Isolating and prokaryotic expression analysis of the PpGAI3 gene in Pyrus pyrifolia

Abstract: 【Objective】Dormancy break is a physiological phenomenon associated with the ability ofplants to cope with changing environmental conditions and adjust their growth habits accordingly. Gibber⁃ellin is an essential plant hormone that can inhibit flower bud dormancy and promote flower bud germina⁃tion. It down-regulates DELLA proteins through protein degradation via the proteasome pathway. In orderto understand the molecular mechanism of bud dormancy for pears, the aim of this study was to isolate thePpGAI3 of DELLA protein-coding gene from Pyrus pyrifolia, and explore the protein expression duringthe dormancy period of pears.【Methods】The buds from the different chilling requirement of Pyrus pyrifo⁃lia‘Mixueli’and‘Huanghuali’were used as the experimental materials. All the samples were collectedfrom the annual branches of the pear tree canopy approximately once a week, from the winter abscissionperiod to the spring germination stage. Total RNA was extracted from the flower buds, and the first-strandcDNA was used as a template after being reverse-transcribed. The PpGAI3 gene was cloned by PCR. Ba⁃sic protein physico-chemical properties of PpGAI3 were predicted by ExPASy ProtParam, and the Phylo⁃genetic tree was constructed by MEGA. Then, it was inserted into the pEASY-Blunt E1 vector to constructthe fusion vector, which was transformed into the BL21（DE3）cell and was induced by IPTG at a tempera⁃ture of 37 ℃. The recombinant expressing plasmid was detected with SDS-PAGE and Western blotting.We used ultrasonic waves to break the cell of both the recombinant pEASY-Blunt E1-PpGAI3mx and E1control group, and then we had the whole cell, the supernatant fluid and the sediment of the cells dividedinto two groups. The polyacrylamide gel electrophoresis (PAGE) was carried out with a 5% stacking geland a 10% separating gel which was used to confirm the protein expression. Finally, the expression of thewhole cell, the supernatant fluid and the sediment of cells in the two groups were further confirmed bymeans of western blotting.【Results】The PpGAI3 genes from the two Pyrus pyrifolia‘Mixueli’and‘Huan⁃ghuali’named PpGAI3mx (GenBank accession number: KU954535) and PpGAI3hh (GenBank accessionnumber: KX078215) were identified and cloned. The open reading frames were 1 641 bp, with an encoding of 546 amino acids. The blast results of the nucleotide sequence showed that the PpGAImx and Pp⁃GAI3hh shared a very high identity, so the DELLA protein-coding gene had a high conservative relation⁃ship between the different chilling requirement varieties of Pyrus pyrifolia. The results of the multiplealignments helped to explain that the DELLA protein-coding gene had a high conservative relationshipbetween different species of fruit plants. Moreover, the N-terminal amino acid sequence of the DELLAprotein-coding genes shared a low homology, but shared a higher homology in the C-terminal amino acidsequence. The PpGAI3 gene had the conserved DELLA domain, a transcriptional regulator DELLA pro⁃tein for regulating the GA signal transduction; and also had a subfamily belonging to the GRAS superfami⁃ly domain, which acted as major players in the GA signaling to regulate the various aspects of plant growthand development. Bioinformatic analysis predicted that the proteins were hydrophily and unstable, the mo⁃lecular masses were 59.636 5, the formulas were C2 626H4 107N729O805S27 and the theoretical isoelectric pointswere 5.37. Phylogenetic tree analysis indicated that the PpGAI3mx and PpGAI3hh were most related to Py⁃rus×bretschneideri. Ligating the target fragment of‘Mixueli’to vector pEASY-Blunt E1, we were then ableto successfully construct the recombinant prokaryotic expressing plasmid of pEASY-Blunt E1-PpGAI3mx.The results of the SDS-PAGE showed that all the whole cell, the supernatant fluid and the sediment ofcells of the recombinant pEASY-Blunt E1-PpGAI3mx had electrophoretic bands at the position of about60 ku compared with the control group. The expressed protein had a closed molecular weight with the pre⁃diction program. It indicated that the expression of recombinant expressing plasmid of the pEASY-BluntE1-PpGAI3mx was successfully induced by IPTG; and the PpGAI3mx of the procaryotic expression existsboth in the supernatant fluid and the sediment. Results obtained from western blotting suggested that thewhole cell, the supernatant fluid and the sedimentof cells containing the recombinant pEASY-Blunt E1-PpGAI3mx had a successful expression of the desired proteins in the correct molecular weight. However,the sediment of the cells containing the recombinant pEASY-Blunt E1-PpGAI3mx had a higher proteinexpression than the supernatant. We could conclude that most of the expressed protein was insoluble andexisted in the form of the inclusion body.【Conclusion】The PpGAI3 isolated from‘Mixueli’and‘Huanghuali’were members of the DELLA protein family, a multi-gene family. The PpGAI3 of the DELLA pro⁃tein-coding genes from Pyrus pyrifolia had high-efficiency expression during the period of bud dormancy,and most of the expression protein was insoluble. But how do they interact with each other or other genesand the precise role they played in GA signal transduction, dormancy breaking, and environmental adaptation will need further investigation.