Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (9): 147-155.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0062
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LIU Wen-jin1(), MA Rui2,3, LIU Sheng-yan3, YANG Jiang-wei1,2, ZHANG Ning1,2, SI Huai-jun1,2()
Received:
2023-01-31
Online:
2023-09-26
Published:
2023-10-24
Contact:
SI Huai-jun
E-mail:1490826168@qq.com;hjsi@gsau.edu.cn
LIU Wen-jin, MA Rui, LIU Sheng-yan, YANG Jiang-wei, ZHANG Ning, SI Huai-jun. Cloning of StCIPK11 Gene and Analysis of Its Response to Drought Stress in Solanum tuberosum[J]. Biotechnology Bulletin, 2023, 39(9): 147-155.
引物名称Primer name | 引物序列Primer sequence(5'-3') | 用途Purpose |
---|---|---|
StCIPK11-F | CGGGGGACGAGCTCGGTACCATGGCCTTATTCTCTTCTTCGG | PCR扩增 PCR amplification |
StCIPK11-R | CCATGTCGACTCTAGATTATGGATCCACCTCTGTTGAAATC | |
StCIPK11-I | GATTCTATTTCCGCGCTAGCCTCTCTCTCTTTTGTATTCC | amiR-StCIPK11 PCR扩增 amiR-StCIPK11 for PCR amplification |
StCIPK11-II | GAGAGGCTAGCGCGGAAATAGAATCAAAGAGAATCAATGA | |
StCIPK11-III | GAGAAGCTAGCGCGGTAATAGATTCACAGGTCGTGATATG | |
StCIPK11-IV | GAATCTATTACCGCGCTAGCTTCTCTACATATATATTCCT | |
StCIPK11-A | CTGCAAGGCGATTAAGTTGGGTAAC | |
StCIPK11-B | GCGGATAACAATTTCACACAGGAAACAG | |
Q- StCIPK11-F | TACGACACCCACACATCGTC | 实时荧光定量PCR Quantitative real-time PCR |
Q-StCIPK11-R | TTTGCGAACAATTCGCCTCC | |
StEf1a-F | CAAGGATGACCCAGCCAAG | 内参基因Reference gene |
StEf1a-R | TTCCTTACCTGAACGCCTGT | |
HYG-F | GTGATTTCATATGCGCGATTGCTG | HYG基因PCR扩增PCR amplification for HYG gene |
HYG-R | ACGAGTGCTGGGGCGTCGGTTTCC | |
NPT II-F | GCTATGACTGGGCACAACAG | NPT II基因PCR扩增PCR amplification for NPT II gene |
NPT II-R | ATACCGTAAAGCACGAGGAA |
Table 1 Primer sequences used in the study
引物名称Primer name | 引物序列Primer sequence(5'-3') | 用途Purpose |
---|---|---|
StCIPK11-F | CGGGGGACGAGCTCGGTACCATGGCCTTATTCTCTTCTTCGG | PCR扩增 PCR amplification |
StCIPK11-R | CCATGTCGACTCTAGATTATGGATCCACCTCTGTTGAAATC | |
StCIPK11-I | GATTCTATTTCCGCGCTAGCCTCTCTCTCTTTTGTATTCC | amiR-StCIPK11 PCR扩增 amiR-StCIPK11 for PCR amplification |
StCIPK11-II | GAGAGGCTAGCGCGGAAATAGAATCAAAGAGAATCAATGA | |
StCIPK11-III | GAGAAGCTAGCGCGGTAATAGATTCACAGGTCGTGATATG | |
StCIPK11-IV | GAATCTATTACCGCGCTAGCTTCTCTACATATATATTCCT | |
StCIPK11-A | CTGCAAGGCGATTAAGTTGGGTAAC | |
StCIPK11-B | GCGGATAACAATTTCACACAGGAAACAG | |
Q- StCIPK11-F | TACGACACCCACACATCGTC | 实时荧光定量PCR Quantitative real-time PCR |
Q-StCIPK11-R | TTTGCGAACAATTCGCCTCC | |
StEf1a-F | CAAGGATGACCCAGCCAAG | 内参基因Reference gene |
StEf1a-R | TTCCTTACCTGAACGCCTGT | |
HYG-F | GTGATTTCATATGCGCGATTGCTG | HYG基因PCR扩增PCR amplification for HYG gene |
HYG-R | ACGAGTGCTGGGGCGTCGGTTTCC | |
NPT II-F | GCTATGACTGGGCACAACAG | NPT II基因PCR扩增PCR amplification for NPT II gene |
NPT II-R | ATACCGTAAAGCACGAGGAA |
Fig. 1 StCIPK11 amplified product bands and amiR-StCIPK11 precursor fragments amplification A: StCIPK11 amplification product bands. B: a, b and c are small fragments. C: d fragment. M: DNA marker DL2000. 1-2: Gene fragment. 3-4: d fragment
Fig. 2 Multiple alignments of homologous sequences between StCIPK11 protein and other species Black: Completely identitical sequence. Pink: Sequence identity above 75%. Blue: Sequence identity above 50%. White: Sequence identity below 30%
Fig. 4 Double-digestion verification of recombinant plasmid A: Double-digestion verification of p1300-StCIPK11. B: Double-digestion verification of pBI121-amiR-StCIPK11. M: DNA marker DL2000. 1-2: Digestion fragment
Fig. 5 RT-qPCR detection of the transgenic plants OE-1, OE-2: The overexpressed plants. Ri-1, Ri-2: The interfering expression plants. NT: Non-transgenic plants. Data are mean ±SD(n=3). LSD post hoc test were used for significant analysis. *: P< 0.05; **: P< 0.01. The same below
Fig. 6 Analysis of physiological indices of transgenic and NT potato plants under PEG6000 treatment A: MDA content; B: proline content; C: SOD activity; D: POD activity
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