocyanidin reductase and anthocyanidin reductase from Populus trichocarpa . PLoS One, 2013, 8(5): e64664. doi:10.1371/journal. Pone. 0064664.
[29] Daniel A H. Biomass allocation is an important determinant of the tannin concentration in growing plants. Annals of Botany, 2007, 99: 111-120.
[1] 陈宝书. 红豆草产量和营养成分的研究. 中国草原, 1983, (2): 36-38.
[2] 陈宝书. 红豆草是优良的豆科牧草和水土保持植物. 中国水土保持, 1984, 29(8): 14-18.
[3] 陈宝书. 红豆草[M]. 兰州: 甘肃科学技术出版社, 1992: 18.
[5] 吴自立, 牛菊兰, 张跃平, 等. 红豆草中丹宁的动态研究. 中国草地学报, 1993, (5): 25-27.
[7] 王玉萍, 杨茁萌, 张博. 顿河红豆草缩合单宁生物合成相关蛋白鉴定. 草地学报, 2000, 8(2): 126-131.
[8] 赵文军, 张迪, 马丽娟, 等. 原花青素的生物合成途径、功能基因和代谢工程. 植物生理学通讯, 2009, 45(5): 509-519.
[15] 萨姆布鲁克J, 拉塞尔D W. 分子克隆实验指南[M]. 2版. 北京: 科学出版社, 1995: 343-392.
[16] 史宝胜, 卓丽环. 紫叶李叶片总RNA提取方法的改进与比较. 分子植物育种, 2006, 4(5): 721-725.
[18] 李宏, 王新力. 植物组织RNA提取的难点及对策. 生物技术通报, 1999, (1): 36-39.
[23] 马婧, 王斌, 代银, 等. 金荞麦无色花色素还原酶基因FdLAR的克隆和表达分析. 药学学报, 2012, 47(7): 953-961.
[25] 沈宝云, 刘玉汇, 张俊莲, 等. 马铃薯块茎GBSSI基因的cDNA克隆及其序列特征分析. 草业学报, 2010, 19(5): 1-8.
[26] 李立芹, 黄玉碧, 王西瑶. 马铃薯WRKY6基因的克隆、序列分析与原核表达研究. 草业学报, 2011, 20(2): 177-183.
[27] 吕慧颖, 李银心, 孔凡江, 等. 植物Na + /H + 逆向转运蛋白研究进展. 植物学通报, 2003, 20(3): 363-369.