草业学报 ›› 2024, Vol. 33 ›› Issue (10): 55-73.DOI: 10.11686/cyxb2023460
周昕越1(), 蒋庆雪1, 贾会丽2, 马琳1, 樊璐1, 王学敏1(
)
收稿日期:
2023-11-09
修回日期:
2024-01-12
出版日期:
2024-10-20
发布日期:
2024-07-15
通讯作者:
王学敏
作者简介:
Corresponding author. E-mail: wangxuemin@caas.cn基金资助:
Xin-yue ZHOU1(), Qing-xue JIANG1, Hui-li JIA2, Lin MA1, Lu FAN1, Xue-min WANG1(
)
Received:
2023-11-09
Revised:
2024-01-12
Online:
2024-10-20
Published:
2024-07-15
Contact:
Xue-min WANG
摘要:
BBX家族转录因子参与光形态建成、成花生理、避荫反应、种子生长发育、激素信号转导及对逆境胁迫的响应等植物重要生长发育过程。紫花苜蓿是耐盐性较强的牧草饲料作物,其品质优良,有“牧草之王”的美誉。发掘并探索紫花苜蓿MsBBX20基因响应非生物胁迫的分子机理,有助于揭示紫花苜蓿抗逆生物学基础,为紫花苜蓿抗逆分子育种提供新的基因资源。通过RT-PCR、3′/5′RACE PCR技术克隆得到MsBBX20基因cDNA序列,生物信息学分析发现其CDS全长834 bp,编码278个氨基酸,该基因编码的蛋白质为锌指结构蛋白家族成员。利用qRT-PCR技术分析MsBBX20在紫花苜蓿不同组织和不同非生物胁迫下的表达模式。通过基因枪轰击技术,在洋葱表皮瞬时表达MsBBX20进行亚细胞定位。克隆得到大小为1737 bp的MsBBX20启动子序列并分析了顺式作用元件,该启动子可驱动GUS报告基因在烟草的叶、茎和根中高效表达。将MsBBX20启动子序列与包含GUS基因的pCAMBIA-1301载体连接,瞬时转化烟草并通过组织化学染色对不同组织进行GUS活性分析。构建pCAMBIA3301-MsBBX20植物超表达载体,通过农杆菌介导的方法遗传转化野生型拟南芥,获得超表达MsBBX20的拟南芥株系。用不同浓度盐溶液(150、200和300 mmol·L-1 NaCl)处理拟南芥转基因株系并分析其MDA含量和抗氧化酶活性。进化分析表明紫花苜蓿MsBBX20蛋白与红三叶TpBBX20的亲缘关系最近。MsBBX20蛋白定位于细胞核。MsBBX20基因在花中表达量最高,且MsBBX20可以响应干旱、盐、冷、脱落酸、赤霉素、光照等多种非生物胁迫。遗传转化获得MsBBX20过表达拟南芥株系,并选择3个高表达阳性株系进行功能验证。在不同浓度NaCl处理条件下,过量表达MsBBX20的拟南芥株系的丙二醛(MDA)含量显著低于对照,而超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和过氧化物酶(POD)活性均显著高于对照。克隆获得紫花苜蓿锌指蛋白转录因子基因MsBBX20,该基因在花中表达量最高,能够响应多种非生物逆境胁迫和外源激素处理,表明MsBBX20可能参与紫花苜蓿的多个逆境响应过程。进一步的研究证明MsBBX20可能通过调节植物的抗氧化酶系统,缓解逆境造成的植物细胞氧化损伤,提高转基因拟南芥对盐胁迫的抗性,进而增强植株的耐盐性。
周昕越, 蒋庆雪, 贾会丽, 马琳, 樊璐, 王学敏. 紫花苜蓿MsBBX20基因克隆及耐盐功能分析[J]. 草业学报, 2024, 33(10): 55-73.
Xin-yue ZHOU, Qing-xue JIANG, Hui-li JIA, Lin MA, Lu FAN, Xue-min WANG. Cloning and salt-tolerance functional analysis of alfalfa MsBBX20 gene[J]. Acta Prataculturae Sinica, 2024, 33(10): 55-73.
引物名称Primer name | 引物序列Primer sequence (5'→3′) | 用途Function |
---|---|---|
BBX-3′-RACE | GGACCCAGATTCATCATCCACCA | 基因克隆Gene cloning |
BBX-5′-RACE | CAGAGCCAATACTGGTGGATGATGA | |
BBX-ORF-F | ATGAAGATCCAATGTGATGTGTGTG | |
BBX-ORF-R | TTATCGAAAGTGTCTAGATTTTTTTATG | |
BBX-Real-up | CCACCAGTATTGGCTCTGAACC | |
BBX-Real-low | CCTGTGTCACTAGCCATGTTGTCT | |
MsBBX20-SP1 | AGGGTAGTCTTTGGAGTTGAGGTGA | 启动子克隆Promoter cloning |
MsBBX20-SP2 | TTGTTGGCACGGTGGATGGTATGAT | |
MsBBX20-SP3 | CCTCAGCTTTCTCACACACATCAC | |
BBX-1301-EcoRⅠ | TATGACCATGATTACGAATTCCGTACCTCACTAGTACCCAATTACA | |
BBX-1301-NcoⅠ | ACCCTCAGATCTACCATGGCCTCAGCTTTCTCACACACATCACA | |
BBX-PA7-GFP-Xho | TACTCGAGATGAAGATCCAATGTGATGTGT | |
BBX-PA7-GFP-SpeⅠ | GCACTAGTTCGAAAGTGTCTAGATTTTTTTA | |
BBX-3301-BglⅡ | TGACCATGGTAGATCTGATGAAGATCCAATGTGATGTGTGTG | |
BBX-3301-BstEⅡ | ATTCGAGCTGGTCACCTTATCGAAAGTGTCTAGATTTTTTTATG | |
35S-3301-F | AACAGAACTCGCCGTAAAGACT | |
MsActin2-F | 内参基因Reference genes | |
MsActin2-R | ||
AtActin-F | GGACAAGTTATCACCATCGG | |
AtActin-R | ATTGGGCAGTTTCTTTGGTAGC |
表1 所用引物信息
Table 1 Primer information used in the experiment
引物名称Primer name | 引物序列Primer sequence (5'→3′) | 用途Function |
---|---|---|
BBX-3′-RACE | GGACCCAGATTCATCATCCACCA | 基因克隆Gene cloning |
BBX-5′-RACE | CAGAGCCAATACTGGTGGATGATGA | |
BBX-ORF-F | ATGAAGATCCAATGTGATGTGTGTG | |
BBX-ORF-R | TTATCGAAAGTGTCTAGATTTTTTTATG | |
BBX-Real-up | CCACCAGTATTGGCTCTGAACC | |
BBX-Real-low | CCTGTGTCACTAGCCATGTTGTCT | |
MsBBX20-SP1 | AGGGTAGTCTTTGGAGTTGAGGTGA | 启动子克隆Promoter cloning |
MsBBX20-SP2 | TTGTTGGCACGGTGGATGGTATGAT | |
MsBBX20-SP3 | CCTCAGCTTTCTCACACACATCAC | |
BBX-1301-EcoRⅠ | TATGACCATGATTACGAATTCCGTACCTCACTAGTACCCAATTACA | |
BBX-1301-NcoⅠ | ACCCTCAGATCTACCATGGCCTCAGCTTTCTCACACACATCACA | |
BBX-PA7-GFP-Xho | TACTCGAGATGAAGATCCAATGTGATGTGT | |
BBX-PA7-GFP-SpeⅠ | GCACTAGTTCGAAAGTGTCTAGATTTTTTTA | |
BBX-3301-BglⅡ | TGACCATGGTAGATCTGATGAAGATCCAATGTGATGTGTGTG | |
BBX-3301-BstEⅡ | ATTCGAGCTGGTCACCTTATCGAAAGTGTCTAGATTTTTTTATG | |
35S-3301-F | AACAGAACTCGCCGTAAAGACT | |
MsActin2-F | 内参基因Reference genes | |
MsActin2-R | ||
AtActin-F | GGACAAGTTATCACCATCGG | |
AtActin-R | ATTGGGCAGTTTCTTTGGTAGC |
生物信息学工具 Bioinformatics tools | 网址 Website | 用途 Purpose |
---|---|---|
Open reading frame finder | https://www.ncbi.nlm.nih.gov/orffinder/ | 开放阅读框分析Open reading frame analysis |
SMART | https://smart.embl.de/ | 蛋白质保守结构域分析Protein conserved domain analysis |
Expasy-ProtParam tool | https://web.expasy.org/protparam/ | 蛋白质理化性质分析Analysis of physical and chemical properties of proteins |
PRABI | https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa%20_sopma.html | 蛋白质二级结构分析Protein secondary structure analysis |
BLAST | https://blast.ncbi.nlm.nih.gov/Blast.cgi | 同源蛋白序列分析Homologous protein sequence analysis |
DNAMAN | https://www.lynnon.com/qa.html | 多序列比对Multiple sequence alignment |
Plant CARE | https://bioinformatics.psb.ugent.be/webtools/plantcare/html/ | 顺式作用元件分析Cis-acting element analysis |
MEGA | https://www.megasoftware.net/ | 系统进化树构建Phylogenetic tree construction |
表2 所用生物信息学工具
Table 2 Bioinformatics tools used
生物信息学工具 Bioinformatics tools | 网址 Website | 用途 Purpose |
---|---|---|
Open reading frame finder | https://www.ncbi.nlm.nih.gov/orffinder/ | 开放阅读框分析Open reading frame analysis |
SMART | https://smart.embl.de/ | 蛋白质保守结构域分析Protein conserved domain analysis |
Expasy-ProtParam tool | https://web.expasy.org/protparam/ | 蛋白质理化性质分析Analysis of physical and chemical properties of proteins |
PRABI | https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa%20_sopma.html | 蛋白质二级结构分析Protein secondary structure analysis |
BLAST | https://blast.ncbi.nlm.nih.gov/Blast.cgi | 同源蛋白序列分析Homologous protein sequence analysis |
DNAMAN | https://www.lynnon.com/qa.html | 多序列比对Multiple sequence alignment |
Plant CARE | https://bioinformatics.psb.ugent.be/webtools/plantcare/html/ | 顺式作用元件分析Cis-acting element analysis |
MEGA | https://www.megasoftware.net/ | 系统进化树构建Phylogenetic tree construction |
图1 MsBBX20的克隆与cDNA全长及对应蛋白序列A:紫花苜蓿MsBBX20基因RACE-PCR结果;M: DL2000;1:3′-RACE 产物;2:5′-RACE 产物;3:ORF扩增产物。A:Results of RACE-PCR for MsBBX20 gene of M. sativa; M: DL2000 DNA marker; 1: 3′-RACE product; 2: 5′-RACE product; 3: ORF amplified product; B: MsBBX20的cDNA全序列 Full cDNA sequence of MsBBX20.
Fig.1 Cloning and full-length cDNA sequence of MsBBX20
图2 MsBBX20蛋白结构特征分析A: MsBBX20蛋白保守结构域 MsBBX20 protein conserved domain; B: MsBBX20蛋白的亲疏水性 The hydrophilicity of MsBBX20 protein; C: MsBBX20蛋白的二级结构;蓝色:α螺旋;绿色:β折叠;紫色:无规则卷曲;红色:延伸链。Secondary structure of MsBBX20 protein; Blue: Alpha helix; Green: Beta fold; Purple: Random curling; Red: Extended chain.
Fig.2 Structural characterization analysis of MsBBX20 protein
图3 MsBBX20编码氨基酸多序列比对Ms:紫花苜蓿;Mt:蒺藜苜蓿;Ca:鹰嘴豆;Gs:野大豆;Tp:红三叶;Va:赤豆;Gm:大豆;Ps:豌豆。下同。Ms: M. sativa; Mt: M. truncatula; Ca: C. arietinum; Gs: G. soja; Tp: T. pratense; Va: V. angularis; Gm: G. max; Ps: P. sativum. The same below.
Fig.3 Multiple sequence alignment of MsBBX20 encoded amino acids
图6 MsBBX20基因启动子的克隆与表达分析A: MsBBX20基因启动子的克隆,M: DL2000;1:MsBBX20基因启动子长度。A: Cloning of the promoter of MsBBX20 gene, M: DL2000; 1: MsBBX20 gene promoter length. B: MsBBX20:GUS转基因烟草的GUS染色;图中比例尺为1 cm; CK为未转基因烟草组织,OE1~OE3为MsBBX20:GUS转基因烟草的不同组织。B: MsBBX20:GUS staining of transgenic tobacco; the scale in the figure is 1 cm; CK is an unmodified tobacco tissue, and OE1-OE3 are different tissues of MsBBX20:GUS transgenic tobacco.
Fig.6 Cloning and expression analysis of MsBBX20 gene promoter
元件名称 Element name | 序列 Sequence | 数量 Amount | 功能 Function |
---|---|---|---|
AE-box | AGAAACTT/AGAAACAT/AGAAACAA | 3 | 光响应模块的一部分Part of a module for light response |
CATT-motif | GCATTC | 2 | 光响应元件的一部分Part of a light responsive element |
G-Box | CACGTT | 2 | 参与光反应的顺式作用调节元件Cis-acting regulatory element involved in light responsiveness |
ABRE | ACGTG | 1 | 参与脱落酸响应的顺式作用元件Cis-acting elements involved in the abscisic acid response |
HSE | CCGAAA | 1 | 参与低温响应的顺式作用元件Cis-acting element involved in low-temperature responsiveness |
MBS | CGGTCA | 1 | MYB结合位点MYB binding site |
MRE | AACCTAA | 1 | 光诱导的MYB响应元件MYB binding site involved in light responsiveness |
Sp1 | CC(G/A)CCC | 1 | 光响应元件Light responsive element |
TATC-box | TATCCCA | 1 | 参与赤霉素反应的顺式作用元件Cis-acting element involved in gibberellin-responsiveness |
TC-rich repeats | ATTCTCTAAC | 1 | 参与防御和胁迫反应的顺式作用元件Cis-acting element involved in defense and stress responsiveness |
Circadian | CAANNNNATC | 2 | 参与昼夜节律顺行调控元件Cis-acting regulatory element involved in circadian control |
表3 MsBBX20基因启动子部分顺式作用元件分析
Table 3 Analysis of cis-acting elements in the promoter region of the MsBBX20 gene
元件名称 Element name | 序列 Sequence | 数量 Amount | 功能 Function |
---|---|---|---|
AE-box | AGAAACTT/AGAAACAT/AGAAACAA | 3 | 光响应模块的一部分Part of a module for light response |
CATT-motif | GCATTC | 2 | 光响应元件的一部分Part of a light responsive element |
G-Box | CACGTT | 2 | 参与光反应的顺式作用调节元件Cis-acting regulatory element involved in light responsiveness |
ABRE | ACGTG | 1 | 参与脱落酸响应的顺式作用元件Cis-acting elements involved in the abscisic acid response |
HSE | CCGAAA | 1 | 参与低温响应的顺式作用元件Cis-acting element involved in low-temperature responsiveness |
MBS | CGGTCA | 1 | MYB结合位点MYB binding site |
MRE | AACCTAA | 1 | 光诱导的MYB响应元件MYB binding site involved in light responsiveness |
Sp1 | CC(G/A)CCC | 1 | 光响应元件Light responsive element |
TATC-box | TATCCCA | 1 | 参与赤霉素反应的顺式作用元件Cis-acting element involved in gibberellin-responsiveness |
TC-rich repeats | ATTCTCTAAC | 1 | 参与防御和胁迫反应的顺式作用元件Cis-acting element involved in defense and stress responsiveness |
Circadian | CAANNNNATC | 2 | 参与昼夜节律顺行调控元件Cis-acting regulatory element involved in circadian control |
图7 MsBBX20在紫花苜蓿各组织部位的表达量不同小写字母表示在P<0.05水平差异显著。Different lowercase letters indicate significant differences at the P<0.05 level.
Fig.7 Expression of MsBBX20 in various tissue parts of M. sativa
图8 MsBBX20在不同逆境胁迫处理下的相对表达量A:干旱胁迫(15% PEG6000)处理下MsBBX20基因的相对表达量 The relative expression level of the MsBBX20 gene under drought stress (15% PEG6000) treatment; B:盐胁迫(250 mmol·L-1 NaCl)处理下MsBBX20基因的相对表达量 The relative expression level of the MsBBX20 gene under salt stress (250 mmol·L-1 NaCl) treatment; C:赤霉素(10 mg·mL-1 GA)胁迫处理下MsBBX20基因的相对表达量 The relative expression level of the MsBBX20 gene under gibberellin stress (10 mg·mL-1 GA) treatment; D:脱落酸(0.1 mmol·L-1 ABA)胁迫处理下MsBBX20基因的相对表达量 The relative expression level of the MsBBX20 gene under abscisic acid stress (0.1 mmol·L-1 ABA) treatment; E:4 ℃冷胁迫处理下MsBBX20基因的相对表达量 The relative expression level of the MsBBX20 gene under cold stress (4 ℃) treatment; F:持续光照处理下MsBBX20基因的相对表达量 The relative expression level of the MsBBX20 gene under continuous light treatment; G:黑暗恢复光照处理下MsBBX20基因的相对表达量 The relative expression level of the MsBBX20 gene under the treatment of dark recovery followed by light illumination; H:不同光周期处理下MsBBX20基因的相对表达量 The relative expression level of the MsBBX20 gene under different light periods treatment;横坐标L和D分别表示光照(Light)和黑暗(Dark) The horizontal coordinates L and D represent light and dark, respectively. 不同小写字母表示在P<0.05水平差异显著。Different lowercase letters indicate significant differences at the P<0.05 level.
Fig.8 Relative expression levels of MsBBX20 under different adverse stress conditions
图9 MsBBX20过表达转化拟南芥株系的分子检测与Southern-blot杂交分析A:转化拟南芥植株的PCR检测 M: DL2000;OE4、OE6和OE7:转基因拟南芥株系。A: PCR detection of transformed A. thaliana plants, M: DL2000; OE4, OE6 and OE7: transgenic Arabidopsis strains. B:转基因拟南芥的Southern杂交检测,CK+:阳性对照,CK-:阴性对照,B: Southern hybridization test of transgenic A. thaliana, CK+: Positive control, CK-: Negative control. C:转基因拟南芥MsBBX20基因的相对表达量。C: Relative expression of transgenic A. thalianaMsBBX20 gene. WT表示野生型拟南芥植株;不同小写字母表示差异达显著水平(P<0.05)。 WT indicates wild-type Arabidopsis plants; Different lowercase letters indicate significant differences at the P<0.05 level.
Fig.9 Molecular detection and Southern-blot hybridization analysis of A. thaliana transgenic lines overexpressing MsBBX20
图10 NaCl胁迫下转MsBBX20基因拟南芥植株生长情况A:盐胁迫(150 mmol·L-1 NaCl)处理4 d的萌发情况 Germination after 4 days of salt stress (150 mmol·L-1 NaCl) treatment; B:盐胁迫(150 mmol·L-1 NaCl)处理7 d的侧根生长情况 Lateral root growth after 7 days of salt stress (150 mmol·L-1 NaCl) treatment; C:盐胁迫(200 mmol·L-1 NaCl)处理0~10 d的萌发情况 Germination after treatment with salt stress (200 mmol·L-1 NaCl) for 0-10 days; D:盐胁迫(200和300 mmol·L-1 NaCl)处理7 d的生长情况 Growth under salt stress (200 and 300 mmol·L-1 NaCl) for 7 days; E:盐胁迫(150 mmol·L-1 NaCl)处理7 d的侧根数量统计 Lateral root number statistics after 7 days of salt stress (150 mmol·L-1 NaCl) treatment; F:盐胁迫(200 mmol·L-1 NaCl)处理下植株鲜重 Plant fresh weight under salt stress (200 mmol·L-1 NaCl); G:盐胁迫(300 mmol·L-1 NaCl)处理下植株叶绿素含量 Chlorophyll content of plants treated with salt stress (300 mmol·L-1 NaCl); H:盐胁迫(300 mmol·L-1 NaCl)处理7 d后的植株存活率。Plant survival rate for 7 days after salt stress (300 mmol·L-1 NaCl). 不同小写字母表示在P<0.05水平差异显著。Different lowercase letters indicate significant differences at the P<0.05 level.
Fig.10 Growth of transgenic Arabidopsis plants with MsBBX20 gene under NaCl stress
图11 盐胁迫下转MsBBX20基因的拟南芥植株生理指标变化情况A:不同NaCl浓度胁迫下转基因拟南芥和WT的POD活性测定 Determination of POD activity of transgenic A. thaliana and WT under different NaCl concentrations of stress; B:不同NaCl浓度胁迫下转基因拟南芥和WT的CAT活性测定 Determination of CAT activity of transgenic A. thaliana and WT under different NaCl concentrations of stress; C:不同NaCl浓度胁迫下转基因拟南芥和WT的SOD活性测定 Determination of SOD activity of transgenic A. thaliana and WT under different NaCl concentrations of stress; D:不同NaCl浓度胁迫下转基因拟南芥和WT的MDA含量测定。Determination of MDA content of transgenic A. thaliana and WT under different NaCl concentrations of stress. 不同小写字母表示在P<0.05水平差异显著。Different lowercase letters indicate significant differences at the P<0.05 level.
Fig.11 Changes of physiological indicators in A. thaliana plants transgenic for MsBBX20 under salt stress
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