向日葵YABBY基因家族鉴定及表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0139

摘要/Abstract

摘要：

【目的】鉴定向日葵中花发育相关的HaYABBY基因，有助于探究其在向日葵花发育过程中的生物学功能和调控机制，为向日葵品种选育提供重要的分子线索。【方法】基于向日葵基因组和转录组数据，使用生物信息学方法，分析向日葵YABBY基因家族成员的染色体定位、系统进化关系、基因结构、保守基序、蛋白结构及理化性质、启动子顺式作用元件和组织表达模式等，并通过RT-qPCR分析HaYABBY基因在WT向日葵和花分生组织决定和花器官分化缺陷突变体cb1中st2-st8时期的表达差异。【结果】在向日葵基因组中鉴定到12个YABBY基因家族成员，分属于4个亚家族，分布在8条染色体上，并且同一亚家族中成员在基因结构和保守基序组成上高度相似。其中8个基因HaYABBY1a、HaYABBY1c、HaYABBY3、HaYABBY4b、HaYABBY5a、HaYABBY5b、HaCRABS CLAWa、HaCRABS CLAWb在向日葵花发育时期特异性高表达。在这8个基因中，HaYABBY3、HaYABBY5a和HaYABBY5d在突变体cb1花发育后期表达上调，且与WT花发育早期水平相当；而HaYABBY4b和HaCRABS CLAWa在cb1中的表达被强烈抑制。顺式作用元件分析显示，HaYABBY均含有多个启动子顺式作用元件，部分元件仅存在于特定基因中。其中，胚乳表达元件存在于HaCRABS CLAWa和HaYABBY4a中；生长素响应元件存在于HaCRABS CLAWa、HaCRABS CLAWb、HaYABBY4a、HaYABBY4b和HaYABBY5c中；分生组织表达元件存在于HaYABBY1a、HaYABBY1c、HaYABBY4b、HaYABBY5a和HaYABBY5d中；赤霉素响应元件存在于HaCRABS CLAWa、HaYABBY5b、HaYABBY5c和HaYABBY5d中；种子特异性调控元件存在于HaYABBY5a、HaYABBY5c和HaYABBY5d中。【结论】HaYABBY3、HaYABBY4b、HaYABBY5a、HaYABBY5d和HaCRABS CLAWa等5个HaYABBY基因在花分生组织的转变和花器官发生过程中可能发挥重要作用，它们在花分生组织转变和花器官发生调控中可能受到生长素响应因子ARF、赤霉素响应因子MYB以及MADS-box等关键因子调节。

关键词: 向日葵, YABBY基因家族, 花发育, 花分生组织转变, 花器官发生, 基因表达

Abstract:

【Objective】The identification of HaYABBY gene related to flower development in sunflower（Helianthus annuus L.）is conductive to exploring its biological function and regulatory mechanism in the floral development of sunflower, and to provide important molecular clues for sunflower variety breeding.【Method】Based on sunflower genome database and our transcriptome data, bioinformatics analysis was conducted to analyze the physicochemical properties, chromosome localization, phylogenetic relationship, gene structure, conserved motif, protein structure, promoter cis-action regulatory element, and tissue expression pattern, etc. RT-qPCR was used to analyze the differences of HaYABBY gene expression between WT sunflower and cb1（crazy broccoli 1）mutant with the defection of meristem determinacy and floral organ differentiation at st2-st8.【Result】Twelve members of YABBY family were identified, belonging to 4 subfamilies, and located in 8 chromosomes； meanwhile, the members in the same subfamily shared similar gene structure and conserved motifs. Results of tissue expression pattern showed that 8 members presented high expressions during flower development in sunflower, including HaYABBY1a, HaYABBY1c, HaYABBY3, HaYABBY4b, HaYABBY5a, HaYABBY5b, HaCRABS CLAWa, and HaCRABS CLAWb, among which HaYABBY3, HaYABBYY5a, and HaYABBYY5d expressed with up-regulation at the late stages of flower development in cb1 mutant; while the expression of HaYABBYY4b and HaCRABS CLAWa was strongly inhibited in cb1 mutants, corresponding to their expressions at early floral developmental in WT plants. The analysis of cis-regulatory elements showed that all the HaYABBY members contained multiple promoter cis-acting elements, some acting elements only existing in specific genes. Among them, endosperm expression elements were presented in HaCRABS CLAWa and HaYABBY4a. Auxin responders were in HaCRABS CLAWa, HaCRABS CLAWb, HaYABBY4a, HaYABBY4b and HaYABBY5c. Meristem expression elements existed in HaYABBY1a, HaYABBY1c, HaYABBY4b, HaYABBY5a, and HaYABBY5d. Gibberellin response elements were included in HaCRABS CLAWa, HaYABBY5b, HaYABBY5c, and HaYABBY5d. Seed-specific regulatory elements presented in HaYABBY5a, HaYABBY5c, and HaYABBY5d.【Conclusion】Five HaYABBY genes of HaYABBY3, HaYABBY4b, HaYABBY5a, HaYABBY5d and HaCRABS CLAW play important roles in the process of floral meristem transformation and floral organ formation in sunflower, and they might depend on the regulation of auxin, gibberellin and meristem expression related regulators during meristem transformation and floral organogenesis, and need the involvement of the auxin response factor ARFs, gibberellin response factor MYBs and flower organ development regulator MADS-boxes.

Key words: sunflower, YABBY gene family, flower development, floral meristem transformation, floral organogenesis, gene expression

王茜, 周家燕, 王倩, 邓玉萍, 张敏慧, 陈静, 杨军, 邹建. 向日葵YABBY基因家族鉴定及表达分析[J]. 生物技术通报, 2024, 40(8): 199-211.

WANG Qian, ZHOU Jia-yan, WANG Qian, DENG Yu-ping, ZHANG Min-hui, CHEN Jing, YANG Jun, ZOU Jian. Identification and Expression Analysis of the YABBY Gene Family in Sunflower[J]. Biotechnology Bulletin, 2024, 40(8): 199-211.

图/表 7

表1 向日葵YABBY基因家族成员信息

Table 1 Information of YABBY gene family members in sunflower

基因 Gene	基因编号 Gene ID	染色体分布Chromosome distribution			氨基酸数目 Number of amino acids	等电点 pI	分子量 Molecular weight/Da	亚细胞定位 Subcellular location
基因 Gene	基因编号 Gene ID	编号No.	开始位置Starting	结束位置Ending	氨基酸数目 Number of amino acids	等电点 pI	分子量 Molecular weight/Da	亚细胞定位 Subcellular location
HaYABBY 1a	HanXRQr2_Chr06g0245541	6	12 754 239	12 758 093	219	7.21	24 420.73	细胞核
HaYABBY 1b	HanXRQr2_Chr13g0613121	13	161 766 031	161 771 918	216	6.72	24 136.35	细胞核
HaYABBY 1c	HanXRQr2_Chr04g0185291	4	195 371 365	195 376 174	219	7.21	24 437.79	细胞核
HaCRABS CLAWa	HanXRQr2_Chr12g0549931	1	81 233 913	81 243 999	173	9.4	19 366.25	细胞核
HaCRABS CLAWb	HanXRQr2_Chr15g0688211	15	39 093 254	39 095 798	180	9.28	19 765.53	细胞核
HaYABBY 3	HanXRQr2_Chr13g0577751	13	57 727 478	57 739 518	225	9.16	24 724.41	细胞核
HaYABBY 4a	HanXRQr2_Chr17g0787111	17	15 387 218	15 388 714	197	4.98	21 878.58	细胞核
HaYABBY 4b	HanXRQr2_Chr06g0253091	6	30 699 177	3 700 575	236	6.4	26 603.38	细胞核
HaYABBY 4c	HanXRQr2_Chr03g0102331	3	78 930 712	78 931 672	99	7.82	10 936.49	细胞核
HaYABBY 5a	HanXRQr2_Chr17g0801501	17	53 868 011	53 872 537	227	8.55	25 164.39	细胞核
HaYABBY 5b	HanXRQr2_Chr10g0462601	10	175 418 675	175 424 195	200	9.74	22 555.59	细胞核
HaYABBY 5c	HanXRQr2_Chr12g0523971	12	5 127 939	5 135 711	186	9.67	21 053.74	细胞核
HaYABBY 5d	HanXRQr2_Chr06g0238981	6	1 174 948	1 181 807	229	8.5	25 139.60	细胞核

图1 向日葵YABBY基因的染色体定位

Fig. 1 Chromosome localization of YABBY genes in sunflower

图2 向日葵YABBY基因家族成员的系统发育树分析 ★代表拟南芥；●代表向日葵；▲代表菊花；■代表莴苣

Fig. 2 Phylogenetic tree analysis of YABBY gene family members in sunflower ★,●,▲ and ■indicate Arabidopsis, sunflower, Chrysanthemum morifolium and Lactuca sativa respectively

图3 YABBY家族成员的基因结构和保守基序 A：YABBY家族成员的保守基序图；B：YABBY家族成员的基因结构图；C：YABBY家族成员核心保守结构域氨基酸列比图

Fig. 3 Gene structure and conserved motifs for YABBY family members A: Mapping of conserved motifs for YABBY family members. B: Mapping of gene structure for YABBY family members. C: Amino acid sequence comparison of core conserved domain of YABBY family members

图4 YABBY基因在野生型（WT）向日葵中的组织表达模式分析 FPKM为YABBY基因家族转录丰度；表达组织：YR：幼根；YS：幼茎；YL：幼叶；R：根；S：茎；L：叶；表达时期：St2：花原基起始期；St3：花器官开始形成期；St4：减数分裂期；St5：花粉成熟期；St6：小花开放期；St7：果实灌浆期；St8：种子饱满期；小写字母表示差异显著（P<0.05）；下同

Fig. 4 Analysis of tissue expression pattern of YABBY gene in WT sunflower FPKM refers to transcriptional abundance of YABBY gene family. Tissues: YR: young root; YS: young stem; YL: young leaf; R: root; S: stem; L: leaf. Expression period: St2: flower primordium initiation stage; St3: flower organ formation stage; St4: meiosis stage; St5: pollen maturation stage; St6: florets opening stage; St7: fruit filling stage and St8 seed full stage. The lowercase letter indicates significant difference at P<0.05 level. The same below

图5 YABBY基因在野生型（WT）向日葵和cb1突变体中的差异表达分析 A：野生型和cb1突变体花序的表型特征；B：野生型和cb1突变体花序st2-st8表型特征；C：YABBY基因在WT和cb1花序St2-St8的相对表达量分析。所用数据为平均值±SD（n=3）；*：显著差异（P<0.05）；**：极显著差异（P<0.01）

Fig. 5 Differential expression analysis of YABBY gene in WT sunflower and cb1 mutant A: Phenotypic characteristics of WT sunflower and cb1 mutant inflorescence. B: Phenotypic characteristics of inflorescences in WT and cb1 mutant sunflower from St2-St8. C: Relative expression of YABBY gene in WT and cb1 during inflorescence development（st2-st8）. All data are mean ±SD（n=3）. *: Significant difference（P<0.05）; **: extremely significant difference（P<0.01）

图6 YABBY家族成员的启动子区域顺式作用元件分析

Fig. 6 Cis-acting element analysis of promoter region in YABBY family members

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