紫花苜蓿酵母表达文库的构建及候选耐盐基因的筛选与鉴定

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

摘要/Abstract

摘要：

目的构建紫花苜蓿（Medicago sativa）盐诱导基因表达文库，筛选候选耐盐基因并进行验证。方法使用1% NaCl处理紫花苜蓿幼苗，收集植物材料并提取总RNA，反转录后构建基因表达酵母文库。将文库质粒与空白质粒（pYES2-NTB）转入酵母菌株，确定用于文库筛选的NaCl浓度及候选耐盐基因。挑选196个单克隆菌落进行耐盐性分析，并进行PCR鉴定和测序分析。选择其中4个候选基因进行转基因功能分析。结果获得高质量RNA，并成功建立紫花苜蓿盐诱导基因表达文库，文库库容为7.68×10⁷ CFU/mL，总克隆数为1.536×10⁸ CFU。文库菌液在SG/-Ura+1.5 mol/L NaCl条件下仍有单克隆酵母菌落存活，而对照组则在1.3 mol/L NaCl培养基上已无法存活。测序结果经序列比对后，鉴定出75个不同基因。筛选并获得4个候选耐盐基因Msa1025270、Msa0320180、Msa0819320、Msa0859120，通过NCBI网站查询得这4个基因的功能与脱落酸受体、ABA反应、脱落酸和环境胁迫诱导、脱水早期反应等蛋白有关。转基因功能鉴定结果显示，4个候选基因表达能够显著提高植物对NaCl的抵抗能力。结论文库菌液具有良好的耐盐抗性，候选耐盐基因转基因植株验证结果表明，Msa1025270、Msa0320180、Msa0819320、Msa0859120 4个基因具有耐盐相关功能。

关键词: 紫花苜蓿, 酵母表达文库, NaCl浓度筛选, 耐盐基因, 转基因功能, 烟草, 基因注释, 农杆菌转化

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

张驰昊, 刘晋囡, 董家乐, 晁跃辉. 紫花苜蓿酵母表达文库的构建及候选耐盐基因的筛选与鉴定[J]. 生物技术通报, 2026, 42(2): 239-249.

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.

图/表 11

表1 引物及序列

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

图1 RNA提取及合成ds cDNA的鉴定A：样品RNA提取（M：Marker；1-2：紫花苜蓿样品）；B：合成ds cDNA扩增结果（M：Marker；1：P1-F/P4-R扩增ds cDNA；2：P2-F/P4-R扩增ds cDNA；3：P3-F/P4-R扩增ds cDNA）；C：纯化与去除小片段（M：Marker，1：3种ds cDNA混合纯化后结果）

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)

图2 大肠杆菌菌落计数及文库鉴定A：文库库容克隆计数；B：文库质量PCR鉴定（M：DNA marker；1‒24：24个不同单克隆菌落）

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)

图3 空载（A）、文库菌液（B）酵母转化PCR验证图A中，M：Marker，1-4：空载酵母转化；图B中，M：DNA marker，1-24：酵母文库菌落

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

图4 NaCl浓度筛选对比A：对照组；B：文库菌液组

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

图5 阳性克隆菌板及回转验证结果A：文库菌液涂布于1.5 mol/L NaCl固体培养基结果图；B：单克隆菌落回转验证结果（144个）；C：单克隆菌落回转验证结果（96个）；：对照组

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

图6 GO分类

Fig. 6 Gene ontology (GO) categorization

图7 COG分类

Fig. 7 Orthologous groups (COG) classification

图8 检测载体菌落PCRA：载体构建菌落PCR；B：农杆菌转化菌落PCR；C：cDNA PCR鉴定

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)

图9 MsG0180、MsG9320、MsG5270、MsG9120表达分析不同字母表示P<0.05水平差异显著，数值为平均值±标准误（n=3）. WT：野生型；1‒8：转基因植株

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

图10 转基因植株耐盐性分析WT：野生型；右侧3株为转基因植株

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

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