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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (11): 2774-2785.doi: 10.3724/SP.J.1006.2022.14189

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

Dirigent gene family identification and expression profiling of Vigna angularis responsive to Uromyces vignae infection

KE Xi-Wang(), YUAN Meng-Qi(), XU Xiao-Dan, YIN Li-Hua, GUO Yong-Xia, ZUO Yu-Hu*()   

  1. College of Agriculture, Heilongjiang Bayi Agricultural University / Heilongjiang Provincial Key Laboratory of Crop-Pest Interaction Biology Ecological Control / National Coarse Cereals Engineering Research Center, Daqing 163319, Heilongjiang, China
  • Received:2021-10-18 Accepted:2022-02-25 Online:2022-11-12 Published:2022-03-23
  • Contact: ZUO Yu-Hu E-mail:kexylh@163.com;2896834372@qq.com;zuoyhu@163.com
  • Supported by:
    The National Natural Science Foundation of China(32102173);The Heilongjiang Provincial Natural Science Foundation of China(YQ2020C034);The Innovative Research Program for Graduates of Heilongjiang Bayi Agricultural University(YJSCX2021-Y50)

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Abstract:

To clarify the role of Dirigent (DIR) gene family of adzuki bean (Vigna angularis) in response to Uromyces vignae infection, genome-wide identification of the DIR family genes of V. angularis (VaDIRs) was conducted. A total of 33 VaDIRs in the V. angularis genome were identified, among which 29 members were located on 8 chromosomes, respectively. Phylogenetic analysis showed that VaDIRs consisted of 4 subfamilies (DIR-a, DIR-b/d, DIR-e, and DIR-f). Cis-acting elements analysis of the promoter of VaDIRs indicated that all members of VaDIRs contained hormones and pathogen responsive elements. Transcriptomic data of the rust resistant cultivar at different times after inoculation revealed that 17 VaDIRs members were differentially expressed, among which 6 VaDIRs were significantly upregulated at 24 hours post inoculation (hpi), and 2 members were highly expressed both at 24 and 48 hpi. The relative expression of the genes mentioned above were further confirmed in different resistant cultivars responsive to U. vignae infection by qRT-PCR. Results demonstrated that VaDIR14, VaDIR16, and VaDIR33 in resistant cultivar were significantly induced than those in the susceptible cultivar during the fungal infection. In short, our results indicated that members of VaDIRs could be used as positive regulators in resistant cultivar to prevent U. vignae infection.

Key words: Vigna angularis, adzuki bean rust, Uromyces vignae, DIR gene, gene family

Table 1

Information of DIR gene family members in Vigna angularis"

基因名称
Gene name		 蛋白序列号
Protein sequence		 基因组注释
Definition		 染色体
Chr.		 分子量
Molecular
weight (kD)		 等电点
pI		 氨基酸数量
Number of the amino acids (aa)
VaDIR1 XP_017433474.1 未知蛋白Uncharacterized protein 01 19.78 9.16 185
VaDIR2 XP_017433817.1 类抗病反应蛋白206
Disease resistance response protein 206-like		 01 20.35 6.28 182
VaDIR3 XP_017434121.1 类引导蛋白11 Dirigent protein 11-like 01 19.98 6.05 180
VaDIR4 XP_017414154.1 类引导蛋白22 Dirigent protein 22-like 02 20.79 7.07 190
VaDIR5 XP_017414268.1 类引导蛋白16 Dirigent protein 16-like 02 25.10 4.87 245
VaDIR6 XP_017412750.1 类引导蛋白16 Dirigent protein 16-like 02 27.37 5.10 266
VaDIR7 XP_017417575.1 类引导蛋白24 Dirigent protein 24-like 03 45.28 4.74 431
VaDIR8 XP_017416488.1 类引导蛋白25 Dirigent protein 25-like 03 24.22 5.78 224
VaDIR9 XP_017417865.1 类引导蛋白22 Dirigent protein 22-like 03 21.15 9.49 194
VaDIR10 XP_017416739.1 类引导蛋白22 Dirigent protein 22-like 03 20.98 9.40 192
VaDIR11 XP_017417602.1 类引导蛋白22 Dirigent protein 22-like 03 20.47 9.01 188
VaDIR12 XP_017417783.1 类引导蛋白2 Dirigent protein 2-like 03 20.34 5.22 186
VaDIR13 XP_017417792.1 类引导蛋白2 Dirigent protein 2-like 03 20.34 5.22 186
VaDIR14 XP_017417756.1 类引导蛋白22 Dirigent protein 22-like 03 24.04 6.59 216
VaDIR15 XP_017417215.1 类引导蛋白22 Dirigent protein 22-like 03 24.02 6.37 216
VaDIR16 XP_017417998.1 类引导蛋白22 Dirigent protein 22-like 03 24.17 7.07 216
VaDIR17 XP_017424454.1 类引导蛋白21 Dirigent protein 21-like 05 20.53 5.67 186
VaDIR18 XP_017426991.1 类引导蛋白11 Dirigent protein 11-like 06 21.11 8.83 196
VaDIR19 XP_017432020.1 类引导蛋白19 Dirigent protein 19-like 08 14.76 6.72 134
VaDIR20 XP_017432805.1 类引导蛋白2 Dirigent protein 2-like 08 19.32 9.45 177
VaDIR21 XP_017432829.1 类引导蛋白2 Dirigent protein 2-like 08 18.78 8.09 175
VaDIR22 XP_017438130.1 类引导蛋白22 Dirigent protein 22-like 10 15.03 6.32 135
VaDIR23 XP_017441922.1 类抗病反应蛋白206
Disease resistance response protein 206-like		 11 21.32 8.58 189
VaDIR24 XP_017441955.1 类抗病反应蛋白206
Disease resistance response protein 206-like		 11 20.22 6.95 183
VaDIR25 XP_017441660.1 类引导蛋白22 Dirigent protein 22-like 11 21.49 9.32 192
VaDIR26 XP_017441835.1 类引导蛋白22 Dirigent protein 22-like 11 21.02 9.25 189
VaDIR27 XP_017442075.1 类引导蛋白22 Dirigent protein 22-like 11 21.17 9.30 192
VaDIR28 XP_017442076.1 类引导蛋白22 Dirigent protein 22-like 11 20.49 9.30 187
VaDIR29 XP_017441889.1 类引导蛋白22 Dirigent protein 22-like 11 20.43 6.05 188
VaDIR30 XP_017411398.1 类引导蛋白9 Dirigent protein 9-like / 26.77 6.30 248
VaDIR31 XP_017411409.1 类引导蛋白10 Dirigent protein 10 like / 31.22 4.88 296
VaDIR32 XP_017405347.1 类引导蛋白22 Dirigent protein 22-like / 21.78 6.97 190
VaDIR33 XP_017411321.1 未知蛋白 Uncharacterized protein / 18.71 6.28 173

Fig. 1

Chromosome distribution of DIR gene family in Vigna angularis Two genes linked by the line indicate a gene pair, and multiple genes linked by the line indicate a gene cluster. NW_016114851.1, NW_016115358.1, and NW_016115450.1 are the scaffolds that are not assembled into chromosomes."

Fig. 2

Phylogenetic tree of DIR family genes from Vigna angularis and other plants constructed by maximum likelihood method Blue represents DIR-a, green represents DIR-b/d, rose red represents DIR-c, purple represents DIR-e, yellow represents DIR-f, and pink represents DIR-g. The number on phylogenetic tree represents the bootstrap value."

Fig. 3

Gene structure and conserved motif distribution of DIR gene family in Vigna angularis A: conserved domain; B: the intron-exon structure of the gene. UTR: non-coding region; CDS: protein-coding sequence; —: intron."

Fig. 4

Distribution of cis-acting elements of promoter of DIR gene family in Vigna angularis TGA-element: auxin-responsive element; Myb: involved in plant cell differentiation, hormonal and environmental factor responses; as-1: auxin and salicylic acid responsive elements; ERE: ethylene response element; ABRE: cis-acting element involved in the abscisic acid responsiveness; TGACG-motif: MeJA-responsive; CGTCA-motif: MeJA-responsive; TC-rich repeats: cis-acting element involved in defense and stress responsiveness; ARE: cis-acting regulatory element essential for the anaerobic induction; W-box: the inducer, injury, and pathogen responses bind to WRKY class transcription factors; TATC-box: cis-acting element involved in gibberellin-responsiveness; P-box: gibberellin-responsive element; WUN-motif: wound-responsive element; LTR: cis-acting element involved in low-temperature responsiveness; TCA-element: cis-acting element involved in salicylic acid responsiveness; GARE-motif: gibberellin-responsive element; SARE: cis-acting element involved in salicylic acid responsiveness; AuxRR-core: cis-acting regulatory element involved in auxin responsiveness."

Fig. 5

Relative expression level analysis of DIR gene family members in Vigna angularis in response to rust infection The value in the figure is the value of log2 ratio, log2 ratio = log2 (post-inoculation expression/control expression), positive value means significantly up-regulated expression at 24 or 48 hours post-inoculation (hpi), negative value means significantly down- regulated expression."

Fig. 6

Symptoms of different adzuki bean varieties at 10 days post inoculated with U. vignae and number of uredia on leaf surface statistics A, B: symptoms of “QH1” and “BQH” at 10 days post inoculation; C: number of the uredia on leaf surface of different varieties at 10 days post inoculation, *: P < 0.05. BQH: Baoqinghong."

Table 2

Primers for VaDIRs and reference genes for qRT-PCR"

基因
Gene		 上游引物
Upstream primer (5°-3°)		 下游引物
Downstream primers (5°-3°)		 产物长度
Product length (bp)
VaDIR9 GGAAAGGCCCAAGGTGTTTA GCGTGCTGCCATTGTATTTC 103
VaDIR12 CGTCACCGCCACTTACTATC GTCGTGGAAGAAGAAGTGAAGA 91
VaDIR14 GCCACCTTCTACCAAACTATCT GGAGGGTTTGTCGTTTGTTATG 105
VaDIR15 GACCCACTGACCATCGGACCTG CTGCGAGCAAGAACAAAGCCAC 260
基因
Gene		 上游引物
Upstream primer (5°-3°)		 下游引物
Downstream primers (5°-3°)		 产物长度
Product length (bp)
VaDIR16 TCTTGTTTCTGGCACCTTCTAC GTCTGATCGTCAATGGGTTCTC 206
VaDIR18 CCCATCTCAGCCACAACAACCG CACTTCCTTCCACGCCATTCAC 129
VaDIR28 AGGTGGTCGGAAGAATTGAG GCTGCTGCCATTGTACTTTC 110
VaDIR33 ATGTTTCAGCAATCGGCGTTCA CAAGACCACGAGCAAAGGCAAA 148
VaACT CTAAGGCTAATCGTGAGAA CGTAAATAGGAACCGTGT 165

Fig. 7

Relative expression level of some members of DIR gene family in Vigna angularis under rust infection R: the highly resistant variety “QH1”, S: the highly susceptible variety “BQH”, Inoculation: the samples inoculated with urediospores of U. vignae, * indicates significant difference at P < 0.05 in different treatments at the same time. BQH: Baoqinghong."

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