Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (5): 882-892.doi: 10.3864/j.issn.0578-1752.2019.05.010

• HORTICULTURE • Previous Articles     Next Articles

Cloning and Expression Analysis of SERK2 Gene in Different Forms of Calli on Peach (Prunus persica L.)

TAN Bin1,2,CHEN TanXing1,HAN YaPing1,ZHANG YaRu1,ZHENG XianBo1,2,CHENG Jun1,2,WANG Wei1,2,FENG JianCan1,2()   

  1. 1 College of Horticulture, Henan Agricultural University, Zhengzhou 450002
    2 Henan Key Laboratory of Fruit and Cucurbit Biology, Zhengzhou 450002
  • Received:2018-10-10 Accepted:2018-11-17 Online:2019-03-01 Published:2019-03-12
  • Contact: JianCan FENG E-mail:jcfeng@henau.edu.cn

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Abstract:

【Objective】The somatic embryogenesis receptor-like kinase 2 (SERK2 ) gene was isolated and cloned from peach (Prunus persica L.). Here, we detected the expression profile of SERK2 in different forms of calli in peach, and analyzed the correlation between SERK2 and the regeneration of embryonic callus. This research will shed on uncovering the molecular mechanism of embryonic callus induction and regeneration in peach with difficulty on tissue culture. 【Method】The full-length cDNA sequence of PpSERK2 gene was obtained by homologous cloning. Then the sequence was analyzed by a series of bioinformatical software packages, including TMPred, DNAMAN and MEGA 5.0 etc. The callus of ‘Qiumihong’ was obtained by using anthers as explants inoculated into NN69 medium supplemented with 2.0 mg·L -1 6-BA and 1.0 mg·L -1 2,4-D. Then the different forms of calli were observed by production of paraffin section. Real-time quantitative PCR (qRT-PCR) was used to analyze the expression of PpSERK2 gene in four forms of ‘Qiumihong’ calli. 【Result】The full-length cDNA sequence of PpSERK2 gene was 1 881 bp. It encodes 626 amino acids and contains SERK conserved function domains. The theoretical isoelectric point of PpSERK2 is 5.38 and its molecular weight is 68.99 KD. The PpSERK2 has the high homology similarity from 67.88% to 92.71% at protein level, especially the highest similarity to Solanum peruvianu and Citrus unshiu . The phylogenetic analysis of SERK proteins from various plant species indicated that PpSERK2, SpSERK1, CitSERK1 and PsSERK2 were clustered together, which showing the consistent result with above protein similarity analysis. Four different forms of calli were obtained from anther of ‘Qiumihong’. The results of histocytology showed that the cells of three forms of calli, including the yellow, loose callus (global embryo), green, compact callus (heart embryo) and pale yellow, transparent callus (suspected torpedo embryo), were small and closely arranged. While the cells of yellow-white, dropsical callus was large and with irregular shapes. Based on histomorphology results, this demonstrated that the yellow, loose callus, green, compact callus and pale yellow, transparent callus were embryonic calli, while yellow-white, dropsical callus was non-embryonic callus. The results of qRT-PCR showed that transcriptional level of PpSERK2 gene in three forms of embryonic calli was remarkably higher than non-embryonic callus. Meanwhile, the transcriptional level of PpSERK2 gene was highest in the yellow, loose callus, followed by the green, compact callus, and lowest in the pale yellow and transparent callus of ‘Qiumihong’. 【Conclusion】The full-length cDNA of the PpSERK2 gene was successfully obtained. According to the results of PpSERK2 expression in four forms calli of ‘Qiumihong’, we speculated that PpSERK2 gene might play a pivotal role on the early stage during the somatic embryogenesis in peach.

Key words: peach, somatic embryogenesis, SERK2, embryonic callus

Fig. 1

Cloning of PpSERK2 A: RNA of shoot tip in ‘Qiumihong’; B: PCR amplification of PpSERK2 , M: DNA marker DL2000"

Fig. 2

The nucleotide sequence of PpSERK2 gene cDNA and its deduced amino acid sequence"

Fig. 3

The diagrams of SERK amino acid sequence in different plants"

Fig. 4

Phylogenetic tree of PpSERK2 and SERK in other plants"

Fig. 5

The morphological and cytological observation of different forms of calli from P. perscia ‘Qiumihong’ A: The yellow, loose embryogenic callus; B: The green, compact embryogenic callus; C: The pale yellow, transparent embryogenic callus; D: The yellow-white, dropsical non-embryonic callus; E: Microscope slide of the yellow, loose embryogenic callus (arrow indicated globular embryo); F: Microscope slide of the green, compact embryogenic callus (arrow indicated heart-shape embryo); G: Microscope slide of the pale yellow, transparent embryogenic callus (arrow indicated not yet fully formed torpedo-shape embryo); H: Microscope slide of the yellow-white, dropsical embryonic callus. A-D: Bar = 1 mm; E-H: Bar = 200 μm"

Fig. 6

The relative of expression of PpSERK2 gene in different forms of calli of P. perscia ‘Qiumihong’ Data are means±standard error (n=3); Different small letters mean significant differences (P <0.05) "

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