Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (11): 328-339.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0049
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YAN Meng-yu(), WEI Xiao-wei, CAO Jing, LAN Hai-yan()
Received:
2023-01-18
Online:
2023-11-26
Published:
2023-12-20
Contact:
LAN Hai-yan
E-mail:1610372465@qq.com;Lanhaiyan@xju.edu.cn
YAN Meng-yu, WEI Xiao-wei, CAO Jing, LAN Hai-yan. Cloning of Basic Helix-loop-helix(bHLH)Transcription Factor Gene SabHLH169 in Suaeda aralocaspica and Analysis of Its Resistances to Drought Stress[J]. Biotechnology Bulletin, 2023, 39(11): 328-339.
名称 Name | 上游引物序列 Forward primer(5'-3') | 下游引物序列 Reverse primer(5'-3') |
---|---|---|
SabHLH169(PCR) | ATGGCTACTCACTTGCAGCAGTTGCTTC | TCAGGCATTAGTTTTTGATTGAAGTAGTTGG |
pSuper1300-SabHLH169 | GCGTCGACATGGCTACTCACTTGCAGCAGTTGCT | CGGGATCCTCAGGCATTAGTTTTTGATTGAAGTA |
pMal-c2X-SabHLH169 | CGGGATCCATGGCTACTCACTTGCAGCAGTTGCTTC | GCGTCGACTCAGGCATTAGTTTTTGATTGAAGTAGTT |
AtHPT | GGTCGCGGAGGCTATGGATGC | GCTTCTGCGGGCGATTTGTGT |
AtRD22(qRT-PCR) | GACTTTCGATTTTACCGACGAG | CGCTACCGGTTTTACCTTTATG |
AtDREB2A(qRT-PCR) | CGTTTCAGGATGAGATGTGTGA | CTCATCGTGCATATAAAACGCA |
AtRD29A(qRT-PCR) | TTCTGTAAGGACGACGTTTACA | CGTACTCGTTACATCCTCTGTT |
AtACTIN(qRT-PCR) | GGTAACATTGTGCTCAGTGGTGG | AACGACCTTAATCTTCATGCTGC |
AtPEPC(qRT-PCR) | AGCCTTCAGGGAACCACAAT | CTCCAAAGACGGGTCGCATG |
AtRubisco(qRT-PCR) | TGGCTTCCTCTATGCTCTCTTC | ACACTTGAGCGGAGTCGGTGCA |
AtLhcb2(qRT-PCR) | ATGTTGGGTGCTCTCGGATG | CGCGTGGATCAAGTTAGGGT |
AtRCA(qRT-PCR) | GGCCGCCGCAGTTTCCACCG | AAAGTTGTAGACACAGGTTCCA |
Table 1 Primer sequences used in this study
名称 Name | 上游引物序列 Forward primer(5'-3') | 下游引物序列 Reverse primer(5'-3') |
---|---|---|
SabHLH169(PCR) | ATGGCTACTCACTTGCAGCAGTTGCTTC | TCAGGCATTAGTTTTTGATTGAAGTAGTTGG |
pSuper1300-SabHLH169 | GCGTCGACATGGCTACTCACTTGCAGCAGTTGCT | CGGGATCCTCAGGCATTAGTTTTTGATTGAAGTA |
pMal-c2X-SabHLH169 | CGGGATCCATGGCTACTCACTTGCAGCAGTTGCTTC | GCGTCGACTCAGGCATTAGTTTTTGATTGAAGTAGTT |
AtHPT | GGTCGCGGAGGCTATGGATGC | GCTTCTGCGGGCGATTTGTGT |
AtRD22(qRT-PCR) | GACTTTCGATTTTACCGACGAG | CGCTACCGGTTTTACCTTTATG |
AtDREB2A(qRT-PCR) | CGTTTCAGGATGAGATGTGTGA | CTCATCGTGCATATAAAACGCA |
AtRD29A(qRT-PCR) | TTCTGTAAGGACGACGTTTACA | CGTACTCGTTACATCCTCTGTT |
AtACTIN(qRT-PCR) | GGTAACATTGTGCTCAGTGGTGG | AACGACCTTAATCTTCATGCTGC |
AtPEPC(qRT-PCR) | AGCCTTCAGGGAACCACAAT | CTCCAAAGACGGGTCGCATG |
AtRubisco(qRT-PCR) | TGGCTTCCTCTATGCTCTCTTC | ACACTTGAGCGGAGTCGGTGCA |
AtLhcb2(qRT-PCR) | ATGTTGGGTGCTCTCGGATG | CGCGTGGATCAAGTTAGGGT |
AtRCA(qRT-PCR) | GGCCGCCGCAGTTTCCACCG | AAAGTTGTAGACACAGGTTCCA |
Fig. 1 Extraction of total RNA and PCR amplification of SabHLH169 ORF in S. aralocaspica A: Total RNA of S. aralocaspica seedlings. 1,2: Two samples of total RNA; B: PCR amplification of SabHLH169 ORF; M1, M2: DL2000, DL5000 DNA markers
Fig. 2 Analysis of amino acid sequences of SabHLH169 in S. aralocaspica N-terminal underline indicates bHLH-MYC_N domain; C-terminal underline indicates HLH domain
Fig. 3 Analysis of the phylogenetic tree and bHLH domain of SabHLH169 in S. aralocaspica A: Analysis of the phylogenetic tree of SabHLH169, which was constructed based on the NJ method using MEGA X software with 1 000 bootstraps. B: Multiple alignment of the bHLH motif of amino acid sequences between SabHLH169 and the closely related species. Different colored letters indicate different degrees of conservation(dark blue > pink red > light blue > grey; the dark background indicates sequence identical); the upper line indicates the HLH domain
Fig. 4 Analysis of the conserved motifs of SabHLH169 in C. quinoa and the closely related species A: Schematic diagram of the base enrichment of the top 10 motifs; boxes in different colors represent motif1-motif10; the height of the letter indicates the frequency of the amino acid, and the higher the height, the greater the frequency. B: Conserved motif analysis of bHLH transcription factors in different species
Fig. 5 Construction of procaryotic expression vector pMal-c2X -SabHLH169 and SDS-PAGE and Western blot analysis of SabHLH169 A: Amplification of SabHLH169. B: PCR identification of pMal-c2X-SabHLH169 transformed colonies; -: negative control. C: Double digestion of recombinant plasmid. D: SDS-PAGE results. M: Protein marker; 1: Before induction of pMal-c2X-SabHLH169; 2-4: after induction of pMal-c2X-SabHLH169(0.5 mmol/L, 0.8 mmol/L, 1 mmol/L IPTG); 5, 6: before and after induction of pMal-c2X. E: Western blot results; 1: After induction of pMal-c2X-SabHLH169(0.5 mmol/L IPTG); 2: before induction of pMal-c2X-SabHLH169; 3: before induction of pMal-c2X
Fig. 6 Construction of plant expression vector pSuper1300-SabHLH169 and identification of transgenic A. thaliana with SabHLH169 overexpression A: Amplification of SabHLH169. B: PCR identification of pSuper1300-SabHLH169 transformed colonies. C: Double digestion of recombinant plasmid. D: Identification of the T0 generation of transgenic A. thaliana overexpress SabHLH169. E, F: PCR and RT-qPCR identification of SabHLH169 in three T1 transgenic lines OE5, OE16, OE20. +: positive control; -: negative control. WT: Wild type A. thaliana; OE5, OE16 and OE20: three transgenic A. thaliana lines with SabHLH169 overexpression. M: DL 5000 DNA marker
株系 Strain | 阳性苗 Positive seedlings | 阴性苗 Negative seedlings | 卡方检验 Chi-square test(P-value) | 株系 Strain | 阳性苗 Positive seedling | 阴性苗 Negative seedling | 卡方检验 Chi-square test(P-value) | |
---|---|---|---|---|---|---|---|---|
OE1 | 134 | 37 | 0.310 | OE13 | 78 | 31 | 0.407 | |
OE5 | 81 | 26 | 0.867 | OE14 | 64 | 27 | 0.304 | |
OE6 | 70 | 33 | 0.099 | OE16 | 65 | 26 | 0.431 | |
OE7 | 79 | 40 | 0.030 | OE17 | 62 | 26 | 0.325 | |
OE8 | 78 | 32 | 0.322 | OE18 | 54 | 25 | 0.173 | |
OE11 | 64 | 29 | 0.169 | OE19 | 54 | 26 | 0.121 | |
OE12 | 61 | 24 | 0.491 | OE20 | 56 | 21 | 0.645 |
Table 2 Antibiotic screening of SabHLH169 transgenic A. thaliana T1 generation for 3∶1 segregation ratio lines
株系 Strain | 阳性苗 Positive seedlings | 阴性苗 Negative seedlings | 卡方检验 Chi-square test(P-value) | 株系 Strain | 阳性苗 Positive seedling | 阴性苗 Negative seedling | 卡方检验 Chi-square test(P-value) | |
---|---|---|---|---|---|---|---|---|
OE1 | 134 | 37 | 0.310 | OE13 | 78 | 31 | 0.407 | |
OE5 | 81 | 26 | 0.867 | OE14 | 64 | 27 | 0.304 | |
OE6 | 70 | 33 | 0.099 | OE16 | 65 | 26 | 0.431 | |
OE7 | 79 | 40 | 0.030 | OE17 | 62 | 26 | 0.325 | |
OE8 | 78 | 32 | 0.322 | OE18 | 54 | 25 | 0.173 | |
OE11 | 64 | 29 | 0.169 | OE19 | 54 | 26 | 0.121 | |
OE12 | 61 | 24 | 0.491 | OE20 | 56 | 21 | 0.645 |
Fig. 7 Seed germination of SabHLH169 transgenic A. thaliana under mannitol treatment A: Phenotype analysis. B: Statistics of seed germination percentage
Fig. 8 Analysis of expression patterns of drought and photosynthesis related genes and PEPC enzyme activity in transgenic A. thaliana A-C: Expressions of drought stress-related genes in transgenic A.thaliana. D-G: Expressions of photosynthesis related genes. H: PEPC enzyme activity in transgenic A. thaliana. Different lowercase letters indicate significant differences in gene expression level at the same condition between different lines(P<0.05)
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