小麦叶枕器官控制因子LIGULELESS 2 的鉴定及其互作蛋白筛选

doi:10.7668/hbnxb.20196290

摘要/Abstract

摘要：

鉴定小麦叶枕控制因子LIGULELESS 2(LG2)并分析其互作蛋白,对探究小麦叶枕器官建成和叶夹角大小调控的分子机制及选育优良株型新品种提供理论依据具有重要意义。首先,通过构建系统发育树,在小麦基因组中鉴定到LG2的2个同源基因,分别命名为TaLG2和TaLG2L,且两者在A、B、D亚基因组均有3个拷贝同源基因,相似性达90%以上;基于转录组数据进行叶枕发育时期和叶片组织特异性表达分析,结果表明,TaLG2和TaLG2L均在叶枕器官中特异表达,且除TaLG2-3A外,其他5个同源基因均在叶枕发育早期S1期特异表达;构建诱饵载体pGBKT7-TaLG2L-1A,进行酵母双杂交(Y2H)筛库,共得到174个阳性克隆,经过测序分析共鉴定到68个小麦TaLG2L-1A的候选互作蛋白;GO功能富集分析显示,这些蛋白主要参与泛素结合、阳离子结合、mRNA结合等过程;进一步利用回转和Y2H一对一互作验证表明,小麦TaLG2L-1A能够分别与生长素响应因子TaARF17和蓝光受体隐花色素TaCRY1发生互作。明确了小麦LG2同源基因的组织特异性表达模式,筛选并获得TaLG2L-1A的关键互作蛋白TaARF17和TaCRY1,揭示了LG2可能通过协同生长素信号和光信号途径调控叶枕器官建成及叶夹角形成的分子机制。

关键词: 小麦, 叶枕, LIGULELESS 2, 表达模式, 酵母双杂交, 互作蛋白

Abstract:

The identification of the wheat lamina joint regulator LIGULELESS 2 (LG2) and analysis of its interacting proteins are of great significance for exploring the molecular mechanisms underlying lamina joint formation and leaf angle regulation, as well as for breeding new varieties with ideal plant architecture. First, two homologous genes of LG2, designated TaLG2 and TaLG2L, were identified in the wheat genome through phylogenetic analysis, each containing three homoeologous copies located in the A, B, and D subgenomes with more than 90% sequence similarity. Transcriptome-based expression profiling during lamina joint development and in different leaf tissues revealed that both TaLG2 and TaLG2L were specifically expressed in the lamina joint. Except for TaLG2-3A, the other five homoeologs exhibited specific expression at the early S1 stage of lamina joint development. The bait vector pGBKT7-TaLG2L-1A was constructed and used for yeast two-hybrid (Y2H) screening yielding 174 positive clones.Sequencing analysis identified 68 candidate interacting proteins of TaLG2L-1A. GO enrichment analysis showed that these proteins were mainly involved in ubiquitin binding, cation binding, and mRNA binding. Further rotation verification and pairwise Y2H assays demonstrated that TaLG2L-1A can interact with the auxin response factor TaARF17 and the blue light receptor cryptochrome TaCRY1, respectively. This study clarifies the tissue-specific expression patterns of wheat LG2 homologs, identifies and obtains key interacting proteins TaARF17 and TaCRY1 of TaLG2L-1A, and reveals the molecular mechanism by which LG2 may coordinate auxin and light signaling pathways to regulate lamina joint formation and leaf angle development.

Key words: Wheat, Lamina joint, LIGULELESS 2, Expression pattern, Yeast two-hybrid, Interaction proteins

中图分类号:

王一墨, 刘籽彤, 牛梓宇, 谷冰艳, 李丽, 宋怡蕾, 牛泽霖, 王荣纳. 小麦叶枕器官控制因子LIGULELESS 2 的鉴定及其互作蛋白筛选[J]. 华北农学报, 2026, 41(4): 24-33. doi: 10.7668/hbnxb.20196290.

WANG Yimo, LIU Zitong, NIU Ziyu, GU Bingyan, LI Li, SONG Yilei, NIU Zelin, WANG Rongna. Identification of LIGULELESS 2 Controlling the Lamina Joints and Screening of Its Interacting Proteins in Wheat[J]. Acta Agriculturae Boreali-Sinica, 2026, 41(4): 24-33. doi: 10.7668/hbnxb.20196290.

http://www.hbnxb.net/CN/Y2026/V41/I4/24

图/表 13

表1 引物信息

Tab.1 Primer information

引物名称 Primer name	序列信息(5'—3') Sequence information(5'—3')	用途 Purpose
TaLG2-F	ATGGTGCAAGGTGAGGAGGC	TaLG2基因克隆
TaLG2-R	TCAAAATCCGGAGAACTGATTCTGTTGC
TaLG2L-F	ATGAGTAGAGTTCTTCCACTCATTGCC	TaLG2L基因克隆
TaLG2L-R	TCAGAGGCCGGAGAACTGG
BD-TaLG2L-1A-F	CATGGAGGCCGAATTCATGAGTAGAGTTCTTCCACTCATTGCC	pGBKT7-TaLG2L-1A载体构建
BD-TaLG2L-1A-R	GCAGGTCGACGGATCCTCCGAGGCCGGAGAACTG
AD-TaWD-F	GGAGGCCAGTGAATTCATGTCCAATTATCACGACGATCAC	pGADT7-TaWD载体构建
AD-TaWD-R	CGAGCTCGATGGATCCTCAAAGCATCGAGTCATGGT
AD-TaCRY1-F	GGAGGCCAGTGAATTCATGTCGGTCTCGTCCTCCTC	pGADT7-TaCRY1载体构建
AD-TaCRY1-R	CGAGCTCGATGGATCCCTAACCGATGAAATTTGGCTGCACG
AD-TaARF17-F	GGAGGCCAGTGAATTCATGAGGCTCTCGTCGCCG	pGADT7-TaARF17载体构建
AD-TaARF17-R	CGAGCTCGATGGATCCTCAGAACTCTACTTCGACCGAGCC

图1 LG2蛋白的系统发育树及TaLG2L和TaLG2的克隆凝胶电泳 A.LG2蛋白在不同物种之间的系统发育树;B.TaLG2L和TaLG2的克隆凝胶电泳。

Fig.1 The phylogenetic tree and cloning of TaLG2L and TaLG2 A.Phylogenetic tree of LG2 proteins in different species;B.Clonal gel electrophoresis of TaLG2L and TaLG2.

图2 ZmLG2、OsLG2与TaLG2-3A、TaLG2-3B、TaLG2-3D的氨基酸序列对比

Fig.2 Amino acid sequence alignment of ZmLG2,OsLG2,TaLG2-3A,TaLG2-3B,and TaLG2-3D

图3 ZmLG2、OsLG2L与TaLG2L-1A、TaLG2L-1B、TaLG2L-1D的氨基酸序列对比

Fig.3 Amino acid sequence alignment of ZmLG2,OsLG2L,TaLG2L-1A,TaLG2L-1B,and TaLG2L-1D

图4 LG2在不同时期和不同组织的差异表达分析转录组数据分析中,FPKM代表Fragments per kilobase of transcript per million mapped reads,用于衡量基因表达水平,每个样品包含3个生物学重复。B和S表示S3时期(萌发后12 d)的叶片和叶鞘;LJ-S1、LJ-S2、LJ-S3表示小麦叶枕发育的S1时期(萌发后4 d)、S2时期(萌发后8 d)、S3时期。不同小写字母表示处理之间的差异显著(P<0.05)。

Fig.4 Differential expression analysis of LG2 in different periods and different tissues In the transcriptome analysis,FPKM(Fragments per kilobase of transcript per million mapped reads)was used to measure gene expression levels,each sample includes three biological replicates.B and S represent the leaf blade and leaf sheath,respectively at the S3 stage(12 d after germination);LJ-S1,LJ-S2,and LJ-S3 represent the S1 stage(4 d after germination),S2 stage(8 d after germination),and S3 stage during lamina joint(LJ)development,respectively.Different lowercase letters indicate significant differences among treatments(P<0.05).

图5 TaLG2L基因扩增产物(A)及酶切验证(B)

Fig.5 PCR detection of TaLG2L gene amplification product(A)and enzyme digestion verification(B)

图6 pGBKT7-TaLG2L-1A诱饵载体自激活检测(A)及其3AT 滴定结果(B)

Fig.6 pGBKT7-TaLG2L-1A decoy vector self-activation detection(A)and the 3AT titration results(B)

图7 TaLG2L-1A文库筛选转化效率

Fig.7 Conversion efficiency of TaLG2L-1A library screening

图8 阳性克隆四缺板(QDO/X)验证结果

Fig.8 The results of positive cloning were verified by four missing plates(QDO/X)

表2 酵母双杂交筛选的候选互作蛋白

Tab.2 Candidate interacting proteins identified through yeast two-hybrid screening

编号 Number	NCBI登录号 Accession	功能描述 Description
1	XM_044599280.1	类转录因子蛋白
2	XM_044592733.1	核糖体蛋白
3	XM_044583174.1	E3泛素蛋白连接酶
4	XM_044582607.1	转录共抑制因子
5	XM_044580925.1	含有Smr结构域的蛋白
6	XM_044568826.1	病原相关蛋白
7	XM_044563894.1	泛素相关蛋白
8	XM_044556763.1	激酶家族蛋白
9	XM_044539035.1	丝氨酸蛋白酶
10	XM_044532185.1	含锌指HIT结构域蛋白1
11	XM_044527595.1	ATP依赖性RNA解旋酶
12	XM_044509258.1	类同源框蛋白BEL1
13	XM_044475164.1	40S核糖体蛋白SA
14	XM_044601792.1	隐花色素-1
15	XM_044580216.1	生长素响应因子17
16	XM_044579331.1	过氧化氢酶
17	XM_044557948.1	含WD重复序列的类蛋白
18	XM_044509917.1	延伸因子
19	XM_044503465.1	锚蛋白重复家族蛋白
20	XM_044487387.1	脱落酸应激成熟蛋白

图9 GO注释分析纵坐标为-log₁₀(P-value),数值越高表示富集显著性越强;黄色柱代表细胞组分,蓝色柱代表分子功能。CC类主要富集条目:1.细胞内解剖结构;2.细胞质;3.核糖体;4.质体基质;5.质体;6.类囊体;7.胞质核糖体;8.类囊体膜;9.细胞器;10.膜结合细胞器;11.非膜结合细胞器;12.细胞内细胞器;13.细胞内膜结合细胞器;14.细胞内非膜结合细胞器;15.核蛋白复合物。MF类主要富集条目:16.泛素结合;17.泛素样蛋白结合;18.阳离子结合;19.mRNA结合;20.聚(A)RNA结合;21.金属离子结合;22.结构分子活性;23.转运金属离子结合;24.离子结合。

Fig.9 GO annotation analysis The ordinate represents-log₁₀(P-value),where a higher value indicates stronger enrichment significance.Yellow bars stand for cellular component(CC),and blue bars represent molecular function(MF).The main enriched terms in CC category include:1.Intracellular anatomical structure;2.Cytoplasm;3.Ribosome;4.Plastid stroma;5.Plastid;6.Thylakoid;7.Cytosolic ribosome;8.Thylakoid membrane;9.Organelle;10.Membrane-bounded organelle;11.Non-membrane-bounded organelle;12.Intracellular organelle;13.Intracellular membrane-bounded organelle;14.Intracellularnon-membrane-bounded organelle;15.Ribonucleoprotein complex.The main enriched terms in MF category include:16.Ubiquitin binding;17.Ubiquitin-like protein binding;18.Cation binding;19.mRNA binding;20.poly(A)RNA binding;21.Metal ion binding;22.Structural molecule activity;23.Transition metal ion binding;24.Ion binding.

图10 回转验证从左到右排列,第一行依次为阳性对照、阴性对照;第二行依次为BD-TaLG2L-1A+AD-TaWD(片段)、BD-TaLG2L-1A+AD-TaCRY1(片段)、BD-TaLG2L-1A+AD-TaARF17(片段)。

Fig.10 Rotation verification From left to right,the first row shows the positive control and negative control;the second row displays BD-TaLG2L-1A+AD-TaWD (fragment),BD-TaLG2L-1A+AD-TaCRY1(fragment),and BD-TaLG2L-1A+AD-TaARF17(fragment),respectively.

图11 TaLG2L-1A与TaWD、TaCRY1、TaARF17一对一互作验证

Fig.11 Pairwise interaction validation between TaLG2L-1A and TaWD,TaCRY1,TaARF17

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