检索
E-mail
RSS
高级检索 图表检索

当期目录 我要投稿 过刊浏览
高级检索 图表检索

作物杂志,2021, 第4期: 1–9 doi: 10.16035/j.issn.1001-7283.2021.04.001

• 遗传育种·种质资源·生物技术 •    下一篇

过表达FtbZIP5提高苦荞毛状根黄酮积累及其耐盐性

翁文凤1,2(), 伍小方2, 张凯旋2, 唐宇3, 江燕1, 阮景军1(), 周美亮2()   

  1. 1贵州大学农学院,550025,贵州贵阳
    2中国农业科学院作物科学研究所,100081,北京
    3四川旅游学院食品学院,610100,四川成都
  • 收稿日期:2021-01-12 修回日期:2021-05-08 出版日期:2021-08-15 发布日期:2021-08-13
  • 通讯作者: 阮景军,周美亮
  • 作者简介:翁文凤,主要从事荞麦品质抗逆研究,E-mail：1332721469@qq.com
  • 基金资助:
    国家重点研发计划(2019YFD1001300);国家重点研发计划(2019YFD1001304)

The Overexpression of FtbZIP5 Improves Accumulation of Flavonoid in the Hairy Roots of Tartary Buckwheat and Its Salt Tolerance

Weng Wenfeng1,2(), Wu Xiaofang2, Zhang Kaixuan2, Tang Yu3, Jiang Yan1, Ruan Jingjun1(), Zhou Meiliang2()   

  1. 1College of Agronomy, Guizhou University, Guiyang 550025, Guizhou, China
    2Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
    3College of Food Science and Technology, Sichuan Tourism University, Chengdu 610100, Sichuan, China
  • Received:2021-01-12 Revised:2021-05-08 Online:2021-08-15 Published:2021-08-13
  • Contact: Ruan Jingjun,Zhou Meiliang

RichHTML

26

PDF (PC)

496

摘要：

bZIP转录因子不仅在植物盐胁迫网络中起着重要的作用,还可调节植物类黄酮的积累。在苦荞品种川荞1号中克隆了1个具有转录激活活性的bZIP家族基因FtbZIP5。FtbZIP5基因能被NaCl和脱落酸（ABA）诱导表达,在茎和叶中的表达高于根中。对过表达FtbZIP5基因毛状根的总黄酮含量进行检测,结果显示,总黄酮含量在过表达株系中显著高于野生株系。同时检测其类黄酮合成途径中关键酶基因的表达量,其中黄烷酮-3-羟化酶基因（F3H）的表达量较高。可推测该过表达毛状根株系中总黄酮的积累与F3H的表达有关。在NaCl（100mmol/L）胁迫下,各株系的总黄酮积累受到抑制,过表达株系的含量减少至0.63mg/g。并且,在这种压力下,F3H的表达水平仍然高于对照。植株在受到胁迫后,对照株系的过氧化氢酶（CAT）活性显著低于过表达株系。随着野生型植株受到胁迫的增强丙二醛（MDA）含量增加,但过表达株系的含量趋于稳定。以上结果表明,在过表达FtbZIP5毛状根中,总黄酮的积累可能是通过关键酶基因F3H的上调表达来调节的。并且FtbZIP5可提高苦荞毛状根耐盐性。通过解析FtbZIP5对苦荞毛状根中总黄酮积累及植株耐盐性的影响,为荞麦耐盐性和解析其耐盐机制研究奠定基础。

关键词: 苦荞, bZIP, 类黄酮, 盐胁迫

Abstract:

The bZIP transcription factors, with an important role in salt stress network, can regulate flavonoid accumulation in plant. In this paper, the bZIP family gene FtbZIP5, with transcriptional activation activity, was cloned from the ‘Chuanqiao 1’. Expression of FtbZIP5 gene in stems and leaves was higher than that in root when treated by NaCl and abscisic acid. Flavonoid detection was carried out on hairy roots of overexpressing the FtbZIP5 gene strain, and results showed that flavonoid accumulation in overexpressing strains was significantly higher than that in wild-type plants. And the expression of key enzyme gene flavanone-3-hydroxylase (F3H) in flavonoid synthesis pathway was high, which can speculate that accumulation of total flavonoids in overexpressed hair root strains was associated with F3H expression. Under the stress of 100mmol/L NaCl, the accumulation of total flavonoids in all lines was suppressed, and the content of total flavonoids in overexpression lines decreased to 0.63mg/g, and the expression of F3H was still higher than that of the control. Catalase activity of the control was significantly lower than that of the overexpressing strains after stressing. After stressing by NaCl, the content of malondialdehyde in wild-type plants increased and that in overexpressing strains was stable. The above results indicated that overexpressing the FtbZIP5 gene in the hairy roots, the increase in total flavonoid content may be regulated by the up-regulation of the key enzyme gene F3H; and FtbZIP5 also improved the salt tolerance of tartary buckwheat roots. This paper analyzed the role of FtbZIP5 in the accumulation of total flavonoids and salt tolerance in the hairy roots of tartary buckwheat, and laid a foundation of studying the salt tolerance of buckwheat and analyzing the salt tolerance mechanism of buckwheat.

Key words: Tartary buckwheat, bZIP, Flavonoids, Salt stress

表1

引物序列汇总

引物名称Primer 引物序列Primer sequence (5′-3′) 用途Function
1307-FtbZIP5-F CTAGAACTAGTGGATCCATGGGAAGTAACCTG 构建过表达载体
1307-FtbZIP5-R GCGGAGTACCCGGGTACCTCACCACACACCAG
pAS-FtbZIP5-F TACCCAGCTTTGACTCATATGATGGGAAGTAACCTG 转录活性分析
pAS-FtbZIP5-R TTAGCTTGGCTGCAGGTCGACTCACCACACACCAG
FtbZIP5-F ATGGGAAGTAACCTG 基因克隆
FtbZIP5-R TCACCACACACCAG
TLF CTCAAGCAA TCAAGCA TTCTAC 转基因毛状根阳性检测
RT1-QF TCAAA TAAGCTCGCCTCCCA
RT1-QR GCTGCA TTTTGTCAAGAGCG
FtbZIP5-QF CAAGTTCCTTTGCAGTCGCC
FtbZIP5-QR TAATCCCTGTGCAACCCACC
FtCHS-QF AGACGCATGTGCGACAAATC
FtCHS-QR CCACTCCTTGATGGCCTTCT
FtCHI-QF GGCACAGTACTCTGAAAAGGT
FtCHI-QR CTTCCCGAATGCAATCCTTAATG
FtF3H-QF AACAGCAGCCGTTTGTCAAT
FtF3H-QR TGCTCCTTAGCCAGCTTCTT
FtPAL-QF ACAAGGCGTTACATGGAGGA
FtPAL-QR CCAAGCTAGGGTTTCTCCCA

表2

在线平台汇总

在线平台Online platform 网址Website 功能Function
Expasy https://web.expasy.org/compute_pi 蛋白理化性质
SOMPA https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html 蛋白二级结构
Swissmodel https://swissmodel.expasy.org/interactive 蛋白三级结构
Cell-ploc 2.0 http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/ 亚细胞定位

图1

苦荞FtbZIP5基因的CDS序列扩增及生物信息学分析 a：FtbZIP5蛋白二级结构预测;α螺旋(蓝色);折叠延伸链(红色);β转角结 FtbZIP5基因编码区序列扩增

图2

FtbZIP5的转录激活分析 1×：原液;10×：稀释10倍

图3

FtbZIP5基因在NaCl和ABA诱导下的表达模式 显著差异性用“**”代表,表示P < 0.01,下同

图4

苦荞毛状根诱导(a)及FtbZIP5基因的表达检测(b) ① 生长10d后的川荞1号无菌苗;② 外植体共培养;③ 诱导10d后的外植体;④ 阳性毛状根

图5

毛状根总黄酮的测定及黄酮合成途径关键酶基因表达量的测定 9PAL：苯丙氨酸解氨酶基因;CHS：查尔酮合成酶基因;CHI：查尔酮异构酶基因;F3H：黄烷酮3-羟化酶基因;RT1：鼠李糖基转移酶基因。“*”表示P < 0.05,下同

图6

盐胁迫对毛状根次生代谢产物的影响

[1] 唐宇, 邵继荣, 周美亮. 中国荞麦属植物分类学的修订. 植物遗传资源学报, 2019,20(3):646-653.
[2] Liu Y X, Cai C Z, Yao Y L, et al. Alteration of phenolic profiles and antioxidant capacities of common buckwheat and tartary buckwheat produced in China upon thermal processing. Journal of the Science of Food and Agriculture, 2019,99(12):5565-5576.
[3] Kanter M, Aktas C, Erboga M. Protective effects of quercetin against apoptosis and oxidative stress in streptozotocin-induced diabetic rat testis. Food and Chemical Toxicology, 2012,50(3/4):719-725.
[4] Wahid A, Ghazanfar A. Possible involvement of some secondary metabolites in salt tolerance of sugarcane. Journal of Plant Physiology, 2006,163(7):723-730.
[5] Annamaria G, Marco M, Moira M, et al. Effect of nitrogen starvation on the phenolic metabolism and antioxidant properties of yarrow (Achillea collina Becker ex Rchb.). Food Chemistry, 2008,114(1):204-211.
[6] Ariel F D, Manavella P A, Dezar C A, et al. The true story of the HD-Zip family. Trends in Plant Science, 2007,12(9):419-426.
[7] Liu M, Wen Y, Sun W, et al. Genome-wide identification,phylogeny,evolutionary expansion and expression analyses of bZIP transcription factor family in tartaty buckwheat. BMC Genomics, 2019,20(1):483.
[8] Guo X L, Hou X M, Fang J, et al. The rice GERMINATION DEFECTIVE 1,encoding a B3 domain transcriptional repressor,regulates seed germination and seedling development by integrating GA and carbohydrate metabolism. The Plant Journal, 2013,75(3):403-416.
[9] Corrêa L G, Riaño-Pachón D M, Schrago C G, et al. The role of bZIP transcription factors in green plant evolution:adaptive features emerging from four founder genes. PLoS ONE, 2008,3(8):e2944.
[10] Li X Y, Gao S Q, Tang Y M, et al. Genome-wide identification and evolutionary analyses of bZIP transcription factors in wheat and its relatives and expression profiles of anther development related TabZIP genes. BMC Genomics, 2015,16:976.
[11] Liu J X, Srivastava R, Che P, et al. Salt stress responses in Arabidopsis utilize a signal transduction pathway related to endoplasmic reticulum stress signaling. The Plant Journal, 2007,51(5):897-909.
[12] Zhang S X, Haider I, Kohlen W, et al. Function of the HD-Zip I gene Oshox22 in ABA-mediated drought and salt tolerances in rice. Plant Molecular Biology, 2012,80(6):571-585.
[13] Lakra N, Nutan K K, Das P, et al. A nuclear-localized histone-gene binding protein from rice (OsHBP1b) functions in salinity and drought stress tolerance by maintaining chlorophyll content and improving the antioxidant machinery. Journal of Plant Physiology, 2015,176:36-46.
[14] Hartmann U, Sagasser M, Mehrtens F, et al. Differential combinatorial interactions of cis-acting elements recognized by R2R3-MYB,BZIP,and BHLH factors control light-responsive and tissue-specific activation of phenylpropanoid biosynthesis genes. Plant Molecular Biology, 2005,57(2):155-171.
[15] Li Q, Zhao H X, Wang X L, et al. Tartary buckwheat transcription factor FtbZIP5,regulated by FtSnRK2.6,can improve salt/drought resistance in transgenic Arabidopsis. International Journal of Molecular Sciences, 2020,21(3):1123.
[16] Malacarne G, Coller E, Czemmel S, et al. The grapevine VvibZIPC22 transcription factor is involved in the regulation of flavonoid biosynthesis. Journal of Experimental Botany, 2016,67(11):3509-3522.
[17] 卢晓玲, 何铭, 张凯旋, 等. 苦荞鼠李糖基转移酶FtF3GT1基因的克隆与转化毛状根研究. 作物杂志, 2020(5):33-40.
[18] 谈天斌, 卢晓玲, 张凯旋, 等. TrMYB308基因的克隆及在苦荞毛状根中的功能分析. 植物遗传资源学报, 2019,20(6):1542-1553.
[19] 李为喜, 朱志华, 李国营, 等. AlCl3分光光度法测定荞麦种质资源中黄酮的研究. 植物遗传资源学报, 2008,9(4):502-505.
[20] Park N I, Li X H, Uddin R M, et al. Phenolic compound production by different morphological phenotypes in hairy root cultures of Fagopyrum tataricum Gaertn. Archives of Biological Sciences, 2011,63(1):193-198.
[21] 苏文华, 张光飞, 李秀华, 等. 植物药材次生代谢产物的积累与环境的关系. 中草药, 2005(9):139-142.
[22] 许盼云, 吴玉霞, 何天明. 植物对盐碱胁迫的适应机理研究进展. 中国野生植物资源, 2020,39(10):41-49.
[23] Zhan X, Shen Q, Chen J, et al. Rice sulfoquinovosyl transferase SQD2.1 mediates flavonoid glycosylation and enhances tolerance to osmotic stress. Plant Cell Environment, 2019,42(7):2215-2230.
[24] Dong N Q, Sun Y, Guo T, et al. UDP-glucosyltransferase regulates grain size and abiotic stress tolerance associated with metabolic flux redirection in rice. Nature Communications, 2020,11(1):2629.
[25] Pedranzani H, Sierra-de-Grado R, Vigliocco A, et al. Cold and water stresses produce changes in endogenous jasmonates in two populations of Pinus pinaster Ait. Plant Growth Regulation, 2007,52:111-116.
[26] Li X H, Kim Y B, Kim Y, et al. Differential stress-response expression of two flavonol synthase genes and accumulation of flavonols in tartary buckwheat. Journal of Plant Physiology, 2013,170(18):1630-1636.
[27] Abdallah S B, Aung B, Amyot L, et al. Salt stress (NaCl) affects plant growth and branch pathways of carotenoid and flavonoid biosyntheses in Solanum nigrum. Acta Physiologiae Plantarum, 2016,38(3):72.
[28] 王琰, 陈建文, 狄晓艳. 水分胁迫下不同油松种源SOD、POD、MDA及可溶性蛋白比较研究. 生态环境学报, 2011,20(10):1449-1453.
[29] 刘会超, 贾文庆. 盐胁迫对白三叶茎的POD、CAT的影响研究. 吉林农业科学, 2009,34(1):43-46.
[1] 贾瑞玲, 赵小琴, 南铭, 陈富, 刘彦明, 魏立平, 刘军秀, 马宁. 64份苦荞种质资源农艺性状遗传多样性分析与综合评价[J]. 作物杂志, 2021, (3): 19–27
[2] 魏艳秋, 景艺卓, 郭笑恒, 张力, 韩丹, 邵惠芳. 外源硒对植物抗盐性的影响研究进展[J]. 作物杂志, 2021, (2): 15–21
[3] 靳建刚, 田再芳. 山西北部地区引种苦荞品种的灰色关联度分析[J]. 作物杂志, 2021, (2): 52–56
[4] 马名川, 刘龙龙, 刘璋, 周建萍, 南成虎, 张丽君. 苦荞全基因组SSR位点特征分析与分子标记开发[J]. 作物杂志, 2021, (1): 38–46
[5] 卢晓玲, 何铭, 张凯旋, 廖志勇, 周美亮. 苦荞鼠李糖基转移酶FtF3GT1基因的克隆与转化毛状根研究[J]. 作物杂志, 2020, (5): 33–40
[6] 杨学乐, 张璐, 李志清, 何录秋. 苦荞种质资源表型性状的遗传多样性分析[J]. 作物杂志, 2020, (5): 53–58
[7] 张自强, 白晨, 张惠忠, 李晓东, 王良, 付增娟, 赵尚敏, 鄂圆圆, 张辉, 张必周. 甜菜耐盐性形态学、生理生化特性及分子水平研究进展[J]. 作物杂志, 2020, (3): 27–33
[8] 李春花, 黄金亮, 尹桂芳, 王艳青, 卢文洁, 孙道旺, 王春龙, 郭来春, 洪波, 任长忠, 王莉花. 苦荞粒形相关性状的遗传分析[J]. 作物杂志, 2020, (3): 42–46
[9] 马成瑞,向达兵,万燕,欧阳建勇,宋月,唐正松,刘建英,赵钢. 不同苦荞品种花和籽粒空间分布特征及差异分析[J]. 作物杂志, 2020, (1): 35–40
[10] 荆培培,任红茹,杨洪建,戴其根. 盐胁迫对2个不同盐敏感性水稻品种（系）叶片光合特性与产量的影响[J]. 作物杂志, 2020, (1): 67–75
[11] 张笛,苗兴芬,王雨婷. 100份谷子品种资源萌发期耐盐性评价及耐盐品种筛选[J]. 作物杂志, 2019, (6): 43–49
[12] 杨甜,张永清,董馥慧,马星星,薛小娇. 不同水分条件下不同抗旱性苦荞根系生长规律研究[J]. 作物杂志, 2019, (6): 76–82
[13] 戈珍梅,刘治国,赵露,张晓宇,刘桂霞. 盐胁迫对膜荚黄芪种子萌发的影响[J]. 作物杂志, 2019, (6): 187–194
[14] 谷娇娇,胡博文,贾琰,沙汉景,李经纬,马超,赵宏伟. 盐胁迫对水稻根系相关性状及产量的影响[J]. 作物杂志, 2019, (4): 176–182
[15] 宋丽芳,冯美臣,张美俊,肖璐洁,王超,杨武德,宋晓彦. 外源硒对苦荞生长发育及子粒硒含量的影响[J]. 作物杂志, 2019, (3): 150–154
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!