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作物学报 ›› 2022, Vol. 48 ›› Issue (11): 2774-2785.doi: 10.3724/SP.J.1006.2022.14189

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

小豆Dirigent基因家族鉴定及锈菌侵染对不同成员表达的影响

柯希望(), 苑梦琦(), 徐晓丹, 殷丽华, 郭永霞, 左豫虎*()   

  1. 黑龙江八一农垦大学农学院 / 黑龙江省作物有害生物互作生物学及生态防控重点实验室 / 国家杂粮工程技术研究中心,黑龙江大庆 163319
  • 收稿日期:2021-10-18 接受日期:2022-02-25 出版日期:2022-11-12 网络出版日期:2022-03-23
  • 通讯作者: 左豫虎
  • 作者简介:第一作者联系方式: 柯希望, E-mail: kexylh@163.com
    苑梦琦: E-mail: 2896834372@qq.com第一联系人：** 同等贡献。
  • 基金资助:
    本研究由国家自然科学基金项目(32102173);黑龙江省自然科学基金项目(YQ2020C034);黑龙江八一农垦大学研究生创新项目(YJSCX2021-Y50)

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 Published:2022-11-12 Published online:2022-03-23
  • Contact: ZUO Yu-Hu
  • 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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摘要：

为明确Dirigent (DIR)基因在小豆抗锈病中的作用,采用生物信息学方法,对小豆DIR基因家族(VaDIRs)进行了全基因组鉴定,结果发现小豆中共有33个DIR基因,其中29个成员分别定位在8条染色体上,系统进化分析表明,VaDIRs包括DIR-a、DIR-b/d、DIR-e和DIR-f四个亚家族,对VaDIRs成员启动子区顺式作用元件的分析发现,VaDIRs启动子区均包含激素、病原体应答等元件。对小豆抗锈病品种接种后不同时间的转录组数据分析发现,差异表达基因中共有17个VaDIRs成员,其中6个于接种后24 h显著上调,2个于接种后24 h和48 h均高量表达。进一步应用qRT-PCR技术对上述8个基因在抗、感品种中应答锈菌侵染的表达分析表明,高抗品种中VaDIR14、VaDIR16和VaDIR33的表达量在锈菌侵染不同阶段均显著高于感病品种。上述结果表明,VaDIRs家族成员可作为正调节因子参与小豆对锈病的抗性。

关键词: 小豆(Vigna angularis), 小豆锈病, 豇豆单胞锈菌(Uromyces vignae), DIR基因, 基因家族

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

表1

小豆DIR基因家族成员信息"

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

图1

小豆DIR基因家族染色体定位 由线相连的2个基因为基因对, 由线相连的多个基因表示多个基因构成的基因簇。NW_016114851.1、NW_016115358.1和NW_016115450.1为未组装到染色体上的scaffolds。"

图2

采用最大似然法构建的小豆与其他植物DIR基因家族成员的系统发育树 蓝色代表DIR-a, 绿色代表DIR-b/d, 玫红色代表DIR-c, 紫色代表DIR-e, 黄色代表DIR-f, 粉色代表DIR-g。系统发育树上的数字为bootstrap值。"

图3

小豆DIR基因家族的基因结构与蛋白保守基序分布 A: 保守结构域; B: 基因内含子-外显子结构。UTR: 非编码区; CDS: 蛋白编码序列; —: 内含子。"

图4

小豆DIR基因家族启动子顺式作用元件分布 TGA-element: 生长素响应元件; Myb: 参与植物的细胞分化、激素和环境因子的应答; as-1: 生长素和水杨酸响应元件; ERE: 乙烯响应元件; ABRE: 脱落酸响应元件; TGACG-motif: 茉莉酸甲酯响应元件; CGTCA-motif: 茉莉酸甲酯响应元件; TC-rich repeats: 防御和应激反应中涉及到的顺式元件; ARE: 厌氧诱导的必须调控元件; W-box: 诱导子、受伤及病原体应答, 结合WRKY类转录因子; TATC-box: 赤霉素响应元件; P-box: 赤霉素响应元件; WUN-motif: 创伤应答元件; LTR: 低温响应元件; TCA-element: 水杨酸响应元件; GARE-motif: 赤霉素响应元件; SARE: 水杨酸响应元件; AuxRR-core: 生长素响应元件。"

图5

小豆DIR基因家族成员在响应锈菌侵染下的表达量分析 图中数值为log2 ratio的值, log2 ratio=log2 (接种后表达量/对照表达量), 正值为接种后24 h或48 h显著上调表达, 负值为显著下调表达。"

图6

抗、感不同小豆品种接种锈菌后10 d的发病情况及叶面夏孢子堆数量统计 A, B: “QH1”和“BQH”接种锈菌后10 d的发病情况; C: 不同品种接种后10 d叶面夏孢子堆数统计, *表示差异显著(P < 0.05)。BQH: 宝清红。"

表2

用于qRT-PCR分析的VaDIRs引物和内参基因的引物"

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

图7

小豆DIR基因家族部分成员在锈菌侵染下的定量表达分析 R: 高抗锈病品种“QH1”; S: 高感品种“BQH”; Inoculation: 接种锈菌夏孢子; *表示同一时间不同处理差异显著(P < 0.05)。BQH: 宝清红。"

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