Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (11): 151-161.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0251
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DANG Yuan(), LI Wei, MIAO Xiang, XIU Yu, LIN Shan-zhi()
Received:
2022-02-28
Online:
2022-11-26
Published:
2022-12-01
Contact:
LIN Shan-zhi
E-mail:9946125098@qq.com;linsz2002@163.com
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.
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 | |
PsOLE2-qf | GAGACGGCGGGCTATTT | PsOLE2基因荧光定量引物 |
PsOLE2-qr | CAGCATCCTGGGACTTATCTAC | |
PsOLE3-qf | GCCTTCCCAGCCAACTTAT | PsOLE3基因荧光定量引物 |
PsOLE3-qr | TTCTTGACTTCCGGTGGATTC | |
PsOLE4-qf | ACGTTAACTGGGACAGTGATG | PsOLE4基因荧光定量引物 |
PsOLE4-qr | GCTGTCAGGAACACGACTATAC | |
PsOLE5-qf | TCTTGGGTCACCAACTACCT | PsOLE5基因荧光定量引物 |
PsOLE5-qr | GTCCCACAAACTCCACCATATC | |
CYP-qf | CAACGGATCTCAGTTCTTCGTCTGC | CYP内参基因荧光定量引物 |
CYP-qr | GACCCAACCTTCTCGATGTTCTTCA | |
UBC-qf | GAGACCAGCAATAACCGTGAA | UBC内参基因荧光定量引物 |
UBC-qr | TCTTGTACTCCGTGGCATCCT | |
PsOLE4-f1 | CTCTTTGAGCAACTAATGACGTACA | PsOLE4克隆引物 |
PsOLE4-r1 | ATTATCCAAACAACCCAACTTACCC | |
PsOLE4-f2 | TGGAGAGAACACGGGGGACTCTAGAATGGCTGATCAATCAAGACACGTC | PsOLE4亚细胞定位引物 |
PsOLE4-r2 | GGTGGCGACCGGTACCCGGGGATCAAAAGGGCAGAAGTACTGCCCATAC | |
PsOLE4-f3 | GAGAGAACACGGGGGACTCTAGAGATGGCTGATCAATCAAGACACGTC | PsOLE4表达载体引物 |
PsOLE4-r3 | GAACGATCGGGGAAATTCGAGCTTCAAAAAGGGCAGAAGTACTGCCCA |
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 | |
PsOLE2-qf | GAGACGGCGGGCTATTT | PsOLE2基因荧光定量引物 |
PsOLE2-qr | CAGCATCCTGGGACTTATCTAC | |
PsOLE3-qf | GCCTTCCCAGCCAACTTAT | PsOLE3基因荧光定量引物 |
PsOLE3-qr | TTCTTGACTTCCGGTGGATTC | |
PsOLE4-qf | ACGTTAACTGGGACAGTGATG | PsOLE4基因荧光定量引物 |
PsOLE4-qr | GCTGTCAGGAACACGACTATAC | |
PsOLE5-qf | TCTTGGGTCACCAACTACCT | PsOLE5基因荧光定量引物 |
PsOLE5-qr | GTCCCACAAACTCCACCATATC | |
CYP-qf | CAACGGATCTCAGTTCTTCGTCTGC | CYP内参基因荧光定量引物 |
CYP-qr | GACCCAACCTTCTCGATGTTCTTCA | |
UBC-qf | GAGACCAGCAATAACCGTGAA | UBC内参基因荧光定量引物 |
UBC-qr | TCTTGTACTCCGTGGCATCCT | |
PsOLE4-f1 | CTCTTTGAGCAACTAATGACGTACA | PsOLE4克隆引物 |
PsOLE4-r1 | ATTATCCAAACAACCCAACTTACCC | |
PsOLE4-f2 | TGGAGAGAACACGGGGGACTCTAGAATGGCTGATCAATCAAGACACGTC | PsOLE4亚细胞定位引物 |
PsOLE4-r2 | GGTGGCGACCGGTACCCGGGGATCAAAAGGGCAGAAGTACTGCCCATAC | |
PsOLE4-f3 | GAGAGAACACGGGGGACTCTAGAGATGGCTGATCAATCAAGACACGTC | PsOLE4表达载体引物 |
PsOLE4-r3 | GAACGATCGGGGAAATTCGAGCTTCAAAAAGGGCAGAAGTACTGCCCA |
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. 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. 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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