巴戟天MoDXS基因及其启动子的克隆与分析

doi:10.16420/j.issn.0513-353x.2020-0334

摘要/Abstract

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。

关键词: 巴戟天, 1-脱氧-D-木酮糖5-磷酸合成酶, 启动子序列, 亚细胞定位

Abstract:

In the root tissue of Morinda officinalis,three full-length cDNA sequences of 1-deoxy-D-xylulose 5-phosphate synthase genes（MoDXS1,MoDXS2-1 and MoDXS2-2）were successfully cloned and their length were 2 676,2 667,and 2 610 bp,respectively. The coding sequence（CDS）of the three cDNAs were 2 154,2 121,and 2 190 bp,encoding 717,706,and 729 amino acid residues,respectively. Sequence comparison by BlastP in NCBI database revealed that MoDXS proteins had high homology with DXS proteins from other species. The result of the phylogenetic tree displayed that MoDXS proteins had the closest relationship with DXS proteins from Coffea canephora and Coffea arabica. Furthermore,MoDXS1,MoDXS2-1,and MoDXS2-2 proteins were grouped into clade 1,clade 3, and clade 2,respectively. MoDXS1 gene had the highest expression level in roots. The expression level of MoDXS2-1 gene had significant differences in three tissues,and the expression level was the highest in leaves. However,the expression level of MoDXS2-2 gene was the highest in roots but lower in stems and leaves. The plantCARE analysis indicated that the upstream regulation sequences of MoDXS1,MoDXS2-1,and MoDXS2-2 were 2 538,732,and 1 744 bp,respectively. The subcellular localization exhibited that the three MoDXS proteins were located on chloroplast.

Key words: Morinda officinalis, 1-deoxy-D-xylulose 5-phosphate synthase, promoter sequence, subcellular localization

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.

图/表 9

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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′

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′

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