Abstract:Lycoris sprengeri is multicolor special flowera with red and blue petals in Lycoris, which flower color formation mechanism is complex, and anthocyanin synthase (ANS) is a key enzyme at the end of plant anthocyanin synthesis pathway. In this study, molecular biological methods were used to analyze the functions of LsANS cDNA and promoter. The results showed, The total length of LsANS cDNA was 1 401 bp (GenBank No. MT664240), ORF 1 068 bp, encoding 355 amino acids; The phylogenetic tree analysis showed that the LsANS had highest homology with ANS gene of L. radiate, which was 98.5%. qRT-PCR showed that the expression of LsANS was the highest in clone H4 and the lowest in clone H1; The expression was the highest at large bud stage and the lowest at full-bloom stage; The consequence of HPLC showed that in different developmental stages, the content of cyanidin-3-O-glucoside and pelargonin-3-O-glucoside in the bud stage was higher than that in other stages; in different clones, pelargonin-3-O-glucose accounted for the highest proportion of anthocyanins measured in clone H4, and the lowest proportion in clone H1; It was speculated that LsANS played an important role in the anthocyanin formation pathway. A 2 114 bp long LsANS promoter sequence was cloned, which contained MYB transcription factor binding site and cis acting elements involved in light, hormone and stress. The PBI121 LsANSpro::GUS fusion expression vector was constructed and transiently transformed into Arabidopsis thaliana, which showed that LsANSpro has the function of promoting downstream genes. By GUS staining assay, promoter activity was detected in the whole roots, leaves, and flowers . The research will provide a theoretical basis for further research the role and mechanism of ANS gene in the flower color formation process of Lycoris.
[1] 安维忠, 刘雅莉, 刘红利, 等. 2015. 葡萄风信子MaANS基因及启动子的克隆与分析[J]. 西北植物学报, 35(9): 1728-1734. (An W J, Liu Y L, Liu H L, et al.2015. Isolation and analysis of Muscari armeniacum MaANS gene and its promoter[J]. Acta Botanica Boreali-Occidentalia Sinica, 35(9): 1728-1734.) [2] 卜星星, 雒晓鹏, 白悦辰, 等. 2014. 金荞麦花青素合酶基因的克隆及其表达与花青素量的相关性研究[J]. 中草药, 45(7): 985-989. (Bu X X, Luo X P, Bai Y C, et al.2014. Gene cloning of anthocyanin synthase in Fagopyrum dibotrys and correlation between its expression level and anthocyanin content[J]. Chinese Traditional and Herbal Drugs, 45(7): 985-989) [3] 曹建军, 梁宗锁. 2008. 欧报春(Primula vulgaris)不同花色与色素关系及花色遗传初步分析[J]. 植物研究, 7(4): 426-432. (Cao J J, Liang Z S, Preliminary analysis on flower color inheritance and relations between flower color and pigments in Primula vulgaris[J]. Bulletin of Botanical Research, 2008, 7(4): 426-432.) [4] 董娇, 周军, 辛培尧, 等. 2010. 不同植物LDOX/ANS基因的生物信息学分析[J]. 基因组学与应用生物学, 29(5): 815-822. (Dong J, Zhou J, Xin P Y, et al.2010. Bioinformatics analysis of LDOX/ANS gene in different plants[J]. Genomics and Applied Biology, 29(5): 815-822.) [5] 樊青玲, 周日宝, 刘湘丹, 等. 2020. 忽地笑不同器官RNA提取方法比较研究[J]. 安徽农业科学, 48(18): 169-172. (Fan Q L, Zou R B, Liu X D, et al.2020. Comparative study on RNA extraction methods in different organs of Lycoris aurea Hurb[J]. Anhui Agricultural Sciences, 48(18): 169-172.) [6] 高燕会, 黄春红, 朱玉球, 等. 2012. 植物花青素苷生物合成及调控的研究进展[J]. 中国生物工程杂志, 32(8): 94-99. (Gao Y H, Huang C H, Zhu Y Q, et al., 2012. Progress on plant anthocyanin biosynthesis and regulation[J]. China Biotechnology, 32(8): 94-99.) [7] 侯朔, 高燕会, 童再康. 2019. 换锦花LsMYB5基因的克隆与表达分析[J].农业生物技术学报, 27(12): 2164-2174. (Hou S, Gao Y H, Tong Z K.2019. Cloning and expression analysis of LsMYB5 gene in Lycoris sprengeri[J]. Journal of Agricultural Biotechnology, 27(12): 2164-2174.) [8] 黄玉, 杨波, 迟小华, 等. 2010. 真核生物启动子的研究及应用[J]. 生物技术通讯, 21(02): 275-279. (Huang Y, Yang B, Chi X H, et al.2010. Investigation and application of promoters of eukaryotic organism[J]. Letters in Biotechnology, 21(02): 275-279.) [9] 蒋婷婷, 高燕会, 童再康. 2015. 石蒜属植物实时荧光定量PCR内参基因的选择[J]. 园艺学报, 42(6): 1129-1138. (Jiang T T, Gao Y H, Tong Z K.2015. Selection of reference genes for quantitative Real-Time PCR in Lycoris[J]. Acta Horticulturae Sinica, 42(6): 1129-1138.) [10] 李小兰, 张明生, 吕享. 2016. 植物花青素合成酶ANS基因的研究进展[J]. 植物生理学报,52(6): 817-827. (Li X L, Zhang M S, Lv X.2016. The research progress on plant anthocyanin synthetase ANS gene[J]. Plant Physiology Journal, 52(6): 817-827.) [11] 梁平, 宋洪元. 2014. 植物花青素生物合成转录调控研究进展[J].南方农业学报, 45(08): 1375-1379. (Liang P, Song H Y, Advances on the transcriptional regulation of anthocyanin biosynthesis in plant[J]. Journal of Southern Agriculture, 45(08): 1375-1379.) [12] 刘平, 任秋婧, 康馨, 等. 2018. 茶树N-甲基转移酶基因启动子克隆及功能分析[J]. 茶叶科学, 38(6): 26-36. (Liu P, Ren Q J, Kang X, et al., 2018. Isolation and functional analysis of promoter for N-methyltransferase gene associated with caffeine biosynthesis in tea plants (Camellia sinensis)[J]. Journal of Tea Science, 38(6): 26-36.) [13] 亓希武, 帅琴, 范丽, 等. 2013. 桑树花青素合成酶(ANS)基因的克隆及在2种果色桑树中的表达特征[J]. 蚕业科学, 39(1): 5-13. (Qi X W, Shuai Q, Fan L, et al.2013. Molecular cloning and expressional pattern gene in two mulberry species with different fruit colors[J]. Acta Sericologica Sinica, 39(1): 5-13.) [14] 王晰, 丁文杰, 李娅, 等. 2018. 长筒石蒜花青素合成酶基因LlANS的克隆与表达分析[J]. 河南农业大学学报, 52(4): 611-617. (Wang X, Ding W J, Li Y, et al.2018. Cloning and expression analysis of anthocyanidin synthase (LIANS) in Lycoris longituba[J]. Journal of Henan Agricultural University, 52(4): 611-617.) [15] 王艳敏, 白卉, 于文喜, 等. 2015. 小黑杨PnsICE1基因启动子克隆及瞬时表达分析[J]. 植物研究, 35(01): 77-83. (Wang Y M, Bai H, Yu W X, et al.2015. Cloning and transient expression analysis of promoter of PnsICE1 from Populus[J]. Bulletin of Botanical Research, 35(01): 77-83.) [16] 许振渊, 高燕会, 周芬静, 等. 2014. 换锦花LsMYB4基因的克隆与表达分析[J]. 园艺学报, 41(11): 2281-2290. (Xu Z Y, Gao Y H, Zhou F J, et al.2014. Cloning and expression analysis of LsMYB4 gene in Lycoris sprengeri[J]. Acta Horticulturae Sinica, 41(11): 2281-2290.) [17] 许志茹, 李春雷, 崔国新, 等. 2009. 芜菁花青素合成酶基因的克隆、序列分析及表达[J]. 生物技术通讯, 20(1): 66-68, 74.(Xu Z R, Li C L, Cui G X, et al. 2009. Cloning, sequence analysis and expression of anthocyanidin synthase gene in turnip[J]. Letters in Biotechnology, 20(1): 66-68, 74.) [18] 杨楠, 2019. 蜡梅花被片花青素合成酶基因CpANS1和转录因子CpMYB2的功能解析[D]. 博士学位论文,华中农业大学, 导师: 陈龙清, 赵凯歌. pp. 56-66. (Yang N.2019. Functional characterization of anthocyanin synrhase CpANS1 and transcription factor CpMUB2 in Chimonanthus praecox tepals[D]. Thesis for Ph.D. Huazhong Agricultural University, Supervisor: Chen L Q, Zhao KG, pp. 56-66) [19] 姚建忠. 2018. 毛白杨花青素合酶基因的克隆与特性分析[J]. 中国细胞生物学学报, 40(3): 349-356. (Yao J Z.2018. Molecular cloning and characterization of two genes encoding anthocyanin synthase from Populus tomentosa[J]. Chinese Journal of Cell Biology, 40(3):349-356.) [20] 姚庆荣, 孙瑞芳. 2008. 花色基因工程育种研究进展[J]. 南方农业(园林花卉版), 2(6): 41-43. (Yao Q R, Sun R F.2008. Research progress of color genetic engineering breeding[J]. South China Agriculture, 2(6): 41-43.) [21] 叶重阳. 2006. 生物技术在花卉产业中的应用[J]. 合肥师范学院学报, 24(3): 78-81. (Ye C Y.2006. Application of biotechnology in flower industry[J]. Journal of hefeei normal university, 24(3): 78-81) [22] 赵蓉蓉, 蒋明, 贺蔡明, 等. 2010. 芥蓝Brassica albograbra花青素合成酶基因BaANS的克隆与序列分析[J]. 浙江农业学报, 22(2): 161-166. (Zhao R R, Jiang M, He C M, et al.2010. Isolation and sequence analysis of anthocyanidin synthase gene BaANS from Brassica albograbra[J]. Acta Agriculturae Zhejiangensis, 22(2): 161-166.) [23] 周生茂, 王玲平, 向珣, 等. 2009. 山药ANS基因的克隆和分子特性及其与花青素积累的关系[J]. 园艺学报, 36(9): 1317-1326. (Zhou S M, Wang L P, Xian X, et al.2009. Cloning and molecular characteristics of ANS gene and its correlations with anthocyanin accumulation in yam[J]. Acta Horticulturae Sinica, 36(9): 1317-1326.) [24] Dong W, You Y, Niu L, et al.2014. Isolation and analysis of the promoter of an anthocyanin synthase gene from purple-fleshed sweet potato tubers[J]. Acta Physiologiae Plantarum, 36(10): 2637-2649. [25] Mizuno T, Yabuya T, KitajimaI J, et al.2013.Identification of novel C-glycosylflavones and their contribution to flower colour of the Dutch iris cultivars[J]. Plant Physioligy and Biochemistry, 72: 116-124. [26] Naing A H, Ji H L, Park K I, et al.2018. Transcriptional control of anthocyanin biosynthesis genes and transcription factors associated with flower coloration patterns in Gerbera hybrida[J]. 3 Biotech, 8(1): 65-76. [27] Putta S, Yarla N S, Peluso I, et al.2017. Anthocyanins: multi-target agents for prevention and therapy of chronic diseases[J]. Current Pharmaceutical Design, 23(41): 6321-6346. [28] Robards K, Antolovich M J A.1997. Analytical chemistry of fruit bioflavonoids a review[J]. Analyst, 122: 11R-34R. [29] Sun H Y, Lim, Jae Kwang Kim, et al.2013. Petal-specific activity of the promoter of an anthocyanidin synthase gene of tobacco (Nicotiana tabacum L.)[J]. Plant Cell Tissue and Organ Culture, 114(3): 373-383. [30] Zhang J, Han, Z Y,Tian, J.et al.2015.The Expression level of anthocyanidin synthase determines the anthocyanin content of crabapple (Malus sp.) petals[J]. Acta Physiologiae Plantarum, 37(6): 109-119. [31] Zheng L, Liu G F, Meng X N, et al.2012. A versatile agrobacterium-mediated transient gene expression system for herbaceous plants and trees[J]. Biochemical Genetics, 50: 9-10.