园艺学报 ›› 2021, Vol. 48 ›› Issue (3): 577-589.doi: 10.16420/j.issn.0513-353x.2020-0334
谢德金1, 周成城2, 杨柯1, 任可1, 杨德明1, 陈凌艳2, 荣俊冬1, 郑郁善1,2,*()
收稿日期:
2020-09-29
出版日期:
2021-03-25
发布日期:
2021-04-02
通讯作者:
郑郁善
E-mail:zys1960@163.com
基金资助:
XIE Dejin1, ZHOU Chengcheng2, YANG Ke1, REN Ke1, YANG Deming1, CHEN Lingyan2, RONG Jundong1, ZHENG Yushan1,2,*()
Received:
2020-09-29
Online:
2021-03-25
Published:
2021-04-02
Contact:
ZHENG Yushan
E-mail:zys1960@163.com
摘要:
从药用植物巴戟天根部组织中克隆了3个1-脱氧-D-木酮糖5-磷酸合成酶基因MoDXS1、MoDXS2-1和MoDXS2-2,其全长cDNA分别为2 676、2 667和2 610 bp,编码区序列长度为2 154、2 121和2 190 bp,编码717、706和729个氨基酸。BlastP序列比对分析表明MoDXS蛋白与其他植物的DXS蛋白高度同源;系统进化发育树分析显示,MoDXS蛋白与中粒咖啡和小粒咖啡DXS蛋白亲缘关系最近。MoDXS1、MoDXS2-1和MoDXS2-2蛋白被分别归为clade 1、clade 3和clade 2。MoDXS1在巴戟天根中表达量最高;MoDXS2-1在巴戟天根、茎、叶中的相对表达量差异显著,叶中最高;MoDXS2-2在根中表达量最高,而在茎和叶中最低。MoDXS1、MoDXS2-1和MoDXS2-2基因的5′端上游启动子序列长度分别为2 538、732和1 744 bp。亚细胞定位预测3个MoDXS均在叶绿体上。
中图分类号:
谢德金, 周成城, 杨柯, 任可, 杨德明, 陈凌艳, 荣俊冬, 郑郁善. 巴戟天MoDXS基因及其启动子的克隆与分析[J]. 园艺学报, 2021, 48(3): 577-589.
XIE Dejin, ZHOU Chengcheng, YANG Ke, REN Ke, YANG Deming, CHEN Lingyan, RONG Jundong, ZHENG Yushan. Cloning and Analysis of MoDXS Gene and Its Promoter in Morinda officinalis[J]. Acta Horticulturae Sinica, 2021, 48(3): 577-589.
用途 Usage | 基因 Gene | 引物序列 Primer sequences | ||||||||
---|---|---|---|---|---|---|---|---|---|---|
cDNA末端快速克隆 5′RACE and 3′RACE | MoDXS1 | 5′RACE-1:5′-TCGTGTAGGGAAACGGCGAAGAA-3′ 5′RACE-2:5′-CCCCTTGGCAACTTCTCTCAGTTCTC-3′ 3′RACE-1:5′-CCAAGGGGGTTACCAAGCAGATT-3′ 3′RACE-2:5′-ATATGCCCGTGGCTTGATTAGTGG-3′ | ||||||||
MoDXS2-1 | 5′RACE-1:5′-CACATTGTGTTGGACCATCCGATC-3′ 5′RACE-2:5′-TCACTGAGAGTGCGAGGCTGGAC-3′ 3′RACE-1:5′-TCACTATGTTTTCCACTCACCTGCGG-3′ 3′RACE-2:5′-TATGAGGCAATGAGTAATGCGGGC-3′ | |||||||||
MoDXS2-2 | 5′RACE-1:5′-AGAACAGGTCCTGGAACAGGCATAG-3′ 5′RACE-2:5′-CAACTGTGGCTGTAGGGAGGGAGAC-3′ 3′RACE-1:5′-CAGGAGAGAAACCAGTTACCCCCG-3′ 3′RACE-2:5′-GGAGGTCACGGATGCACACGATC-3′ | |||||||||
克隆 Cloning | MoDXS1 | F:5′-ATGGGTCGCGGATCCGAATTCATGGCTCTGAGTGCATTCGC-3′ R:5′-TTGTCGACGGAGCTCGAATTCCTATGACATGATTTCCAGAGCTTCTC-3′ | ||||||||
MoDXS2-1 | F:5′-ATGGGTCGCGGATCCGAATTCATGGGCAGTGCAATTTTTGAG-3′ R:5′-TTGTCGACGGAGCTCGAATTCTTAGCACATCAGAAGAAGAGCCTC-3′ | |||||||||
MoDXS2-2 | F:5′-ATGGGTCGCGGATCCGAATTCATGGCTTCAGCATCATATGGCG-3′ R:5′-TTGTCGACGGAGCTCGAATTCTTATAGATTGAGGAGGTGTAAACTTTCCC-3′ | |||||||||
qRT-PCR | MoDXS1 | F:5′-ATGTTGTCACCGAGAAAGGC-3′;R:5-′TGGCACTGGCTTTGAATTGC-3′ | ||||||||
MoDXS2-1 | F:5′-ACGTTCATGTCCTGTTTGCC-3′;R:5′-TTGCCACCATATGAGCAAGC-3′ | |||||||||
MoDXS2-2 | F:5′-AGGCGTTGTTGAGCTTACTG-3′;R:5′-AACGTTTGTCGGATCGTGTG-3′ | |||||||||
内参基因 Reference gene | ef1α | F:5′-GGCTGCCGAGATGAACAA-3′;R:5′-CTCAAACTTCCACAAGGCAATA-3′ | ||||||||
热不对称交错PCR Thermal asymmetric interlaced PCR | MoDXS1 | SP1:5′-TGACAATGATGCCCGAACTCTAC-3′ SP2:5′-ATGACCCCCAGTCTTTGAAACG-3′ SP3:5′-GGCATTTTGTCTCTTCTCCCAGTCAA-3′ | ||||||||
MoDXS2-1 | SP1:5′-ACGAACAGATTCCTCGGAAGCA-3′ SP2:5′-CGCAGGTGAGTGGAAAACATAGTGAA-3′ SP3:5′-GCAACAATGCGGTCTCGCTTTC-3′ | |||||||||
MoDXS2-2 | SP1:5′-GCTTCACTATTCTCCCTTTGAGCAG-3′ SP2:5′-ACGGGGGTAACTGGTTTCTCTC-3′ SP3:5′-ATGCCCACCGATCTTGGACACT-3′ | |||||||||
亚细胞定位 Subcellular localization | MoDXS1 | F:5′-ATTTGGAGAGGACAGGGTACCATGGCTCTGAGTGCATTCGC-3′ R:5′-AGTGTCGACTCTAGAGGATCCTGACATGATTTCCAGAGCTTCTCT-3′ | ||||||||
MoDXS2-1 | F:5′-ATTTGGAGAGGACAGGGTACCATGGGCAGTGCAATTTTTGAG-3′ R:5′-AGTGTCGACTCTAGAGGATCCGCACATCAGAAGAAGAGCCTCAC-3′ | |||||||||
MoDXS2-2 | F:5′-ATTTGGAGAGGACAGGGTACCATGGCTTCAGCATCATATGGC-3′ R:5′-AGTGTCGACTCTAGAGGATCCTAGATTGAGGAGGTGTAAACTTTCCTT-3′ |
表1 引物序列
Table 1 Primer sequences
用途 Usage | 基因 Gene | 引物序列 Primer sequences | ||||||||
---|---|---|---|---|---|---|---|---|---|---|
cDNA末端快速克隆 5′RACE and 3′RACE | MoDXS1 | 5′RACE-1:5′-TCGTGTAGGGAAACGGCGAAGAA-3′ 5′RACE-2:5′-CCCCTTGGCAACTTCTCTCAGTTCTC-3′ 3′RACE-1:5′-CCAAGGGGGTTACCAAGCAGATT-3′ 3′RACE-2:5′-ATATGCCCGTGGCTTGATTAGTGG-3′ | ||||||||
MoDXS2-1 | 5′RACE-1:5′-CACATTGTGTTGGACCATCCGATC-3′ 5′RACE-2:5′-TCACTGAGAGTGCGAGGCTGGAC-3′ 3′RACE-1:5′-TCACTATGTTTTCCACTCACCTGCGG-3′ 3′RACE-2:5′-TATGAGGCAATGAGTAATGCGGGC-3′ | |||||||||
MoDXS2-2 | 5′RACE-1:5′-AGAACAGGTCCTGGAACAGGCATAG-3′ 5′RACE-2:5′-CAACTGTGGCTGTAGGGAGGGAGAC-3′ 3′RACE-1:5′-CAGGAGAGAAACCAGTTACCCCCG-3′ 3′RACE-2:5′-GGAGGTCACGGATGCACACGATC-3′ | |||||||||
克隆 Cloning | MoDXS1 | F:5′-ATGGGTCGCGGATCCGAATTCATGGCTCTGAGTGCATTCGC-3′ R:5′-TTGTCGACGGAGCTCGAATTCCTATGACATGATTTCCAGAGCTTCTC-3′ | ||||||||
MoDXS2-1 | F:5′-ATGGGTCGCGGATCCGAATTCATGGGCAGTGCAATTTTTGAG-3′ R:5′-TTGTCGACGGAGCTCGAATTCTTAGCACATCAGAAGAAGAGCCTC-3′ | |||||||||
MoDXS2-2 | F:5′-ATGGGTCGCGGATCCGAATTCATGGCTTCAGCATCATATGGCG-3′ R:5′-TTGTCGACGGAGCTCGAATTCTTATAGATTGAGGAGGTGTAAACTTTCCC-3′ | |||||||||
qRT-PCR | MoDXS1 | F:5′-ATGTTGTCACCGAGAAAGGC-3′;R:5-′TGGCACTGGCTTTGAATTGC-3′ | ||||||||
MoDXS2-1 | F:5′-ACGTTCATGTCCTGTTTGCC-3′;R:5′-TTGCCACCATATGAGCAAGC-3′ | |||||||||
MoDXS2-2 | F:5′-AGGCGTTGTTGAGCTTACTG-3′;R:5′-AACGTTTGTCGGATCGTGTG-3′ | |||||||||
内参基因 Reference gene | ef1α | F:5′-GGCTGCCGAGATGAACAA-3′;R:5′-CTCAAACTTCCACAAGGCAATA-3′ | ||||||||
热不对称交错PCR Thermal asymmetric interlaced PCR | MoDXS1 | SP1:5′-TGACAATGATGCCCGAACTCTAC-3′ SP2:5′-ATGACCCCCAGTCTTTGAAACG-3′ SP3:5′-GGCATTTTGTCTCTTCTCCCAGTCAA-3′ | ||||||||
MoDXS2-1 | SP1:5′-ACGAACAGATTCCTCGGAAGCA-3′ SP2:5′-CGCAGGTGAGTGGAAAACATAGTGAA-3′ SP3:5′-GCAACAATGCGGTCTCGCTTTC-3′ | |||||||||
MoDXS2-2 | SP1:5′-GCTTCACTATTCTCCCTTTGAGCAG-3′ SP2:5′-ACGGGGGTAACTGGTTTCTCTC-3′ SP3:5′-ATGCCCACCGATCTTGGACACT-3′ | |||||||||
亚细胞定位 Subcellular localization | MoDXS1 | F:5′-ATTTGGAGAGGACAGGGTACCATGGCTCTGAGTGCATTCGC-3′ R:5′-AGTGTCGACTCTAGAGGATCCTGACATGATTTCCAGAGCTTCTCT-3′ | ||||||||
MoDXS2-1 | F:5′-ATTTGGAGAGGACAGGGTACCATGGGCAGTGCAATTTTTGAG-3′ R:5′-AGTGTCGACTCTAGAGGATCCGCACATCAGAAGAAGAGCCTCAC-3′ | |||||||||
MoDXS2-2 | F:5′-ATTTGGAGAGGACAGGGTACCATGGCTTCAGCATCATATGGC-3′ R:5′-AGTGTCGACTCTAGAGGATCCTAGATTGAGGAGGTGTAAACTTTCCTT-3′ |
图1 MoDXS1,MoDXS2-1和MoDXS2-2基因的PCR扩增产物 M1:1 kb plus DNA marker;M2:1 kb DNA marker;T:TaKaRa PCR扩增产物;K:KOD PCR扩增产物。
Fig. 1 PCR amplification product of MoDXS1,MoDXS2-1,and MoDXS2-2 genes M:1 kb plus DNA marker;M2:1 kb DNA marker;T:PCR amplification product of TaKaRa;K:PCR amplification product of KOD.
图2 MoDXS氨基酸序列与其他植物的DXS序列比对 Mo:巴戟天;Ca:小粒咖啡;Cc:中粒咖啡。
Fig. 2 Multiple amino acid sequence alignments of DXS between M. officinalis and other plants Mo:Morinda officinalis;Ca:Coffea arabica;Cc:Coffea canephora.
蛋白名称 Protein name | 分子量/kD Molecular weight | 等电点 pI | 不稳定系数 Instability index | 总平均疏水系数 Grand average of hydropathicity |
---|---|---|---|---|
MoDXS1 | 77.30 | 6.43 | 41.72 | -0.072 |
MoDXS2-1 | 77.07 | 6.18 | 46.82 | -0.013 |
MoDXS2-2 | 78.87 | 6.62 | 40.63 | -0.095 |
表2 MoDXS1、MoDXS2-1和MoDXS2-2蛋白的理化参数
Table 2 Physical and chemical parameters of MoDXS1,MoDXS2-1,and MoDXS2-2 proteins
蛋白名称 Protein name | 分子量/kD Molecular weight | 等电点 pI | 不稳定系数 Instability index | 总平均疏水系数 Grand average of hydropathicity |
---|---|---|---|---|
MoDXS1 | 77.30 | 6.43 | 41.72 | -0.072 |
MoDXS2-1 | 77.07 | 6.18 | 46.82 | -0.013 |
MoDXS2-2 | 78.87 | 6.62 | 40.63 | -0.095 |
蛋白二级结构 Protein secondary structure | α螺旋(H) Alpha helix(H) | β折叠(E) Extended strand(E) | β转角(T) Beta turn(T) | 无规则卷曲(C) Random coil(C) |
---|---|---|---|---|
MoDXS1 | 39.61 | 15.06 | 8.09 | 37.24 |
MoDXS2-1 | 39.80 | 16.15 | 6.09 | 37.96 |
MoDXS2-2 | 37.45 | 15.23 | 7.54 | 39.78 |
表3 MoDXS1、MoDXS2-1和MoDXS2-2蛋白的二级结构预测
Table 3 Protein secondary structure prediction of MoDXS1,MoDXS2-1,and MoDXS2-2 proteins %
蛋白二级结构 Protein secondary structure | α螺旋(H) Alpha helix(H) | β折叠(E) Extended strand(E) | β转角(T) Beta turn(T) | 无规则卷曲(C) Random coil(C) |
---|---|---|---|---|
MoDXS1 | 39.61 | 15.06 | 8.09 | 37.24 |
MoDXS2-1 | 39.80 | 16.15 | 6.09 | 37.96 |
MoDXS2-2 | 37.45 | 15.23 | 7.54 | 39.78 |
图4 巴戟天DXS基因的亚细胞定位 A:488 nm激发光下MoDXS-eGFP的绿色荧光;B:555 nm激发光下叶绿体的自发荧光;C:明场;D:图像叠加。
Fig. 4 Subcellular localization of MoDXS-eGFP A:Fluorescence microscope images of MoDXS-eGFP at 488 nm;B:Chlorophyll autofluorescence at 555 nm;C:Bright field;D:Merged image.
图6 巴戟天不同组织中MoDXS1,MoDXS2-1和MoDXS2-2基因的相对表达量 同一基因不同字母表示在不同组织中的表达量差异显著(P < 0.05)。
Fig. 6 Relative expression of MoDXS1,MoDXS2-1,and MoDXS2-2 in different tissues from Morinda officinalis In same gene,different letters indicate there are significant difference among different tissues(P < 0.05).
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