生物技术通报 ›› 2024, Vol. 40 ›› Issue (11): 192-201.doi: 10.13560/j.cnki.biotech.bull.1985.2024-0423
收稿日期:
2024-05-08
出版日期:
2024-11-26
发布日期:
2024-12-19
通讯作者:
林善枝,男,博士,教授,研究方向:植物分子生物学;E-mail: linsz2002@163.com作者简介:
王紫睿,女,硕士研究生,研究方向:植物分子生物学;E-mail: 1290459671@qq.com
基金资助:
WANG Zi-rui(), LIU Xiao-han, XIU Yu, LIN Shan-zhi()
Received:
2024-05-08
Published:
2024-11-26
Online:
2024-12-19
摘要:
【目的】 转运蛋白PsMATE40在山杏种子苦杏仁苷转运中起到重要作用。克隆PsMATE40基因特异启动子并分析其驱动基因表达活性,为PsMATE40基因及其启动子的应用奠定基础。【方法】 依据前期克隆得到的PsMATE40基因,以山杏种子基因组DNA为模板,通过染色体步移克隆得到PsMATE40基因特异启动子全长序列(命名为ProPsMATE40),预测分析其顺式调控元件,采用GUS组织化学染色分析检测启动子活性;利用无缝克隆方法分别构建组成型启动子CaMV35S和特异启动子ProPsMATE40驱动的PsMATE40基因的植物表达载体,开展农杆菌介导的烟草瞬时转化,利用RT-qPCR技术比较分析它们驱动PsMATE40基因的转录表达。【结果】 山杏种子PsMATE40基因启动子全长序列为1 964 bp,含有2种启动子核心元件(CAAT-box 和TATA-box)、多种激素信号(生长素、茉莉酸和水杨酸等)响应元件和胁迫(光、干旱和损伤等)响应元件以及种子发育调控元件等,自身特异启动子ProPsMATE40驱动的PsMATE40基因表达水平明显高于CaMV35S。【结论】 山杏种子PsMATE4基因表达可能受植物激素以及生物与非生物胁迫等多种因素的复杂调控,而自身启动子可有效促进PsMATE40基因的转录表达。
王紫睿, 刘潇菡, 修宇, 林善枝. 山杏种子PsMATE40基因启动子的克隆及驱动表达分析[J]. 生物技术通报, 2024, 40(11): 192-201.
WANG Zi-rui, LIU Xiao-han, XIU Yu, LIN Shan-zhi. Cloning of the Promoter of MATE40 Gene from Prunus sibirica Seeds and Analysis of Gene Expression Driven by Promoter[J]. Biotechnology Bulletin, 2024, 40(11): 192-201.
PCR扩增轮数Rounds of PCR amplification | PCR扩增程序PCR amplification procedure |
---|---|
第一轮PCR | 94℃ 1 min; 98℃ 1 min; 94℃ 30 s, 65℃ 1 min, 72℃ 4 min, 5个循环; 94℃ 30 s, 25℃ 3 min, 72℃ 2 min; 94℃ 30 s, 65℃ 1 min, 72℃ 2 min, 15个循环; 72℃ 10 min |
第二轮PCR | 94℃ 30 s, 65℃ 1 min, 72℃ 2 min, 98℃ 30 s, 65℃ 1 min, 72℃ 2 min, 94℃ 30 s, 65℃ 1 min, 72℃ 2 min, 15个循环; 72℃ 10 min |
第三轮PCR | 94℃ 30 s, 65℃ 1 min, 72℃ 2 min, 98℃ 30 s, 65℃ 1 min, 72℃ 2 min, 94℃ 30 s, 65℃ 1 min, 72℃ 2 min, 15个循环; 72℃ 10 min |
表1 Genome Walking kit三轮PCR扩增程序
Table 1 Genome Walking kit three-round PCR amplification procedure
PCR扩增轮数Rounds of PCR amplification | PCR扩增程序PCR amplification procedure |
---|---|
第一轮PCR | 94℃ 1 min; 98℃ 1 min; 94℃ 30 s, 65℃ 1 min, 72℃ 4 min, 5个循环; 94℃ 30 s, 25℃ 3 min, 72℃ 2 min; 94℃ 30 s, 65℃ 1 min, 72℃ 2 min, 15个循环; 72℃ 10 min |
第二轮PCR | 94℃ 30 s, 65℃ 1 min, 72℃ 2 min, 98℃ 30 s, 65℃ 1 min, 72℃ 2 min, 94℃ 30 s, 65℃ 1 min, 72℃ 2 min, 15个循环; 72℃ 10 min |
第三轮PCR | 94℃ 30 s, 65℃ 1 min, 72℃ 2 min, 98℃ 30 s, 65℃ 1 min, 72℃ 2 min, 94℃ 30 s, 65℃ 1 min, 72℃ 2 min, 15个循环; 72℃ 10 min |
表达载体 Expression vector | 引物Primer | 引物序列Primer sequence(5'-3') |
---|---|---|
pBI121-ProPsMATE40::GUS | Q121 F | TATGACCATGATTACGCCAAGCTT CACATCTTCCTCCAAATC |
Q121 R | CTGACCACCCGGGGATCCTCTAGACATTTAGGGAAGAAGCCA | |
pBI121-35S::PsMATE40av | 35S-121 F | TGGAGAGAACACGGGGGACTCTCCCTGCTTAATTTGCTCCACCCC |
35S-121 R | TGGTGGCGACCGGTACCCGGGCGCTCAAGTTGCTGTCCATCTGTTT | |
pBI121-ProPsMATE40::PsMATE40 | MATE-121 F | GAACACGGGGGACTCTAGAGCTCCCTGCTTAATTTGCTCCACCCC |
MATE-121 R | TAAGGGACTGACCACCCGGGCGCTCAAGTTGCTGTCCATCTGTTT |
表2 PsMATE40基因启动子无缝克隆所需的特异性引物
Table 2 Specific primers for seamless cloning of the promoter of PsMATE40 gene
表达载体 Expression vector | 引物Primer | 引物序列Primer sequence(5'-3') |
---|---|---|
pBI121-ProPsMATE40::GUS | Q121 F | TATGACCATGATTACGCCAAGCTT CACATCTTCCTCCAAATC |
Q121 R | CTGACCACCCGGGGATCCTCTAGACATTTAGGGAAGAAGCCA | |
pBI121-35S::PsMATE40av | 35S-121 F | TGGAGAGAACACGGGGGACTCTCCCTGCTTAATTTGCTCCACCCC |
35S-121 R | TGGTGGCGACCGGTACCCGGGCGCTCAAGTTGCTGTCCATCTGTTT | |
pBI121-ProPsMATE40::PsMATE40 | MATE-121 F | GAACACGGGGGACTCTAGAGCTCCCTGCTTAATTTGCTCCACCCC |
MATE-121 R | TAAGGGACTGACCACCCGGGCGCTCAAGTTGCTGTCCATCTGTTT |
基因 Gene | 引物 Primer | 序列 Sequence(5'-3') |
---|---|---|
GUS | GUS-F | GAATACGGCGTGGATACGTTAG |
GUS-R | GATCAAAGACGCGGTGATACA | |
PsMATE40 | PsMATE40-F | AACAATGGCATCCAAGTTTTCGCCT |
PsMATE40-R | GGAGCACACCAGTCAAAGTTAAGAG | |
Actin | NtActin-F | CTGCTGGAATTCACGAAACA |
NtActin-R | GCCACCACCTTGATCTTCAT |
表3 PsMATE40和GUS基因的RT-qPCR检测所需的引物
Table 3 Primers for the RT-qPCR detections of PsMATE40 and GUS genes
基因 Gene | 引物 Primer | 序列 Sequence(5'-3') |
---|---|---|
GUS | GUS-F | GAATACGGCGTGGATACGTTAG |
GUS-R | GATCAAAGACGCGGTGATACA | |
PsMATE40 | PsMATE40-F | AACAATGGCATCCAAGTTTTCGCCT |
PsMATE40-R | GGAGCACACCAGTCAAAGTTAAGAG | |
Actin | NtActin-F | CTGCTGGAATTCACGAAACA |
NtActin-R | GCCACCACCTTGATCTTCAT |
图1 山杏种子PsMATE40基因5'端已知序列的扩增验证 A:山杏种子PsMATE40基因5'端序列的PCR扩增电泳图谱,M为2000 DNA marker,1为PsMATE40基因扩增片段;B:山杏种子PsMATE40基因5'端测序序列与已知序列比对结果,测序序列和已知序列为PsMATE40基因5'端ORF序列
Fig. 1 Amplified identification of the known sequence at the 5' end for PsMATE40 gene from P. sibirica seeds A: PCR amplification electrophoretic map of the sequence at the 5' end of PsMATE40 gene from P. sibirica seeds, M is 2000 DNA marker, and 1 is PsMATE40 gene fragment. B: Alignment results of sequenced sequences and known sequences at the 5' end of PsMATE40 gene. Sequenced sequence and known sequence are the ORF at the 5' end of PsMATE40 gene
图2 山杏种子PsMATE40基因启动子全长序列的PCR扩增 A:第一次山杏种子PsMATE40基因启动子三轮步移电泳图谱,AP1,AP2,AP3和AP4为简并上游引物,M为2000 DNA分子标准,1为第一轮步移结果,2为第二轮步移结果,3为第三轮步移结果;B:第二次山杏种子PsMATE40基因启动子三轮步移电泳图谱,AP1,AP2,AP3和AP4为简并上游引物,M为2000 DNA分子标准,1为第一轮步移结果,2为第二轮步移结果,3为第三轮步移结果;C:PsMATE40基因启动子的扩增电泳图谱,M为2000 DNA分子标准,1为PsMATE40基因启动子的扩增片段
Fig. 2 PCR amplification of full-length sequence of PsMATE40 gene promoter from P. sibirica seeds A: The first three-round step electrophoretic map for PsMATE40 gene promoter from P. sibirica seeds, AP1, AP2, AP3 and AP4 are the upstream degenerate primers, M was 2000 DNA marker, and 1, 2 and 3 are the results of the 1st, 2nd and 3rd PCR, respectively. B: The second three-round step electrophoretic map for PsMATE40 gene promoter from P. sibirica seeds, AP1, AP2, AP3 and AP4 are the upstream degenerate primers, M is 2000 DNA marker, and 1, 2 and 3 are the results of the 1st, 2nd and 3rd PCR, respectively. C: Amplification electrophoretic map for the promoter of PsMATE40 gene from P. sibirica seeds, M is 2000 DNA marker, and 1 is PsMATE40 gene fragment
图3 山杏种子PsMATE40基因启动子序列分析 转录起始位点定义为+1;不同颜色代表不同顺式作用元件
Fig. 3 Sequence analysis of the PsMATE40 gene promoter from P. sibirica seeds The transcription start site is defined as +1, and the putative cis-acting regulatory elements is marked with different colors
顺式作用元件名称 Cis-acting element name | 序列 Sequence | 位置 Position/bp | 数目 Amount | 功能 Function |
---|---|---|---|---|
核心启动子元件 | ||||
TATA-box | ATTATA | -75/-69, -250/-244, -476/-472, -509/-503, -720/-714, -746/-742, -1215/-1211, -1784/-1774 | 8 | 转录起始核心元件 |
CAAT-box | CAAT | -42/-36, -89/-84, -236/-232, -278/-274, -382/-378, -616/-611, -655/-650, -736/-732, -935/-930, -1140/-1136, -1262/-1258, -1499/-1495, -1952/-1947 | 13 | 启动子核心作用元件 |
激素响应元件 | ||||
ERE | ATTTCATA | -211/-203, -1836/-1828 | 2 | 参与乙烯响应 |
CATATG-box | CATATG | -341/-335, -1790/-1784 | 2 | 参与生长素响应 |
CGTCA-motif | CGTCA | -554/-549, -1054/-1049 | 2 | 参与茉莉酸甲酯响应 |
TCA | TCATCTTCAT | -413/-403 | 1 | 参与水杨酸响应 |
胁迫响应元件 | ||||
TC-rich repeats | GTTTTCTTAC | -1301/-1291 | 1 | 防御和应激反应 |
W-box | TGACC | -751/-747, -1128/-1123, -1332/-1328, -1753/-1748 | 4 | 结合WRKY转录因子 |
MYB | CAACAG | -843/-837, -1393/-1387 | 2 | 结合MYB 转录因子 |
MYC | CAATTG | -676/-670, -1079/-1073, -1088/-1082, -1400/-1394 | 4 | 干旱响应 |
WRE3 | CCACCT | -1644/-1638 | 1 | 损伤响应 |
AE-box | AGAAACTT | -1747/-1739 | 1 | 光响应 |
Box 4 | ATTAAT | -394/-388, -594/-584 | 2 | 光响应 |
GATA-motif | GATA | -539/-535, -1205/-1201, -1759/-1755, -1795/-1791 | 4 | 光响应 |
GT1-motif | GGTTAA | -790/-784 | 1 | 光响应 |
种子或胚乳调控元件 | ||||
DOF | AAAG | -854/-850, -918/-914, -1681/-1677, -1699/-1695 | 4 | 种子特异表达调控 |
SEF1-motif | ATATTTAA | -143/-135 | 1 | 种子发育相关 |
PYRIMIDINE-box | CCTTTT | -1377/-1371 | 1 | 种子发育相关 |
ACGTC-box | ACGTC | -1543/-1538 | 1 | 种子发育相关 |
PROLAMIN-box | TGCAAAG | -930/-923 | 1 | 胚乳特异表达调控 |
DPBF | ACACAGG | -1735/-1728 | 1 | 胚乳发育相关 |
表4 山杏种子PsMATE40基因启动子元件分析
Table 4 Analyses of cis-acting regulatory elements in the PsMATE40 gene promoter from P. sibirica seeds
顺式作用元件名称 Cis-acting element name | 序列 Sequence | 位置 Position/bp | 数目 Amount | 功能 Function |
---|---|---|---|---|
核心启动子元件 | ||||
TATA-box | ATTATA | -75/-69, -250/-244, -476/-472, -509/-503, -720/-714, -746/-742, -1215/-1211, -1784/-1774 | 8 | 转录起始核心元件 |
CAAT-box | CAAT | -42/-36, -89/-84, -236/-232, -278/-274, -382/-378, -616/-611, -655/-650, -736/-732, -935/-930, -1140/-1136, -1262/-1258, -1499/-1495, -1952/-1947 | 13 | 启动子核心作用元件 |
激素响应元件 | ||||
ERE | ATTTCATA | -211/-203, -1836/-1828 | 2 | 参与乙烯响应 |
CATATG-box | CATATG | -341/-335, -1790/-1784 | 2 | 参与生长素响应 |
CGTCA-motif | CGTCA | -554/-549, -1054/-1049 | 2 | 参与茉莉酸甲酯响应 |
TCA | TCATCTTCAT | -413/-403 | 1 | 参与水杨酸响应 |
胁迫响应元件 | ||||
TC-rich repeats | GTTTTCTTAC | -1301/-1291 | 1 | 防御和应激反应 |
W-box | TGACC | -751/-747, -1128/-1123, -1332/-1328, -1753/-1748 | 4 | 结合WRKY转录因子 |
MYB | CAACAG | -843/-837, -1393/-1387 | 2 | 结合MYB 转录因子 |
MYC | CAATTG | -676/-670, -1079/-1073, -1088/-1082, -1400/-1394 | 4 | 干旱响应 |
WRE3 | CCACCT | -1644/-1638 | 1 | 损伤响应 |
AE-box | AGAAACTT | -1747/-1739 | 1 | 光响应 |
Box 4 | ATTAAT | -394/-388, -594/-584 | 2 | 光响应 |
GATA-motif | GATA | -539/-535, -1205/-1201, -1759/-1755, -1795/-1791 | 4 | 光响应 |
GT1-motif | GGTTAA | -790/-784 | 1 | 光响应 |
种子或胚乳调控元件 | ||||
DOF | AAAG | -854/-850, -918/-914, -1681/-1677, -1699/-1695 | 4 | 种子特异表达调控 |
SEF1-motif | ATATTTAA | -143/-135 | 1 | 种子发育相关 |
PYRIMIDINE-box | CCTTTT | -1377/-1371 | 1 | 种子发育相关 |
ACGTC-box | ACGTC | -1543/-1538 | 1 | 种子发育相关 |
PROLAMIN-box | TGCAAAG | -930/-923 | 1 | 胚乳特异表达调控 |
DPBF | ACACAGG | -1735/-1728 | 1 | 胚乳发育相关 |
图4 山杏种子PsMATE40基因启动子驱动活性分析 A: 烟草叶片瞬时表达GUS组织化学染色,WT为野生型植株,35S::GUS为阳性对照,ProPsMATE40::GUS为PsMATE40基因启动子表达叶片;B: GUS基因RT-qPCR分析,以PsMATE40基因启动子驱动的GUS基因表达量标准化校正为1
Fig. 4 Driven activity analysis of PsMATE40 gene promoter from P. sibirica seeds A: Histochemical staining analysis of GUS in transformed tobacco leaves, WT is wild type plant, 35S::GUS is positive control, and ProPsMATE40::GUS is tobacco leaf with GUS expression driven by PsMATE40 gene promoter. B: Detection of GUS expression by RT-qPCR, the expression of GUS gene in the leaves driven by PsMATE40 gene promoter is arbitrarily set to 1 for standardization
图5 特异启动子ProPsMATE40驱动PsMATE40基因表达分析 A:阳性菌落PCR鉴定,M为2000 DNA 分子标准,1与2为pBI121-ProPs-MATE40::PsMATE40阳性菌落PCR产物,3与4为pBI121-35S::PsMATE40阳性菌落PCR产物;B:PsMATE40基因瞬时转化烟草叶片的表达分析,以35S启动子驱动的PsMATE40基因表达量标准化校正为1
Fig. 5 Expression analysis of PsMATE40 driven by specific promoter ProPsMATE40 gene from P. sibirica seeds A: PCR identification of the positive colonies, M is 2000 DNA marker; 1 and 2 are PCR products of the positive colonies of pBI121-ProPsMATE40::GUS expression vector, and 3 and 4 are PCR products of positive colonies of pBI121-35S::PsMATE40 expression vector. B: Expression analysis of PsMATE40 gene transiently transformed in tobacco leaves, the expression of PsMATE40 gene in the leaves driven by 35S promoter is arbitrarily set to 1 for standardization
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