Biotechnology Bulletin ›› 2021, Vol. 37 ›› Issue (7): 107-117.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0599
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GAO Bo(), MA Juan, LI Xiu-hua, LI Jiao-sheng, WANG Rong-yan, CHEN Shu-long()
Received:
2021-05-08
Online:
2021-07-26
Published:
2021-08-13
Contact:
CHEN Shu-long
E-mail:gaobo89@163.com;chenshulong65@163.com
GAO Bo, MA Juan, LI Xiu-hua, LI Jiao-sheng, WANG Rong-yan, CHEN Shu-long. Cloning and Functional Analysis of Dd-mel-26 Gene from Ditylenchus destructor[J]. Biotechnology Bulletin, 2021, 37(7): 107-117.
引物Primer | 序列信息Sequence(5'-3') | 备注Remarks |
---|---|---|
mel-26F | GATGCCTTCTGAATCTGTTG | cDNA中间区段扩增 |
mel-26R | GCAGTAAGAACCAGGAACAG | |
mel26-5GSP1 | GTCGGAATGTGACGGGTTTG | 5'RACE引物 |
mel26-5GSP2 | TTTCCGTTTGTGAGGCGTTG | |
mel26-3GSP1 | ATCGGGCAGATTTGTTACGC | 3'RACE引物 |
mel26-3GSP2 | CCCGCAGAGCTTTCTTCGTC | |
QT | CCAGTGAGCAGAGTGACGAGGACTCGAGCTCAAGCTTTTTTTTTTTTTTTTT | RACE加帽引物 |
QO | CCAGTGAGCAGAGTGACG | |
QI | GAGGACTCGAGCTCAAGC | |
mel-26RT | CACAAGAACGGTGATGCTAT | 5'RACE反转录引物 |
mel-26GF | GATGCCTTCTGAATCTGTTG | 基因组序列扩增 |
mel-26GR | ACATCACAGACATAGCCAGA | |
26T7aF | TAATACGACTCACTATAGGGTGCCTTCTGAATCTGTTGCC | dsRNA合成模板扩增 |
26T7aR | TAATACGACTCACTATAGGGGAATGTGACGGGTTTGGATG | |
26T7bF | TAATACGACTCACTATAGGGTCCAAACCCGTCACATTCCG | |
26T7bR | TAATACGACTCACTATAGGGACGAGCTGCGTCCGCAATAA | |
26T7cF | TAATACGACTCACTATAGGGCGTCCGAAATCAACGAACTT | |
26T7cR | TAATACGACTCACTATAGGGCGGAGATGGAACTTAGAGGG | |
GFP_L1 | TAATACGACTCACTATAGGGGACTTCTTCAAGTCCGCCAT | 阴性对照dsRNA模板合成引物 |
GFP_R1 | TAATACGACTCACTATAGGGAAGTTCACCTTGATGCCGTT | |
mel26q_L | TTGAAATGCGAATTCATCCA | qPCR扩增引物 |
mel26q_R | TCATAATTGCAAACGCCAAG | |
actin_L | AGGACTTGTACGCCAACACC | |
actin_R | ACATTTGCTGGAAGGTGGAG | |
mel26-probe-F | TCCAAACCCGTCACATTCCG | Southern blotting探针引物 |
mel26-probe-R | ACGAGCTGCGTCCGCAATAA |
Table 1 Primer information used in this study
引物Primer | 序列信息Sequence(5'-3') | 备注Remarks |
---|---|---|
mel-26F | GATGCCTTCTGAATCTGTTG | cDNA中间区段扩增 |
mel-26R | GCAGTAAGAACCAGGAACAG | |
mel26-5GSP1 | GTCGGAATGTGACGGGTTTG | 5'RACE引物 |
mel26-5GSP2 | TTTCCGTTTGTGAGGCGTTG | |
mel26-3GSP1 | ATCGGGCAGATTTGTTACGC | 3'RACE引物 |
mel26-3GSP2 | CCCGCAGAGCTTTCTTCGTC | |
QT | CCAGTGAGCAGAGTGACGAGGACTCGAGCTCAAGCTTTTTTTTTTTTTTTTT | RACE加帽引物 |
QO | CCAGTGAGCAGAGTGACG | |
QI | GAGGACTCGAGCTCAAGC | |
mel-26RT | CACAAGAACGGTGATGCTAT | 5'RACE反转录引物 |
mel-26GF | GATGCCTTCTGAATCTGTTG | 基因组序列扩增 |
mel-26GR | ACATCACAGACATAGCCAGA | |
26T7aF | TAATACGACTCACTATAGGGTGCCTTCTGAATCTGTTGCC | dsRNA合成模板扩增 |
26T7aR | TAATACGACTCACTATAGGGGAATGTGACGGGTTTGGATG | |
26T7bF | TAATACGACTCACTATAGGGTCCAAACCCGTCACATTCCG | |
26T7bR | TAATACGACTCACTATAGGGACGAGCTGCGTCCGCAATAA | |
26T7cF | TAATACGACTCACTATAGGGCGTCCGAAATCAACGAACTT | |
26T7cR | TAATACGACTCACTATAGGGCGGAGATGGAACTTAGAGGG | |
GFP_L1 | TAATACGACTCACTATAGGGGACTTCTTCAAGTCCGCCAT | 阴性对照dsRNA模板合成引物 |
GFP_R1 | TAATACGACTCACTATAGGGAAGTTCACCTTGATGCCGTT | |
mel26q_L | TTGAAATGCGAATTCATCCA | qPCR扩增引物 |
mel26q_R | TCATAATTGCAAACGCCAAG | |
actin_L | AGGACTTGTACGCCAACACC | |
actin_R | ACATTTGCTGGAAGGTGGAG | |
mel26-probe-F | TCCAAACCCGTCACATTCCG | Southern blotting探针引物 |
mel26-probe-R | ACGAGCTGCGTCCGCAATAA |
Fig. 1 Amplification results of the cDNA and genomic sequence of Dd-mel-26 A:3' RACE;B:5' RACE;C:intermediate region of the cDNA;D:Dd-mel-26 genomic sequence
Fig. 4 Tertiary structure of Dd-MEL-26 protein The orange monomer is 26_ A,the violet monomer is 26_ B,the two red α-helix structures are 3-box domains,and the butterfly-like structure rich in β-strand in the upper part of the structure is the MATH domain(MATH_ A and MATH_B),and the butterfly-like structure rich in α-helix in the lower part is BTB domain(BTB_ A and BTB_B)
Fig. 5 Neighbor-Joining tree calculated from the sequences of Dd-mel-26 amino acid sequence Only bootstrap values more than 70% are listed at the branches points
Fig. 9 Relative expressions of target gene(A),average passing rate in sand column(B),and average reproduction(C)in the Dd-mel-26 dsRNA-treated nematodes
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