Cloning of Oleosin Gene PsOLE4 from Prunus sibirica and Its Regulatory Function Analysis for Oil Accumulation

doi:10.13560/j.cnki.biotech.bull.1985.2022-0251

Abstract

Abstract:

Oleosin（OLE）plays an important role in regulating synthesis and accumulation of plant lipids. Based on our recent mRNA transcriptomics sequencing data for the seeds of Prunus sibirica from different developing stages，a total of 5 OLE family genes with complete open reading frame and typical conserved oleosin domain were screened by functional annotation，and importantly，RT-qPCR and differential transcription profile were used to determine the PsOLE4 with abundant transcript closely related to development and oil accumulation of P. sibirica seeds as research objects. Hence，the PsOLE4 gene was cloned from P. sibirica seeds，and then the analyses of bioinformatics，tissue-specific expression，subcellular localization and ectopic expression in Arabidopsis thaliana as well as oil content and fatty acid composition of transgenic seeds were conducted. The results showed that the full-length cDNA of PsOLE4 contained an open reading frame of 378 bp in length，encoding a protein of 125 amino acid residues with a molecular mass of 13 kD. The PsOLE4 protein，without signal peptide，was a non-secretory and hydrophobic protein containing 18 phosphorylation sites，2 transmembrane domains and a highly conserved proline junction domain（PX₅SP₃P），which was mainly localized on cell membrane. The expressions of PsOLE4 was much higher in the seeds of P. sibirica than in the stems，leaves and fruits，and PsOLE4 expression efficiently enhanced the contents of oils and its five predominant fatty acids in A. thaliana seeds，revealing that PsOLE4 with seed-expressed specificity may contribute to the oil accumulation of transgenic A. thaliana seeds. In conclusion，our results could provide important foundation for the further studies of functional identification of PsOLE4 and its application for oil yield improvement.

Key words: oleosin, oil, Prunus sibirica seeds, PsOLE4 gene cloning, tissue specific expression, subcellular localization, genetic transformation, function analysis

DANG Yuan, LI Wei, MIAO Xiang, XIU Yu, LIN Shan-zhi. Cloning of Oleosin Gene PsOLE4 from Prunus sibirica and Its Regulatory Function Analysis for Oil Accumulation[J]. Biotechnology Bulletin, 2022, 38(11): 151-161.

Figures/Tables 9

Table 1 Primers and sequences used in the study

引物 Primer	序列 Sequence（5'-3'）	备注 Annotation
PsOLE1-qf	CTTTGACCATCGCCACTCCG	PsOLE1基因荧光定量引物
PsOLE1-qr	CGCCACACCGAATCCACC	PsOLE1基因荧光定量引物
PsOLE2-qf	GAGACGGCGGGCTATTT	PsOLE2基因荧光定量引物
PsOLE2-qr	CAGCATCCTGGGACTTATCTAC	PsOLE2基因荧光定量引物
PsOLE3-qf	GCCTTCCCAGCCAACTTAT	PsOLE3基因荧光定量引物
PsOLE3-qr	TTCTTGACTTCCGGTGGATTC	PsOLE3基因荧光定量引物
PsOLE4-qf	ACGTTAACTGGGACAGTGATG	PsOLE4基因荧光定量引物
PsOLE4-qr	GCTGTCAGGAACACGACTATAC	PsOLE4基因荧光定量引物
PsOLE5-qf	TCTTGGGTCACCAACTACCT	PsOLE5基因荧光定量引物
PsOLE5-qr	GTCCCACAAACTCCACCATATC	PsOLE5基因荧光定量引物
CYP-qf	CAACGGATCTCAGTTCTTCGTCTGC	CYP内参基因荧光定量引物
CYP-qr	GACCCAACCTTCTCGATGTTCTTCA	CYP内参基因荧光定量引物
UBC-qf	GAGACCAGCAATAACCGTGAA	UBC内参基因荧光定量引物
UBC-qr	TCTTGTACTCCGTGGCATCCT	UBC内参基因荧光定量引物
PsOLE4-f1	CTCTTTGAGCAACTAATGACGTACA	PsOLE4克隆引物
PsOLE4-r1	ATTATCCAAACAACCCAACTTACCC	PsOLE4克隆引物
PsOLE4-f2	TGGAGAGAACACGGGGGACTCTAGAATGGCTGATCAATCAAGACACGTC	PsOLE4亚细胞定位引物
PsOLE4-r2	GGTGGCGACCGGTACCCGGGGATCAAAAGGGCAGAAGTACTGCCCATAC	PsOLE4亚细胞定位引物
PsOLE4-f3	GAGAGAACACGGGGGACTCTAGAGATGGCTGATCAATCAAGACACGTC	PsOLE4表达载体引物
PsOLE4-r3	GAACGATCGGGGAAATTCGAGCTTCAAAAAGGGCAGAAGTACTGCCCA	PsOLE4表达载体引物

Fig. 1 Analysis of conserved domain for PsOLEs and dynamic transcriptional expression of PsOLEs in P. sibirica seeds A：Analysis of conserved domain for PsOLE proteins in P. sibirica seeds. B：Expressions level of PsOLE family genes by mRNA high-throughput sequencing，the values were normalized as log10（FPKM）. C：Determination of relative expressions of PsOLE family genes by RT-qPCR，the values were normalized as log10（relative expression）. The bar in the figure refers to the standard deviation

Fig. 2 Amplification electrophoretic map of the PsOLE4 gene from P. sibirica seeds M：2000 DNA marker. 1：PsOLE4 gene fragment

Fig. 3 Bioinformatics analysis for PSOLE4 protein from P. sibirica seeds A：Analysis of phosphorylation site for PsOLE4 protein from P. sibirica seeds. B：Analysis of hydrophilicity/hydrophobicity for PsOLE4. C：Analysis of signal peptide for PsOLE4. D：Analysis of transmembrane region for PsOLE4. E：Prediction of secondary structure for PsOLE4. F：Prediction of tertiary structure for PsOLE4

Fig. 4 Multiple sequence alignment of PsOLE4 protein from P. sibirica seeds

Fig. 5 Analysis of phylogenetic tree for PsOLE4 protein from P. sibirica seeds

Fig. 6 Analysis of relative expression for PsOLE4 gene in the different tissues of P. sibirica The lowest expression of PsOLE4 gene in the leaves was taken as control，and standardized calibration is 1

Fig. 7 Subcellular localization of PsOLE4 from P. sibirica seeds Dark is defined as dark-field fluorescence image. Bright is defined as bright-field image. Merged is defined as the merged image in both bright and dark fields

Fig. 8 Analyses for growth status in PsOLE- transgenic A. thaliana and fatty acid content and composition of their seeds A：PCR identification for transgenic A. thaliana，M：DNA marker，1：PCR product from empty vector transgenic plant（empty），2-4：PCR product from PsOLE4 transgenic plants（35S∷PsOLE4 #1、35S∷PsOLE4 #2和35S∷PsOLE4 #3）. B：Growth status of WT，empty and PsOLE4 overexpressing A. thaliana lines，scale bar=5 cm. C：Fatty acid contents in the seeds of WT，empty and PsOLE4 overexpressing transgenic A. thaliana lines. D：Fatty acid composition in seeds of A. thaliana. The asterisks（*）above the columns indicate significant differences at P<0.05. C16∶0：Palmitic acid. C18：0：Stearic acid. C18：1：Oleic acid. C18：2：Linoleic acid. C18：3：Linolenic acid. C20：0：Arachidic acid. C20：1：Eicosenoic acid

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