Cloning and Analysis of MoDXS Gene and Its Promoter in Morinda officinalis

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

Acta Horticulturae Sinica ›› 2021, Vol. 48 ›› Issue (3): 577-589.doi: 10.16420/j.issn.0513-353x.2020-0334

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Cloning and Analysis of MoDXS Gene and Its Promoter in Morinda officinalis

XIE Dejin¹, ZHOU Chengcheng², YANG Ke¹, REN Ke¹, YANG Deming¹, CHEN Lingyan², RONG Jundong¹, ZHENG Yushan¹^,²^,^*()

¹College of Forestry,Fujian Agriculture and Forestry University,Fuzhou 350002,China
²College of Landscape,Fujian Agriculture and Forestry University,Fuzhou 350002,China

Received:2020-09-29 Online:2021-03-25 Published:2021-04-02
Contact: ZHENG Yushan E-mail:zys1960@163.com

Abstract

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

CLC Number:

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.

Figures/Tables 9

References 28

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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

Cloning and Analysis of MoDXS Gene and Its Promoter in Morinda officinalis

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