Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (1): 148-159.doi: 10.3864/j.issn.0578-1752.2020.01.014

• SPECIAL FOCUS: MOLECULAR BIOLOGY OF CUCUMBER • Previous Articles     Next Articles

Cloning and Functional Analysis of CsRPL1/2 in Cucumber

WeiYuan SONG,Yu HOU,JianYu ZHAO,XiaoFeng LIU,XiaoLan ZHANG()   

  1. College of Horticulture, China Agricultural University, Beijing 100089
  • Received:2019-06-03 Accepted:2019-07-02 Online:2020-01-01 Published:2020-01-19
  • Contact: XiaoLan ZHANG E-mail:zhxiaolan@cau.edu.cn

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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

Table 1

Primers sequences"

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

Fig. 1

The phylogenetic analysis and gene structure of cucumber CsRPL1/2 and Arabidopsis AtRPL A: Phylogenetic analysis of BELL subfamily gene in cucumber and Arabidopsis thaliana, AtRPL and its homologous gene CsRPL1/2 in the red box. BEL1 (AT5G41410); ATH1 (AT4G32980); BLH1 (AT2G35940); BLH2/SAW1 (AT4G36870); BLH3 (AT1G75410); BLH4/SAW2 (AT2G23760); BLH5 (AT2G27220); BLH6 (AT4G34610); BLH7 (AT2G16400); BLH8/PNF (AT2G27990); BLH9/RPL (AT5G02030); BLH10 (AT1G19700); BLH11 (AT1G75430). B: Purple squares represent 5' UTR, 3' UTR; orange squares represent exons; straight lines represent introns"

Fig. 2

Sequence alignment of cucumber CsRPL with RPL homologs from other species A: BELL-Domain in the orange box, Homeodomain in the blue box, and EAR-Motifs in the red dotted box. AtRPL: Arabidopsis RPL（NP_195823.1）; GmRPL: Melon RPL (XP_008448414.1); VvRPL: Grape RPL (XP_010654234.1); PpRPL: Peach RPL (XP_007208167.1); GhRPL: Cotton RPL (XP_016738976.1); MdRPL: Apple RPL (XP_008363517.1); GmRPL: Soybean RPL (XP_003516903.1); SlRPL: Tomato RPL (XP_004246395.1); ZmRPL: Corn RPL: (NP_001168681.1); TaRPL: Wheat RPL (BAJ04689. 1); OsRPL: Rice RPL (XP_015641948.1); B-E: Amino acid conservation analysis of BELL-Domain (B), Homeodomain (C) and EAR-Motif (D and E); VvRPL, GhRPL and monocotyledon ZmRPL, TaRPL and OsRPL was not included; Asterisks represent the completely conserved amino acids"

Fig. 3

Gene expression pattern of CsRPL1/2 A: Relative expression of CsRPL1/2 in various parts of cucumber; B-F: In situ hybridization analysis of CsRPL1 (B, C) and CsRPL2 (D, E) in cucumber fruits and shoot tips; B, D: CsRPL1/2 expressed in placenta C, E: CsRPL1/2 signal was enriched in the central zone of the meristem in the shoot tips; White dotted box represents the signal enrichment region; F: CsRPL-SP6 probe was used as negative control, and no hybridization signal was found. Scale bar: 100 μm"

Fig. 4

PCR identification of transgenic Arabidopsis M: 2000 bp marker; Plasmid: positive control plasmid; Col: wild type Arabidopsis"

Fig. 5

Phenotypic observation and statistical analysis of ectopic expression of CsRPL1/2 in Arabidopsis A: The phenotype of wild type and transgenic plants (Col: wild type; #1-1: CsRPL1 overexpressing line; #2-1: CsRPL2 overexpressing line, scale bar: 10 cm); B: Seeds development in the fruits, white arrows pointed to abortive seeds (scale bar: 2 mm); C: The character of silique length (scale bar: 1 cm) D: Statistical data of silique length; E: Pollen viability test (scale bar 1 mm); F: Statistical data of seed number per silique; G: Statistical data of pollen viability test; H: Expression analysis of CsRPL1/2 in wild type and transgenic plants"

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