中国农业科学 ›› 2022, Vol. 55 ›› Issue (24): 4912-4926.doi: 10.3864/j.issn.0578-1752.2022.24.010
李雨泽1(),朱嘉伟1,林蔚1,2,蓝茉莹1,夏黎明1,张艺粒1,罗聪1,黄桂香1,何新华1()
收稿日期:
2022-03-14
接受日期:
2022-05-25
出版日期:
2022-12-16
发布日期:
2023-01-04
通讯作者:
何新华
作者简介:
李雨泽,E-mail:基金资助:
LI YuZe1(),ZHU JiaWei1,LIN Wei1,2,LAN MoYing1,XIA LiMing1,ZHANG YiLi1,LUO Cong1,HUANG Gui Xiang1,HE XinHua1()
Received:
2022-03-14
Accepted:
2022-05-25
Online:
2022-12-16
Published:
2023-01-04
Contact:
XinHua HE
摘要:
【目的】以香水柠檬(Citrus limon (L.) Burm. F.)为研究对象,分析两个RHF2A的时空表达规律,利用酵母双杂交技术和双分子荧光互补试验(BiFC)筛选、验证其互作蛋白,为深入研究RHF2A在香水柠檬自交不亲和过程中的分子作用机制奠定基础。【方法】从前期转录组和泛素修饰组(未公开)筛选获得2个E3泛素连接酶RHF2A(RING-H2 Zinc Finger2A)基因RHF2A-1、RHF2A-2,并克隆其全长序列,通过生物信息学分析2个RHF2A的序列和蛋白质结构,预测其启动子顺式作用元件,构建35S-RHF2A-GFP融合蛋白表达载体用于亚细胞定位分析,采用实时荧光定量PCR分析两个RHF2A的时空表达模式,构建酵母双杂诱饵载体从香水柠檬酵母文库筛选互作蛋白。构建BiFC载体,在洋葱活体细胞内对目标蛋白进行互作验证。【结果】从香水柠檬克隆获得RHF2A-1、RHF2A-2,ORF全长分别为1 161和1 134 bp;NCBI结构域预测发现其具有Ring/U-box结构域。启动子分析发现其具有花粉特异性表达相关元件POLLEN1LELAT52、GTGANTG10。组织表达分析发现RHF2A-1在花粉特异性表达,RHF2A-2在叶中特异性表达;时空表达分析结果显示,RHF2A-1在自交柱头表达量从第1天开始升高,第3天达到峰值,是杂交柱头表达量的5倍以上。亚细胞定位显示RHF2A-1和RHF2A-2定位在细胞核。经过Uniprot网站预测其互作蛋白显示RHF2A能与KRP6、AT3G57370、UBA1、FBL17、SK11蛋白相互作用,推测其参与自交不亲和泛素化反应途径、配子体发育调控、花粉的生长发育等生物学过程。通过酵母双杂交技术筛选出72个克隆,对其测序并进行Blast比对后排除重复克隆,最终得到ABCF3等20个候选互作蛋白。经过一对一互作验证、双分子荧光互补实验确定RHF2A-1与ABCF3-2存在互作关系。【结论】RHF2A-1的时空表达规律与自交不亲和过程中花粉在雌蕊上的萌发规律相吻合;筛选得到柠檬授粉过程中直接影响花粉生长发育的互作候选蛋白,初步证明RHF2A-1在自交不亲和过程中具有重要作用。
李雨泽,朱嘉伟,林蔚,蓝茉莹,夏黎明,张艺粒,罗聪,黄桂香,何新华. 香水柠檬RHF2A的克隆与互作蛋白的筛选[J]. 中国农业科学, 2022, 55(24): 4912-4926.
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.
表1
PCR引物列表"
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 |
表3
RHF2A-1所有候选蛋白信息"
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 |
表4
RHF2A-2所有候选蛋白信息"
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 |
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