香水柠檬RHF2A的克隆与互作蛋白的筛选

doi:10.3864/j.issn.0578-1752.2022.24.010

Abstract

Abstract:

【Objective】Xiangshui lemon (Citrus limon (L.) Burm. F.) was used to study the expression of two RHF2A genes, and to screen and verify their interaction proteins by yeast two-hybrid technology and BiFC, so as to lay a foundation for further studying the molecular mechanism of RHF2A in the process of lemon self-incompatibility. 【Method】Two E3 ubiquitin ligase RHF2A (RING-H2 Zinc Finger2A) genes including RHF2A-1 and RHF2A-2 of Xiangshui lemon were screened from the transcriptome and ubiquitin modification group, and their full-length sequences were cloned. The sequence and protein structure of two RHF2A genes were analyzed by bioinformatics to predict the cis acting elements of their promoters. 35S-RHF2A-GFP fusion protein expression vector was constructed for subcellular localization analysis. The temporal and spatial expression patterns of two RHF2A were analyzed by real-time fluorescence quantitative PCR. The yeast two-hybrid bait vector was constructed to screen the interaction proteins from the lemon yeast library. The BiFC vector was constructed to verify the interaction of the target protein in onion living cells. 【Result】The RHF2A-1 and RHF2A-2 genes were obtained from ‘Xiangshui’ lemon, and the total length of ORF was 1 161 and 1 134 bp, respectively. NCBI domain prediction found that it had a Ring/U-box domain. Promoter analysis showed that there were POLLEN1LELAT52 and GTGANTG10 related to pollen specific expression elements. Tissue expression analysis showed that RHF2A-1 gene was specifically expressed in pollen and RHF2A-2 was specifically expressed in leaves; the results of temporal and spatial expression analysis showed that the expressed of RHF2A-1 in self-stigma tended to increase from the first day and reached the peak on the third day, which was more than 5 times that of hybrid stigma. Subcellular localization showed that RHF2A-1and RHF2A-2 were localized in the nucleus. The interaction protein predicted by Uniprot website showed that RHF2A could interact with KRP6, AT3G57370, UBA1, FBL17 and SK11proteins, and RHF2A gene was involved in biological processes such as self-incompatibility ubiquitination reaction pathway, gametophyte development regulation and pollen growth and development. 72 clones were screened by yeast two-hybrid technology. After sequencing and blast comparison, the repetitive clones were excluded. Finally, 20 candidate interaction proteins such as ABCF3 were obtained. Through one-to-one interaction verification and BiFC, it was determined that there was an interaction relationship between RHF2A-1 and ABCF3-2. 【Conclusion】The temporal and spatial expression of RHF2A-1 gene was consistent with the germination of pollen on pistil in the process of self-incompatibility; the interaction candidate proteins directly affecting pollen growth and development during the pollination were screened, which preliminarily proved that RHF2A-1 gene played an important role in the process of self-incompatibility.

Key words: lemon, RHF2A, gene cloning, spatiotemporal expression, yeast two hybrid technology

LI YuZe,ZHU JiaWei,LIN Wei,LAN MoYing,XIA LiMing,ZHANG YiLi,LUO Cong,HUANG Gui Xiang,HE XinHua. Cloning and Interaction Protein Screening of RHF2A Gene from Xiangshui Lemon[J].Scientia Agricultura Sinica, 2022, 55(24): 4912-4926.

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Figures/Tables 16

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Table 3

The candidate proteins interacted with RHF2A-1"

编号 Number	基因描述 Gene description	分子功能 Molecular function
A1	S-腺苷甲硫氨酸合酶2 Adenosylmethionine synthase 2	影响水杨酸、油菜素内酯，种子萌发、花粉萌发、花粉管伸长，细胞壁 It affects salicylic acid, brassinolide, seed germination, pollen germination, pollen tube elongation and cell wall
A2	赤霉素受体GID1B Gibberellin receptor GID1B	具有可溶性赤霉素(GA)受体的功能 Functions as soluble gibberellin (GA) receptor
A3	异质核核糖核蛋白1 Heterogeneous nuclear ribonucleoprotein 1	参与前mRNA的处理 Involved in the packaging of pre-mRNA
A4	E3泛素蛋白连接酶RGLG2 E3 ubiquitin-protein ligase RGLG2	能够调节生长素转运蛋白丰度 Acting on the auxin transport proteins abundance
A5	bZIP转录因子17 bZIP transcription factor 17	转录激活剂参与盐和渗透应激反应 Transcriptional activator involved in salt and osmotic stress responses
A6	天冬氨酸蛋白酶A1 Aspartic proteinase A1	体外具有天冬氨酸蛋白酶活性 Possesses aspartic protease activity in vitro
A7	茎特异蛋白TSJT1 Stem-specific protein TSJT1
A8	L-抗坏血酸氧化酶同系物 L-ascorbate oxidase homolog	可能参与花粉管生长的氧化还原酶 Probable oxidoreductase that may be involved in pollen tube growth.
A9	水稻开花位点T1 Rice Flowering Locus T1	移动式促花信号，可诱导开花 Probable mobile flower-promoting signal (florigen)
A10	苹果酸脱氢酶 Malate dehydrogenase	可能参与器官间的中心代谢和氧化还原稳态 It may be involved in central metabolism and redox homeostasis between organs
A11	天冬氨酸蛋白酶 Aspartic proteinase	具有天冬氨酸蛋白水解活性,有助于花粉和胚珠的发育，包括生长的花粉管顶端细胞壁的构成 Displays aspartic proteolytic activity, contributes to pollen and ovule development, including the apical cell wall constitution of the growing pollen tubes
A12	FBA6	以氧化还原的方式与GAPC1结合到线粒体外膜，导致肌动蛋白的结合和捆绑 Associates with GAPC1 to the outer mitochondrial membrane, in a redox-dependent manner, leading to binding and bundling of actin
A13	RING-H2 finger protein ATL39	E2泛素结合酶 E2 ubiquitin binding enzyme
A14	E3 ubiquitin-protein ligase RNF14	E3泛素连接酶 E3 ubiquitin binding enzyme
A15	ABCF3转运蛋白 ABC transporter F family member 3	具有跨膜运输功能的转运蛋白，可能与S-RNase跨膜运输和盐胁迫有关 Transporters with transmembrane transport function may be related to transmembrane transport of S-RNase and salt stress
A16	AP-1 Complex subunit mu-2	在跨高尔基体网络和早期内体（TGN/EE）的蛋白质分类中起作用 Plays a role in protein sorting at the trans-Golgi network and early endosomes (TGN/EE)
A17	KH结构域蛋白HEN4 KH domain-containing protein HEN4	可能与花发育相关 It may be related to flower development
A18	SEC10b	参与极化细胞生长和器官形态发生。在胞质分裂过程中，参与了细胞板的形成、细胞板的成熟和新的原代细胞壁的形成 Involved in polarized cell growth and organ morphogenesis. During cytokinesis, involved in cell plate initiation, cell plate maturation and formation of new primary cell wall
A19	TBCC	参与微管蛋白的质量控制 Participate in the quality control of tubulin
A20	蛋白磷酸酶2C 56 Protein phosphatase 2C 56	脱落酸(ABA)信号通路的关键成分和抑制因子，调控大量ABA反应 Abscisic acid (ABA) is a key component and inhibitor of signaling pathway, which regulates a large number of ABA responses

Table 3

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References 34

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引物 Primer	引物序列 Primer sequence (5′-3′)
ClRHF2A-1	F: GATGGAGGAGGGCAAGAAAC; R: CAAAATCGCCAAACGTAGGA
ClRHF2A-2	F: GCAGGACCATCAGATTTCCA; R: CTGCTGTTGTTGTCCCTGGT
qRTClRHF2A-1	F: GCAAGTGATACTGACCTTGA; R: GACTAGCACCAGCAAGTGTA
qRTClRHF2A-2	F: CGTTGACTAGTTGCAGGCAT; R: CCTGTAACTCAAAGTCTCCCA
qRTClABCF1	F: GAAGTGCGGCTGATAAAGCT; R: GCACCCTATTGCAGTCAAGA
qRTClABCF3-1	F: GCGGCTGGATTACTGGATAAGT; R: CCAGCCGTTAGGAAAGTCCCAT
qRTClABCF3-2	F: GGAAGGCATTATGGGCTTGT; R: GCGCCATTGCTCTTTTCAGTATC
qRTClABCF4	F: CTGGTGTAGCCGCAAAGTCT; R: GGCGGCATCAATCTCTTCATCT
qRTClABCF5	F: CTCCTCCTTCTTCAGCACTT; R: GGCGCAGATGAAATACTCGAA
ClActin	F: CGTATGAGCAAGGAAATCACAG; R: ATTGATCCTCCAATCCAAACAC

基因名称 Gene ID	分子式 Formula	脂肪系数 Aliphatic index	亲水性 GRAVY	分子质量 Molecular weight (kD)	等电点 pI	不稳定系数 Instability index
RHF2A-1	C₁₇₅₄H₂₇₄₀N₅₄₄O₅₉₈S₁₉	49.90	-0.706	41.63	5.86	58.24
RHF2A-2	C₁₇₁₅H₂₇₀₄N₅₃₂O₆₀₈S₁₇	51.19	-0.807	41.05	5.10	65.95

编号 Number	基因描述 Gene description	分子功能 Molecular function
B1	糖蛋白葡萄糖基转移酶 UDP-glucose:glycoprotein glucosyltransferase
B2	液泡相关蛋白13F Putative vacuolar protein sorting-associated protein 13F	与水杨酸相关 Related to salicylic acid
B3	bZIP转录因子17 bZIP transcription factor 17	参与盐和渗透胁迫反应的转录激活因子 Transcriptional activators involved in salt and osmotic stress response
B4	生长素诱导蛋白PCNT115 Auxin-induced protein PCNT115	参与植物生长素调控 Participate in plant auxin regulation
B5	光系统II反应中心PSB28蛋白 Photosystem II reaction center PSB28 protein	参与植物光合作用 Participate in plant photosynthesis
B6	AP-1 complex subunit mu-2	参与细胞内蛋白质转运 Participate in intracellular protein transport
B7	30S核糖体蛋白3-1 30S ribosomal protein 3-1	可能是核糖体蛋白或核糖体相关蛋白 It may be ribosomal protein or ribosomal associated protein
B8	蛋白质LEO1同系物 Protein LEO1 homolog	参与调节开花时间 Participate in regulating flowering time
B9	泛素结合酶E2 Probable ubiquitin-conjugating enzyme E2 C	E2泛素结合酶 E2 ubiquitin binding enzyme
B10	S-腺苷甲硫氨酸合酶 2 S-adenosylmethionine synthase 2	影响水杨酸、油菜素内酯，种子萌发、花粉萌发、花粉管伸长，细胞壁 It affects salicylic acid, brassinolide, seed germination, pollen germination, pollen tube elongation and cell wall

Cloning and Interaction Protein Screening of RHF2A Gene from Xiangshui Lemon

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