Scientia Agricultura Sinica ›› 2018, Vol. 51 ›› Issue (8): 1598-1606.doi: 10.3864/j.issn.0578-1752.2018.08.017

• RESEARCH NOTES • Previous Articles     Next Articles

Molecular Cloning of Leaf Developmental Gene EjGRF5, Its Promoter and Expression Analysis in Different Ploidy Loquat (Eriobotrya japonica (Thunb.) Lindl.)

LIU Chao1, WANG Lingli2, WU Di1, DANG Jiangbo1, SHANG Wei1, GUO Qigao1, LIANG Guolu1   

  1. 1College of Horticulture and Landscape Architecture, southwest university, Chongqing 400715; 2Technical Advice Station of Economic Crop, Chongqing 401120
  • Received:2017-09-01 Online:2018-04-16 Published:2018-04-16

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Abstract: 【Objective】 In order to provide more details for further studying the mechanisms of EjGRF5 gene in regulating the growth vigor of different ploidy loquat leaf, the aims of this study are to isolate the code region of EjGRF5 gene which is involved in the regulation of leaf development and its promoter sequence, and illustrate the expression pattern of the EjGRF5 in different ploidy loquat. 【Method】 The primers were designed by using the EjGRF5 reference sequence obtained from the RNA-Seq, and the full length of EjGRF5 was cloned by using the genome DNA of Longquan-1 tetraploid, and then the full length and reference sequences were compared to obtain the targeted sequence. The Bioedit7.2 and SignalP4.1 were used to analyze the structure of the EjGRF5 CDS and the physical and chemical properties of EjGRF5; Mega7.0 was used to construct the EjGRF5 phylogenetic tree. The online software of LocTree3 and SoftBerry ProtComp9.0 was adopted to predict the subcellular location of EjGRF5. The genome walking technique was employed to amplify the promoter sequence, and the online software PlantCARE was adopted to analyze the structure of the promoter. The expression patterns of EjGRF5 in triploid loquat and their parents (4x, 2x) were analyzed preliminarily. 【Result】 When Comparing the sequenced data with the reference sequence of EjGRF5, the results showed that the full length of EjGRF5 is 1368 bp and it contains three extron and two intron sequences. The CDS length of EjGRF5 is 987 bp. The results of phylogenetic analysis revealed that the EjGRF5 protein is highly homologous with some other species in Rosaceae and is closest to Pyrus bretschneideri. The result of subcellular localization prediction showed that EjGRF5 protein is located in the nucleus. The promoter analysis indicated that there were multiple putative cis-acting elements involved in the responsive elements, including abscisic acid (ABA), ethylene, heat, anaerobic inductive, gibberellin (GA) and light. Moreover, the number of light responsiveness element has reached 11. QRT-PCR result showed that the expression level of EjGRF5 exhibit varying degrees of up-regulation in almost all of the triploids except the hybrids A-6 and B-3 compared with the middle-parent value (MPV). Among the hybrids, the expression of A-3 was 20 times higher than that of MPV and A-5 was about 18 times higher than that of MPV. 【Conclusion】 The coding region and CDS sequence of EjGRF5 gene which is related to the development of loquat leaves were isolated, and the qRT-PCR results indicated that the expression of EjGRF5 in triploid loquat exhibited a trend of up-regulation.

Key words: loquat, EjGRF5, gene cloning, promoter cloning, expression analysis

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