黄瓜CsRPL1/2的克隆及其功能分析

doi:10.3864/j.issn.0578-1752.2020.01.014

摘要/Abstract

摘要：

【目的】AtRPL是拟南芥果实发育调控网络中的重要基因,参与胎座框的形成。同源克隆黄瓜的RPL,进行表达模式分析,在拟南芥中异源过表达CsRPL,探究CsRPL的生物学功能。【方法】根据拟南芥AtRPL信息在黄瓜基因组数据库中比对,克隆得到黄瓜CsRPL;利用MEGA5.2对CsRPL与其他物种同源蛋白进行氨基酸序列比对;利用实时荧光定量PCR（qRT-PCR）及原位杂交技术检测CsRPL在黄瓜中的表达模式;在拟南芥中异源表达CsRPL,并对转基因植株进行表达和表型分析。【结果】在黄瓜中存在两个RPL,分别命名为CsRPL1和CsRPL2,均含有BELL-Domain、Homeodomain保守域和两个EAR-Motif。CsRPL1在黄瓜各个部位均有表达,在开放的雄花中表达量最高,且在果实生长前期,其表达量随着果实的发育逐渐降低;CsRPL2在黄瓜各部位表达量均显著低于CsRPL1。原位杂交显示CsRPL1/2在黄瓜果实的胎座框中表达,且杂交信号在茎尖分生组织（SAM）的Central zone（CZ）区域富集。拟南芥异源过表达CsRPL1/2转基因植株的果荚变短,花粉育性降低,且种子发育受到抑制。【结论】CsRPL1/2参与生殖器官的发育,且可能存在功能冗余,在正常生长条件下,CsRPL1优先发挥功能。相比于AtRPL,CsRPL1/2的功能并不完全保守。

关键词: 黄瓜, CsRPL, 转基因植株, 表达分析, 果实

Abstract:

【Objective】AtRPL is an important gene regulating Arabidopsis fruit development and mediating replum formation. Using homologous cloning, the RPL homolog in cucumber was identified, the biological function of CsRPL by expression analysis and ectopic transformation in Arabidopsis was explored.【Method】We cloned the CsRPL gene by performing a BLAST search of AtRPL in cucumber genome. Then we performed amino acid sequence alignment of CsRPL and RPL homologs from other species by MEGA5.2. CsRPL expression pattern in cucumber was detected by real-time quantitative PCR (qRT-PCR) and in situ hybridization. Expression and phenotypic analysis of transgenic Arabidopsis upon ectopic expression of CsRPL were performed as well.【Result】Two RPL genes, named CsRPL1 and CsRPL2, were identified in cucumber with the conserved BELL domain and Homeodomain, and two EAR-Motifs. CsRPL1 was expressed in all organs of cucumber, with the highest expression level in male flowers at anthesis. During early stages of fruit development, the CsRPL1 expression decreased gradually. The expression level of CsRPL2 was significantly lower than that of CsRPL1. In situ hybridization revealed that signals of CsRPL1/2 were detected in the placenta of fruits and the central zone (CZ) of the shoot apical meristem (SAM). Ectopic overexpression of CsRPL1/2 into Arabidopsis resulted in shorter siliques, reduced pollen fertility, and inhibited seed development.【Conclusion】CsRPL1/2 were involved in the development of reproductive organs and might have functional redundancy in cucumber. CsRPL1 might play the primary role under normal growth condition. The function of CsRPL1/2 was not fully conserved as compared to that of AtRPL.

Key words: cucumber, CsRPL, transgenic plant, expression analysis, fruit

宋维源,侯钰,赵剑宇,刘小凤,张小兰. 黄瓜CsRPL1/2的克隆及其功能分析[J]. 中国农业科学, 2020, 53(1): 148-159.

WeiYuan SONG,Yu HOU,JianYu ZHAO,XiaoFeng LIU,XiaoLan ZHANG. Cloning and Functional Analysis of CsRPL1/2 in Cucumber[J]. Scientia Agricultura Sinica, 2020, 53(1): 148-159.

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引物名称 Primer name	引物序列Primer sequence (5′-3′)
CsRPL1-F	ATGGCTGAGGGTATTGAATCCTAC
CsRPL1-R	TCAGCCCACAAAGTCATGTAACAGC
CsRPL2-F	ATGGCGGAGGGTTTTGAAGTTTAC
CsRPL2-R	TCAAAACCACTCATTTACAAACCCT
CsRPL1-1300-F	TACACCAAATCGACTCTAGAATGGCTGAGGGTATTGAATC
CsRPL1-1300-A	CCTTGCTCACCATGGTACCGCCCACAAAGTCATGTAACA
CsRPL2-1300-F	TACACCAAATCGACTCTAGAATGGCGGAGGGTTTTGAAGT
CsRPL2-1300-A	CCTTGCTCACCATGGTACCAAACCACTCATTTACAAACC
35S-F	GTAAGGGATGACGCACAATC
CsRPL1-OV-R	ATTGAGAGAAGCGTTGATTTATGAG
CsRPL2-OV-R	TACTCAAATGTCGTAACCACTGCTT
UBI-F	CACCAAGCCCAAGAAGATC
UBI-R	TAAACCTAATCACCACCAGC
Actin-F	CCTTCGTCTTGATCTTGCGG
Actin-R	AGCGATGGCTGGAACAGAAC
CsRPL1-qPCR-F	CCGCTCACAATGGAAAACCC
CsRPL1-qPCR-R	AGGATACATTCAAGGGCTCG
CsRPL2-qPCR-F	TACGCCTCCTTTCGTTCCACT
CsRPL2-qPCR-R	TTGTCGTCGGAGATGTTAGGG
CsRPL1-SP6	GATTTAGGTGACACTATAGAATGCTACCCGCTCACAATGGAAAAC
CsRPL1-T7	TGTAATACGACTCACTATAGGGGTCCACCCATCACATACCCA
CsRPL2-SP6	GATTTAGGTGACACTATAGAATGCTCTTCTTCTTTTTTTTCACCCG
CsRPL2-T7	TGTAATACGACTCACTATAGGGCTTCCAAGGAGTCAAAAACGG