Construction of Alfalfa Yeast Expression Library and Screening and Identification of Salt-tolerance Genes

doi:10.13560/j.cnki.biotech.bull.1985.2025-0844

Abstract

Abstract:

Objective To construct a cDNA library of salt-induced genes in alfalfa (Medicago sativa ), screen the candidate salt-tolerant genes and verify it. Method Alfalfa seedlings were treated with 1% NaCl, after which plant materials were collected and total RNA was extracted. Following reverse transcription, a yeast cDNA expression library was constructed. The library plasmids and the empty plasmids (pYES2-NTB) were transformed into yeast strains to determine the NaCl concentration for library screening and to identify candidate salt-tolerant genes. A total of 196 single yeast colonies were selected for salt-tolerance assays, followed by PCR identification and sequencing analysis. Four of the candidate genes were chosen for transgenic functional analysis. Result High-quality RNA was obtained, and a cDNA library of salt-induced gene expression in alfalfa was successfully established. The library capacity was 7.68×10⁷ CFU/mL and the total number of clones is 1.536×10⁸ CFU. The cDNA library and blank plasmid were transferred into the yeast strain INVSC1. The monoclonal yeast colonies still survived in the library culture medium under SG/-Ura+1.5 mol/L NaCl, while that in the control group no longer survived on 1.3 mol/L NaCl culture medium. After sequence alignment, 75 different genes were identified, the four candidate salt-tolerant genes were screened out, Msa1025270, Msa0320180, Msa0819320, and Msa0859120. Through the NCBI website, it was found that the functions of these four genes were related to proteins such as abscisic acid receptors, response to ABA, abscisic acid and environmental stress induction, and early dehydration response. The results of transgenic functional identification showed that the expressions of the four candidate genes significantly enhanced the resistance of plants to NaCl. Conclusion The library bacterial liquid has good salt resistance, and the verified results of salt-tolerant gene transgenic plants show that the four gene Msa1025270, Msa0320180, Msa0819320, and Msa0859120 have the function related to salt-tolerance.

Key words: Medicago sativa L., yeast expression library, NaCl concentration screening, salt-tolerant gene, transgenic verification, tobacco, gene annotation, Agrobacterium transformation

ZHANG Chi-hao, LIU Jin-nan, DONG Jia-le, CHAO Yue-hui. Construction of Alfalfa Yeast Expression Library and Screening and Identification of Salt-tolerance Genes[J]. Biotechnology Bulletin, 2026, 42(2): 239-249.

Figures/Tables 11

Table 1 Primers and sequences

引物名称 Primer name	序列 Sequence （5′‒3′）	用途 Usage
CDS III/3' PCR Primer	GACGATAAGGTACCTAAGGATCCACCGGG	逆转录 Reverse transcription
SMART IV Oligonucleotide	GTGCTGGATATCTGCAGAATTCCATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN	逆转录 Reverse transcription
P1-F	GATCTGTACGACGATGACGATAAGGTACCTAAGGATCCAGGG	ds cDNA扩增 ds cDNA amplification
P2-F	GATCTGTACGACGATGACGATAAGGTACCTAAGGATCCAAGGG
P3-F	GATCTGTACGACGATGACGATAAGGTACCTAAGGATCCAAAGGG
P4-R	ACTCGAGCGGCCGCCACTGTGCTGGATATCTGCAGAATTCCA
pYES2-NTB-F	CAGCTGTAATACGACTCACTATAGG	文库菌落PCR鉴定 PCR identification of library colonies
pYES2-NTB-R	AGGGTTAGGGATAGGCTTACCTTCG	文库菌落PCR鉴定 PCR identification of library colonies
NtActin-F	AGGTGGAGACATGGGTGGTG	烟草内参基因表达分析 Analysis of tobacco internal reference gene expression
NtActin-R	TCATTAGGCACACAGATCTCTG
MsG5270-qRT-F	CGGCTTGCTAACTACCGTTCT	基因表达分析 Gene expression analysis
MsG5270-qRT-R	TTGATTTGATCTCGTTGAGGC
MsG0180-qRT-F	GCTGATGAAATCGTCCCA
MsG0180-qRT-R	CAACCCTGTTCCTCCCAC
MsG9320-qRT-F	CTCTTGGCTATGGTTCTTC
MsG9320-qRT-R	TTGGCATCGTTTACTTCA
MsG9120-qRT-F	TCCAATGTTCGTCCCACT
MsG9120-qRT-R	CTCTTTCCCATCTTCGTG
MsG5270-F	TCACCATTTACGAACGATACATGTTACCAAACCCAACAAC	克隆及表达载体构建 Construction of cloning and expression vectors
MsG5270-R	ATGGTGATGCATTCCCGGGTTTATCGTTGATTTGATCTCG
MsG9320-F	TCACCATTTACGAACGATACATGGATTCGAGAAAAGCAAT
MsG9320-R	ATGGTGATGCATTCCCGGGTTCAATTCTGAGTGTTGTCCT
MsG9120-F	TCACCATTTACGAACGATACATGGAAGTTCTTTCTGCTAC
MsG9120-R	ATGGTGATGCATTCCCGGGTCTACCTGGGCTGATGAATAG
MsG0180-F	TCACCATTTACGAACGATACATGGGTGTTTTTACTTTCAA
MsG0180-R	ATGGTGATGCATTCCCGGGTCTAGTAATTAGGGTTTGCTA
3302Y-F	TGACGCACAATCCCACTATCCTT	植物表达载体PCR鉴定 PCR identification of plant expression vector
NOS181-R	CGTATTAAATGTATAATTGCGGGAC	植物表达载体PCR鉴定 PCR identification of plant expression vector

Fig. 1 RNA extraction and identification of synthesized ds cDNAA: Sample RNA extraction (M: Marker, 1‒2: alfalfa (Medicago sativa) samples). B: Amplified results of synthesized ds cDNA (M: Marker; 1: P1-F/P4-R amplified ds cDNA; 2: P2-F/P4-R amplified ds cDNA; 3: P3-F/P4-R amplified ds cDNA). C: Purified and removed small fragments (M: Marker; 1: results after the three ds cDNAs were mixed and purified)

Fig. 2 Count and library identification of Escherichia coli colonyA: Library capacity and clone count. B: PCR identification of library quality (M: DNA marker; 1‒24: 24 different cloned colonies)

Fig. 3 PCR identification of yeast transformed with empty vector (A) and library cultures (B)In Fig. A, M: marker, 1-4: yeast transformation empty vector. In Fig. B, M: DNA marker, 1-24: yeast library colonies

Fig. 4 NaCl concentration screening and comparisonA: Control group. B: Library bacterial solution group

Fig. 5 Positive clone plate and results of rotation verificationA: The result of applying the library bacterial solution on 1.5 mol/L NaCl solid culture medium. B: Verification results of monoclonal colony rotation (144). C: Verification results of monoclonal colony rotation (96). : Control group

Fig. 6 Gene ontology (GO) categorization

Fig. 7 Orthologous groups (COG) classification

Fig. 8 Detection vector colony PCRA: Vector construction colony PCR (1: pBⅠ-MsG5270; 2: pBⅠ-MsG0180; 3: pBⅠ-MsG9320; 4: pBⅠ-MsG9120). B: Agrobacterium transformed colony PCR (1: pBⅠ-MsG5270; 2: pBⅠ- MsG0180; 3: pBⅠ-MsG9320; 4: pBⅠ-MsG9120). C: cDNA PCR identification (1: pBⅠ-MsG5270; 2: pBⅠ-MsG0180; 3: pBⅠ-MsG9320; 4: pBⅠ-MsG9120)

Fig. 9 Expression analysis of MsG0180, MsG9320, MsG5270, and MsG9120Different letters indicate significant differences at P<0.05 level. The data are in means ±SE (n=3), WT: Wild type; 1‒8: transgenic plants

Fig. 10 Analysis of salt tolerances in transgenic plantsWT: Wild type; three plants on the right are transgenic ones

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