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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (3): 645-655.doi: 10.3724/SP.J.1006.2024.34084

• CROP GENETICS & BREEDINGZ·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2024-03-12 Published: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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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

Table S1

All primers used in this study"

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

Fig. 1

Cloning and sequence analysis of HcKAN4 gene in kenaf a: the electrophoretic map of HcKAN4 gene cloning. M: DL2000 DNA marker; 1 and 2 represent cloning products. b: the sequence alignment diagram of HcKAN4 protein with homologous proteins of other species. The red box represents MYB conserved domain, HcKAN4 represents kenaf KAN4 protein, HsKAN4 represents hibiscus KAN4 protein, GhKAN4 represents cotton KAN4 protein, DzKAN4 represents durian KAN4 protein, and VvKAN4 represents grape KAN4 protein. c: the schematic diagram of HcKAN4 gene structure."

Fig. S1

Phylogenetic tree of KAN4 proteins in different plants CaKAN4: pepper KAN4 protein; ZmKAN4, maize KAN4 protein; BaKAN4, rapeseed KAN4 protein; CsKAN4 cucumber protein; VvKAN4, grape KAN4 protein; HsKAN4: hibiscus KAN4 protein; ATKAN4, Arabidopsis KAN4 protein; HcKAN4: kenaf KAN4 protein; GaKAN4: cotton KAN4 protein; DzKAN4: durian KAN4 protein."

Fig. S2

Prediction of the transmembrane structure of Hc.KAN4 protein"

Fig. 2

Relative expression level of HcKAN4 genes of different tissues at different stages in kenaf root-60 d, stem-stick-60 d, leaf-60 d, stem-bark-60 d, and stem-bark- 120 d represent root, stem stick, leaf, stem bark after 60 days of seed germination, and stem bark after 120 days of seed germination, respectively. R, B3, B5, and B7 represent round leaf, tri-lobed leaf, penta-lobed leaf, and septi-lobed leaf after 120 days of seed germination, respectively."

Fig. 3

PCR detection of Hc.KAN4 recombinant plasmid transformation in kenaf M: DL2000 DNA marker. a: P represents plasmid, 1-4 represents Agrobacterium pTRV2-KAN4 to be tested; b: 1-5 represents Agrobacterium pTRV1 to be tested; c: 1-5 represents Agrobacterium pTRV2 (mock) to be tested."

Fig. 4

cDNA and amino acid sequences of HcKAN4 in kenaf The silent fragment is underlined by the red line."

Fig. 5

Silencing effect detection of individuals after HcKAN4 gene silencing in kenaf a: the relative expression level of HcKAN4 genes in leaves after VIGS silencing; b: the relative expression level of HcKAN4 genes in stem bark after VIGS silencing. Different lowercase letters above the bars indicate significant difference at the 0.05 probability level in the relative expression level of the same gene at different individuals."

Table 1

Genes related to flavonoid synthesis and their domains identified by NCBI (CDD)"

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

Fig. 6

Relative expression level of genes related to flavonoid biosynthesis at different stages in kenaf Hypocotyl-10 d: hypocotyl of 10 days after seed; stem-bark60 d: stem barks of 60 days after seed; stem-bark-120 d: stem barks of 120 days after seed. Different lowercase letters above the bars indicate that there is significant difference at the 0.05 probability level in the relative expression level of the same gene at different stages."

Fig. 7

Relative expression level of genes related to cellulose and lignin biosynthesis at different stages in kenaf a: the relative expression level of lignin synthesis-related genes in stem bark at different stages; b: the relative expression level of cellulose synthesis related genes in stem bark at different stages; Hypocotyl-10 d: hypocotyl of 10 days after seed; stem-bark60 d: stem barks of 60 days after seed; stem-bark-120 d: stem barks of 120 days after seed. Different lowercase letters above the bars indicate that there is significant difference at the 0.05 probability level in the relative expression level of the same gene at different stages."

Fig. 8

Relative expression level of flavonoid synthesis-related genes after VIGS silencing of HcKAN4 gene in kenaf Each group of data represents the mean of 3 technical replicates, and the error is expressed in SD; * and ** mean significant differences at the 0.05 and 0.01 probability levels, respectively. HcCHS: chalcone synthetase gene; HcF3'H: flavonoid 3’-hydroxylase gene; HcF3'5H: flavonoid 3’5’-hydroxylase gene; HcANS: anthocyanin synthase gene; HcANR: anthocyanin reductase gene."

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