Cloning of Basic Helix-loop-helix（bHLH）Transcription Factor Gene SabHLH169 in Suaeda aralocaspica and Analysis of Its Resistances to Drought Stress

doi:10.13560/j.cnki.biotech.bull.1985.2023-0049

Abstract

Abstract:

The basic helix-loop-helix（bHLH）transcription factor（TF）is one of the largest TF families in eukaryotes, its members are widely involved in plant growth, development and response to stress. In our previous work of studying photosynthesis key enzyme PEPC-1 in Suaeda aralocaspica, a bHLH TF（SabHLH169）potentially interacting with the SaPEPC-1 promoter was screened. In the present study, the biological characteristics and preliminary function of this gene was explored, which may provide evidence for further studies of bHLH related genes in S. aralocaspica. The gene was obtained by homogous cloning, and the drought-resistant properties of SabHLH169 gene were characterized by bioinformatics and qRT-PCR analyses. The results showed that the open reading frame（ORF）of SabHLH169 contained 2100 bp nucleotides, encoding an protein containing 699 amino acids. The protein had a typical bHLH structural domain at the C-terminus. The prokaryotic expression and Western blot of the recombinant protein showed that the protein was expressed correctly, and the size was consistent with the predicted result. Arabidopsis thaliana transgenic lines of SabHLH169 presented higher drought tolerance at germination stage. Under drought stress, three drought-responsive genes（AtRD22, AtRD29A and AtDREB2A）and three photosynthesis key enzyme genes（AtLhcb2, AtRubicso and AtPEPC）were up-regulated in transgenic A. thaliana. Meanwhile, the PEPC enzyme activity was significantly enhanced. It is speculated that SabHLH169 gene may be involved in response to drought stress.

Key words: Suaeda aralocaspica, bHLH, drought stress, transgenic A. thaliana, photosynthesis key enzyme

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.

Figures/Tables 10

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

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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