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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

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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

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