Home > Archive>Volume 32, Issue 2, 2024 >257-263. DOI:10.11926/jtsb.4746
PDF HTML XML Export Cite reminder
Analysis of AN2 Upstream Promoter Activity Based on Tail-PCR
Author:
  • Article
    • | |
  • Metrics
    • |
  • Reference [32]
    • |
  • Related [20]
    • | | |
  • Comments
    Abstract:

    Lycium ruthenicum is rich in anthocyanin, and AN2 is the main gene regulating anthocyanin anmeta- bolism. In order to analyze the activity difference of AN2 gene promoter, the upstream sequence of AN2 gene start codon about 1 686 bp (LrAN2p) and 1 495 bp (LbAN2p) of L. barbarum were cloned by Tail-PCR method, respectively. Plant CARE predicted that there were 133 and 137 cis-acting elements in sequences of LbAN2p and LrAN2p, respectively, among which 11 and 15 cis-acting elements were involved in photoregulation, and 13 and 16 cis-elements involved in hormone response, respectively. The plant expression vectors pKGWFS7:LbAN2p and pKGWFS7:LrAN2p were constructed, and the transgenic tobacco was obtained by using Agrobacterium- mediated tobacco genetic transformation system. The results of GUS staining showed that LrAN2p could drive the expression of GUS in tobacco, and the leaves were blue, with stronger starting activity than LbAN2p. The results of qRT-PCR showed that GUS gene in LrAN2p transgenic tobacco had high transcription level, which might lead to high expression of AN2 gene in L. ruthenicum, and activating the anthocyanin anabolic pathway. Thus, these would provide a theoretical basis for understanding the mechanism of fruit color formation and AN2 gene expression.

    Reference
    [1] GUO B Z. Economic Flora of Qinghai[M]. Xining: Qinghai People’s Press, 1987.[郭本兆. 青海经济植物志[M]. 西宁: 青海人民出版社, 1987.]
    [2] LIU Z G, DONG B M C L, LIU C, et al. Variation of anthocyanin content in fruits of wild and cultivated Lycium ruthenicum[J]. Ind Crops Prod, 2020, 146: 112208. doi: 10.1016/j.indcrop.2020.112208-16.
    [3] ZHENG J, DING C X, WANG L S, et al. Anthocyanins composition and antioxidant activity of wild Lycium ruthenicum Murr. from Qinghai-Tibet Plateau[J]. Food Chem, 2011, 126(3): 859–865. doi: 10.1016/j.foodchem.2010.11.052.
    [4] Rukeya JIAPAER, SUN Y J, ZHONG L Z, et al. A review of phytochemical composition and bio-active of Lycium barbarum fruit (Goji)[J]. J Chin Inst Food Sci Technol, 2013, 13(8): 161–172.[如克亚·加帕尔, 孙玉敬, 钟烈州, 等. 枸杞植物化学成分及其生物活性的研究进展[J]. 中国食品学报, 2013, 13(8): 161–172. doi: 10.16429/j.1009-7848.2013.08.001.]
    [5] JIANG X F. Overview on chemical constituents and pharmacological research of medlar[J]. J Jiangxi Univ TCM, 2013, 25(3): 98–100.[江旭锋. 枸杞子化学成分及其药理学研究概况[J]. 江西中医学院学报, 2013, 25(3): 98–100. doi: 10.3969/j.issn.1005-9431.2013.03.032.]
    [6] LI J, YANG F, LUAN G X, et al. Research progress on ingredient and pharmacological activities of anthocyanins from Lycium ruthenicum[J]. W China J Pharm Sci, 2022, 37(3): 331–336.[李婧, 杨芳, 栾广祥, 等. 黑果枸杞的花青素类成分及其药理作用的研究进展[J]. 华西药学杂志, 2022, 37(3): 331–336. doi: 10.13375/j.cnki.wcjps.2022.03.022.]
    [7] TANAKA Y, SASAKI N, OHMIYA A. Biosynthesis of plant pigments: anthocyanins, betalains and carotenoids[J]. Plant J, 2008, 54(4): 733-749. doi: 10.1111/j.1365-313X.2008.03447.x.
    [8] AMRHEIN N. Novel inhibitors of phenylpropanoid metabolism in higher plants[M]//LUCKNER M, SCHREIBER K. Regulation of Secondary Product and Plant Hormone Metabolism. Oxford: Pergamon Press, 1979: 173–182. doi: 10.1016/B978-0-08-023179-2.50020-4.
    [9] BOSS P K, DAVIES C, ROBINSON S P. Analysis of the expression of anthocyanin pathway genes in developing Vitis vinifera L. cv Shiraz grape berries and the implications for pathway regulation[J]. Plant Physiol, 1996, 111(4): 1059–1066. doi: 10.1104/pp.111.4.1059.
    [10] DONG Y H, BEUNING L, DAVIES K, et al. Expression of pigmentation genes and photo-regulation of anthocyanin biosynthesis in developing Royal Gala apple flowers[J]. Funct Plant Biol, 1998, 25(2): 245–252. doi: 10.1071/PP97108.
    [11] KREYNES A E, YONG Z H, ELLIS B E. Developmental phenotypes of Arabidopsis plants expressing phosphovariants of AtMYB75[J]. Plant Signal Behav, 2021, 16(1): 1836454-68. doi: 10.1080/15592324.2020.1836454.
    [12] CHHON S, JEON J, KIM J, et al. Accumulation of anthocyanins through overexpression of AtPAP1 in Solanum nigrum Lin. (black nightshade)[J]. Biomolecules, 2020, 10(2): 277–293. doi: 10.3390/biom10020277.
    [13] GERATS A G, FARCY E, WALLROTH M, et al. Control of anthocyanin synthesis in Petunia hybrida by multiple allelic series of the genes An1 and An2[J]. Genetics, 1984, 106(3): 501–508. doi: 10.1093/genetics/106.3.501.
    [14] BAO X M, ZONG Y, HU N, et al. Functional R2R3-MYB transcription factor NsMYB1, regulating anthocyanin biosynthesis, was relative to the fruit color differentiation in Nitraria sibirica Pall.[J]. BMC Plant Biol, 2022, 22(1): 186-198. doi: 10.1186/s12870-022-03561-5.
    [15] ZONG Y, ZHU X B, LIU Z G, et al. Functional MYB transcription factor encoding gene AN2 is associated with anthocyanin biosynthesis in Lycium ruthenicum Murray[J]. BMC Plant Biol, 2019, 19(1): 169-178. doi: 10.1186/s12870-019-1752-8.
    [16] ZONG Y, LI S M, XI X Y, et al. Comprehensive influences of overexpression of a MYB transcriptor regulating anthocyanin biosynthesis on transcriptome and metabolome of tobacco leaves[J]. Int J Mol Sci, 2019, 20(20): 5123–5136. doi: 10.3390/ijms20205123.
    [17] SHEN Z Y, ZHANG C, DONG B, et al. Cloning and expression analysis of the promoters of OfLCYB and OfLCYE in Osmanthus fragrans[J]. Biotechnol Bull, 2018, 34(1): 137–143.[沈子又, 张超, 董彬, 等. 桂花OfLCYB和OfLCYE启动子的克隆和活性分析[J]. 生物技术通报, 2018, 34(1): 137–143. doi: 10.13560/j.cnki.biotech. bull.1985.2017-0706.]
    [18] LIU Y G, WHITTIER R F. Thermal asymmetric interlaced PCR: Automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking[J]. Genomics, 1995, 25(3): 674–681. doi: 10.1016/0888-7543(95)80010-J.
    [19] GAO L, TIAN Y, CHEN M C, et al. Cloning and functional characterrization of epidermis-specific promoter MtML1 from Medicago truncatula[J]. J Biotechnol, 2019, 300: 32–39. doi: 10.1016/j.jbiotec.201.
    [20] HORSCH R B, FRY J E, HOFFMANN N L, et al. A simple and general method for transferring genes into plants[J]. Science, 1985, 227(4691): 1229–1231. doi: 10.1126/science.227.4691.1229.
    [21] Fernández-Piñán S, López J, Armendariz I, et al. Agrobacterium tumefaciens and Agrobacterium rhizogenes-mediated transformation of potato and the promoter activity of a suberin gene by GUS staining[J]. JoVE, 2019(145): e59119–e59128. doi: 10.3791/59119.
    [22] LAI Y S, SHIMOYAMADA Y, NAKAYAMA M, et al. Pigment accumulation and transcription of LhMYB12 and anthocyanin biosynthesis genes during flower development in the Asiatic hybrid lily (Lilium spp.)[J]. Plant Sci, 2012, 193–194: 136–147. doi: 10.1016/j.plantsci.2012.05.013.
    [23] LU J, ZHAO H Y, HE Y K, et al. Advances in promoters in higher plants and their applications[J]. Prog Nat Sci, 2004, 14(8): 856–862.[路静, 赵华燕, 何奕昆, 等. 高等植物启动子及其应用研究进展[J]. 自然科学进展, 2004, 14(8): 856–862. doi: 10.3321/j.issn:1002-008X.2004.08.003.]
    [24] WANG F, WANG X J, ZHAO S N, et al. Light regulation of anthocyanin biosynthesis in horticultural crops[J]. Sci Agric Sin, 2020, 53(23): 4904–4917.[王峰, 王秀杰, 赵胜男, 等. 光对园艺植物花青素生物合成的调控作用[J]. 中国农业科学, 2020, 53(23): 4904-4917. doi: 10.3864/j.issn.0578-1752.2020.23.015.]
    [25] WANG H X, LIU T Y, ZHUANG W B, et al. Research advances in the function of anthocyanin in plant stress response[J]. J Agric Biotechnol, 2020, 28(1): 174–183.[王鸿雪, 刘天宇, 庄维兵, 等. 花青素苷在植物逆境响应中的功能研究进展[J]. 农业生物技术学报, 2019, 28(1): 174–183. doi: 10.3969/j.issn.1674-7968.2020.01.018.]
    [26] LIU L, DU H, TANG X F, et al. The roles of MYB transcription factors on plant defense responses and its molecular mechanism[J]. Hereditas, 2008, 30(10): 1265–1271.[刘蕾, 杜海, 唐晓凤, 等. MYB转录因子在植物抗逆胁迫中的作用及其分子机理[J]. 遗传, 2008, 30(10): 1265–1271. doi 10.3724/SP.J.1005.2008.01265.]
    [27] LIU H J, LIU Y, LIU L. Progress of research on the influence of abscisic acid in plant resistance[J]. Biotechnol Bull, 2008(6): 7–9.[刘红娟, 刘洋, 刘琳. 脱落酸对植物抗逆性影响的研究进展[J]. 生物技术通报, 2008(6): 7–9.]
    [28] SHI X W. Identification and analysis of MicroRNA related to adversity stress and anthocyanin biosynthesis in sweet potato[D]. Jinzhong: Shanxi Agricultural University, 2018.[石晓雯. 甘薯逆境胁迫和花青素合成相关microRNA及其靶基因的鉴定和分析[D]. 晋中: 山西农业大学, 2018.]
    [29] SA Q L, LI W B, SUN Y R. Transcriptional regulation of the G-box and G-box-binding proteins in plant gene expression[J]. Plant Physiol Commun, 2003, 39(1): 89–92.[萨其拉, 李文彬, 孙勇如. G-box和G-box结合蛋白在植物基因诱导表达中的转录调控作用[J]. 植物生理学通讯, 2003, 39(1): 89–92. doi: 10.13592/j.cnki.ppj.2003.01.028.]
    [30] XUE H M, ZHOU Y L, GAO Y H. Cloning and promoter function analysis of the anthocyanins synthase gene (LsANS) in Lycoris sprengeri[J]. Chin J Agric Biotechol, 2022, 30(8): 1468–1479.[薛惠敏, 周洋丽, 高燕会. 换锦花花青素合成酶基因(LsANS)的克隆及启动子功能分析[J]. 农业生物技术学报, 2022, 30(8): 1468–1479. doi: 10.3969/j.issn.1674-7968.2022.08.003.]
    [31] LUO X Y, ZONG Y, CAO D, et al. Cloning and functional analysis of promoter of TaMYC1 gene in common wheat[J]. Mol Plant Breed, 2022, 20(8): 2456–2462.[罗香怡, 宗渊, 曹东, 等. 普通小麦中TaMYC1基因启动子的克隆和功能分析[J]. 分子植物育种, 2022, 20(8): 2456–2462. doi: 10.13271/j.mpb.020.002456.]
    [32] CHEN J, HU R, LIU Y, et al. Cloning and expression analysis of BnaFIL gene promoter in Brassica napus L.[J]. Acta Agric BorealiSin, 2022, 37(3): 53–59.[陈静, 胡蓉, 刘勇, 等. 甘蓝型油菜BnaFIL基因启动子的克隆与表达分析[J]. 华北农学报, 2022, 37(3): 53–59. doi: 10.7668/hbnxb.20192788.]
    Cited by
    Comments
    Comments
    分享到微博
    Submit
Get Citation

宗渊,韦国,石光禹,刘宝龙,包雪梅.基于Tail-PCR分析AN2基因上游启动子活性[J].热带亚热带植物学报,2024,32(2):257~263

Copy
Share
Article Metrics
  • Abstract:198
  • PDF: 418
  • HTML: 273
  • Cited by: 0
History
  • Received:November 07,2022
  • Online: March 22,2024
  • Published: March 20,2024
Article QR Code
You are the first4648536 Visitors
Governing Body：中国科学院
Organizer：中国科学院华南植物园 广东省植物学会
Address：Xingke Road 723,Tianhe District,Guangzhou,China
Phone：+86 (0)20-3725 2514
Journal of Tropical and Subtropical Botany ® 2025 All Rights Reserved