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作物学报 ›› 2024, Vol. 50 ›› Issue (3): 645-655.doi: 10.3724/SP.J.1006.2024.34084

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

红麻HcKAN4基因克隆、表达及在类黄酮合成中的功能

吴法轩1,2(), 李秦1,2, 杨昕1,2, 李新根1,2, 徐建堂1,2, 陶爱芬1,2, 方平平1,2, 祁建民1,2, 张立武1,2,*()   

  1. 1福建农林大学作物遗传育种与综合利用教育部重点实验室 / 农业农村部闽台作物生物育种重点实验室 / 福建省作物设计育种重点实验室, 福建福州 350002
    2福建农林大学农业农村部东南黄红麻实验观测站 / 福建省麻类种质资源共享平台 / 福建省南方经济作物遗传育种与多用途开发国际科技合作基地, 福建福州 350002
  • 收稿日期:2023-05-17 接受日期:2023-09-13 出版日期:2024-03-12 网络出版日期:2023-09-28
  • 通讯作者: *张立武, E-mail: lwzhang@fafu.edu.cn; zhang_liwu@hotmail.com
  • 作者简介:E-mail: 2534995528@qq.com
  • 基金资助:
    国家自然科学基金项目(31972968);财政部和农业农村部国家现代农业产业技术体系建设专项(CARS-16)

Cloning, expression, and function of HcKAN4 gene of kenaf in flavonoid synthesis

WU Fa-Xuan1,2(), LI Qin1,2, YANG Xin1,2, LI Xin-Gen1,2, XU Jian-Tang1,2, TAO Ai-Fen1,2, FANG Ping-Ping1,2, QI Jian-Min1,2, ZHANG Li-Wu1,2,*()   

  1. 1Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops, Fujian Agriculture and Forestry University / Key Laboratory of Ministry of Agriculture and Rural Affairs for Biological Breeding of Fujian and Taiwan Crops / Fujian Key Laboratory for Crop Breeding by Design, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
    2Experiment Station of Ministry of Agriculture and Rural Affairs for Jute and Kenaf in Southeast China, Fujian Agriculture and Forestry University / Public Platform of Fujian for Germplasm Resources of Bast Fiber Crops / Fujian International Science and Technology Cooperation Base for Genetics, Breeding and Multiple Utilization Development of Southern Economic Crops, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
  • Received:2023-05-17 Accepted:2023-09-13 Published:2024-03-12 Published online:2023-09-28
  • Contact: *E-mail: lwzhang@fafu.edu.cn; zhang_liwu@hotmail.com
  • Supported by:
    National Natural Science Foundation of China(31972968);China Agriculture Research System of MOF and MARA(CARS-16)

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摘要：

MYB类转录因子KAN4 (KANADI4)在红麻类黄酮合成和纤维发育中发挥着重要作用。本研究以红麻品种‘福红952’为材料, 对HcKAN4基因进行克隆和表达模式分析, 探讨TRV-VIGS诱导该基因沉默对类黄酮合成途径中关键酶基因表达量改变的影响。基因克隆显示, HcKAN4基因开放阅读框(open reading frame, ORF)全长为966 bp, 编码322个氨基酸, 包含一个MYB保守结构域。进化树分析发现, 其与拟南芥和木槿KAN4s的亲缘关系较近。表达分析表明, 该基因在红麻不同组织中均有表达, 且转录水平随着植物生长而递增。VIGS诱导基因沉默显示, 6株HcKAN4的转录水平显著下调, 达到基因沉默效果。进一步实时荧光定量PCR检测发现, 类黄酮合成相关基因HcCHS、HcF3’5’H、HcANS、HcANR的转录水平显著下调, 分别是对照组的0.51、0.14、0.23、0.11倍, 表明HcKAN4基因可调控红麻类黄酮生物合成相关基因。这些结果为阐明红麻MYB转录因子调控类黄酮合成提供了依据, 同时为改善纤维品质提供了研究思路。

关键词: 红麻, HcKAN4, 类黄酮合成, VIGS

Abstract:

MYB-like transcription factor KAN4 (KANADI4) plays an important role in flavonoid synthesis and fiber development in kenaf. In this study, the variety “Fuhong 952” was used as the experimental material to clone and analyze the relative expression pattern of HcKAN4 genes, and to investigate the effect of TRV-VIGS-induced silencing of HCKAN4 gene on the expression of key enzyme genes in flavonoid synthesis pathway in kenaf, the variety ‘Fuhong 952’ was used as the experimental material. Gene cloning showed that the total length of ORF of HcKAN4 gene was 966 bp, encoding 322 amino acids and containing a conserved domain of MYB. Phylogenetic tree revealed that it was closely related to KAN4s of Arabidopsis and Hibiscus. The relative expression pattern indicated that the gene was expressed in different plant tissues, and the transcription level increased with plant growth in kenaf. VIGS-induced gene silencing revealed that the transcription level of 6 HcKAN4 individuals was significantly down-regulated, indicating gene silencing successfully. Real-time quantitative PCR demonstrated that the relative level of flavonoid synthesis-related genes, HcCHS, HcF3'5'H, HcANS, and HcANR, were significantly down-regulated, which were 0.51, 0.14, 0.23, and 0.11 times of those in the control group, respectively, suggesting that the relative expression level of HcKAN4 gene can regulate the biosynthesis of flavonoid in kenaf. These results provide a basis for clarifying the regulation of flavonoid synthesis by MYB transcription factor and give research ideas for improving fiber quality in kenaf.

Key words: kenaf, HcKAN4, flavonoid synthesis, VIGS

附表1

本试验所用引物"

引物名称
Prime name		 引物序列
Prime sequences (5°-3°)		 扩增长度Amplification length (bp) 用途
Usage
HcKAN4-F ACACTTGAGAAGAGACAACTCGT 966 基因克隆
Gene cloning
HcKAN4-R ATGATGAGAACCGCACTTTT
HcKAN4-SF GGGGACCACTTTGTACAAGAAAGCTGGGTCTCCATCCAAATCAACAATCC 254 基因沉默片段克隆
Fragment cloning of gene silencing
HcKAN4-SR GGGGACAAGTTTGTACAAAAAAGCAGGCTTCAAGCGTCAGAGCTCCCAG
pDONR207-F GTAACATCAGAGATTTTGAGACAC 250 载体检测
Vector detection
pDONR207-R TCGCGTTAACGCTAGCATGGATCTC
ATTB1-F GGGGACAAGTTTGTACAAAAAAGCAGGCTTC 250 pTRV1 and pTRV2
ATTB1-R GGGGACCACTTTGTACAAGAAAGCTGGGTC
Hc18sRNA-F GGTTCACCTACGGAAACCTTG 175 qRT-PCR检测引物
Detection primers of qRT-PCR
Hc18sRNA-R CTACGTCCCTGCCCTTTGTA
HcKAN4-q-F TTTTGCCTGATCTATCCCTGC 145
HcKAN4-q-R AAATCGCTTCCACTACTCCCA
HcCHS-F GCACAAAGAGCCGAGGGTC 103
HcCHS-R TTCGCTGTTGGTGATACGGA
HcF3’5’H-F GCTAAGGCAGGCAGAAAGAGG 237
HcF3’5’H-R AGTGGGAAAAACGCCAAAATC
HcF3’H-F TTCAAGAACTGAGCCGCCA 136
HcF3’H-R TTGCCGCATATGAGCCCTA
HcANS-F GAGCAAAGGTACGAGGAGGGA 173
HcANS-R CAGCAACGGCAAGTTCAAGAG
HcANR-F CCCTTGCCCTTCAAATACTCC 171
HcANR-R GTTCCTCCACAAAAGATACCCG

图1

红麻HcKAN4基因克隆及序列分析 a: HcKAN4基因克隆电泳图。其中, M表示DL2000 DNA marker, 1和2表示克隆产物。b: HcKAN4蛋白与其他物种同源蛋白序列比对图。其中红框表示MYB保守结构域, HcKAN4表示红麻KAN4蛋白, HsKAN4表示木槿KAN4蛋白, GhKAN4表示棉花KAN4蛋白, DzKAN4表示榴莲KAN4蛋白, VvKAN4表示葡萄KAN4蛋白。c: HcKAN4基因结构示意图。"

附图1

不同植物KAN4蛋白系统进化树 CaKAN4: 辣椒KAN4蛋白; ZmKAN4: 玉米KAN4蛋白; BaKAN4: 油菜KAN4蛋白; CsKAN4: 黄瓜蛋白; VvKAN4: 葡萄KAN4蛋白; HsKAN4: 木槿KAN4蛋白; ATKAN4: 拟南芥KAN4蛋白; HcKAN4: 红麻KAN4蛋白; GaKAN4: 棉花KAN4蛋白; DzKAN4: 榴莲KAN4蛋白。"

附图2

Hc.KAN4蛋白跨膜结构预测"

图2

红麻HcKAN4基因在不同时期不同组织中的表达分析 root-60 d、stem-stick-60 d、leaf-60 d、stem-bark-60 d、stem-bark- 120 d分别代表种子萌发60 d后根、茎骨、叶、茎皮、和种子萌发120 d后茎皮; R、B3、B5、B7分别代表种子萌发120 d后圆叶、三裂叶、五裂叶和七裂叶。"

图3

红麻Hc.KAN4基因重组载体质粒转化后菌液PCR检测 M: DL2000 DNA marker。a: P代表质粒, 1~4代表pTRV2-KAN4待测农杆菌; b: 1~5代表pTRV1待测农杆菌; c: 1~5代表pTRV2 (空载)待测农杆菌。"

图4

红麻HcKAN4的cDNA和氨基酸序列 红色线标出沉默片段。"

图5

HcKAN4基因沉默后红麻植株的沉默效果检测 a: VIGS沉默后叶片HcKAN4基因的qRT-PCR分析; b: VIGS沉默后茎皮HcKAN4基因的qRT-PCR分析。柱上不同小写字母表示同一基因在不同植株表达量差异达到显著水平(P < 0.05)。"

表1

NCBI (CDD)鉴定红麻类黄酮合成基因及其结构域"

基因简称
Gene abbreviation		 红麻数据库编号
Database number of H. cannabinus L.		 结构域
Domain		 CDD (蛋白Protein)
区间 Interval E值 E-value
Hc.CHS Hc.02G012910 PLN03172 super family 36-424 0
Hc.F3’H Hc.17G017480 p450 super family 23-510 0
Hc.ANR Hc.12G017890 PLN00198 super family 1-335 0
Hc.F3’5’H Hc.10G006460 p450 super family 6-508 0
Hc.ANS Hc.11G024480 PLN03178 2-355 0

图6

红麻类黄酮合成相关基因在不同时期茎皮的表达分析 Hypocotyl-10 d: 种子萌发后10 d的下胚轴; stem-bark-60 d: 种子萌发后60 d的茎皮; stem-bark-120 d: 种子萌发后120 d的茎皮。柱上不同小写字母表示同一基因在不同时期表达量差异达到显著水平(P < 0.05)。"

图7

红麻纤维素和木质素合成相关基因在不同时期茎皮的表达量 a: 表示木质素合成相关基因在不同时期茎皮的表达量; b: 表示纤维素合成相关基因在不同时期茎皮的表达量; Hypocotyl-10 d: 种子萌发后10 d的下胚轴; stem-bark-60 d: 种子萌发后60 d的茎皮; stem-bark-120 d: 种子萌发后120 d的茎皮。柱上不同小写字母表示同一基因在不同时期表达量差异达到显著水平(P < 0.05)。"

图8

红麻HcKAN4基因VIGS沉默后类黄酮合成相关基因的qRT-PCR分析 每组数据代表3次技术重复的平均值, 误差以SD表示; *表示在0.05概率水平差异显著, **表示在0.01概率水平差异显著。HcCHS: 查尔酮合成酶基因; HcF3’H: 类黄酮3’-羟化酶基因; HcF3’5’H: 类黄酮3’5’-羟化酶基因; HcANS: 花青素合成酶基因; HcANR: 花青素还原酶基因。"

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