Bioinformatics and Expression of Hydroxycinnamyl transferase Gene Family from Dendrobium officinale
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摘要:
目的 对铁皮石斛HCT（Hydroxycinnamoyl transferase）基因家族进行特征鉴定和表达模式分析，为研究铁皮石斛黄酮类化合物和石斛酚的生物合成奠定研究基础。 方法 通过NCBI在线网站获得铁皮石斛的基因数据信息，利用生物信息学方法分析铁皮石斛DoHCT基因家族成员系统进化树、DoHCTs保守基序和保守结构域，并且预测DoHCT家族成员的蛋白二级和三级结构；同时利用逆转录PCR的方法分析DoHCTs在不同胁迫条件下以及在根茎叶中的差异表达情况。 结果 铁皮石斛DoHCTs共有 8个家族成员(DoHCT 1~8)，保守结构域分析结果显示：DoHCT7~8属于转移酶超家族，DoHCT 1~6属于PLN02481超家族。该家族基因的表达可受到非生物胁迫不同程度的影响，其中高温条件下DoHCT家族基因均下调表达；组织表达检测结果显示除DoHCT7外，其余基因均在根茎叶中差异表达。 结论 在全基因组水平对DoHCT家族进行了系统的生物信息学分析，显示DoHCTs 在不同胁迫下以及铁皮石斛根茎叶中存在差异表达，证实其在非生物胁迫应答中起到调控作用。 Abstract:Objective Characterization and the expressions of hydroxycinnamyl transferase (HCT) gene family from Dendrobium officinale were studied to reveal the biosynthesis of flavonoids and dendrophenol in the plant. Method Relevant gene sequences were obtained from the NCBI online website. Conserved motifs and domains, evolutionary tree, and protein secondary and tertiary structures of each member of DoHCTs were analyzed using bioinformatics methods. Expressions of the genes under stresses and in roots, stems, and leaves were determined using RT-PCR. Result The gene family had 8 members, DoHCT1–8. Their conserved domains showed that DoHCT7/8 belonged to the transferase superfamily and DoHCT1–6 to the PLN02481 superfamily. Abiotic stresses could affect the expressions, such as, elevated temperatures downregulated them. Except DoHCT7, the genes were differentially expressed in the roots, stems, and leaves. Conclusion The comprehensive bioinformatics and expression analyses on the 8 members of the DoHCT family from D. officinale revealed that SoHCTs were with differental expression under different abiotic stress or tissues, and proved to play regulatory role in abiotic stress. -
Key words:
- Dendrobium officinale /
- hydroxycinnamoyl transferase /
- expression analysis /
- flavonoid /
- dendrophenol
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图 1 铁皮石斛DoHCT家族系统进化分析
注：At 拟南芥；Do铁皮石斛；Gm 大豆 ；Td 小麦；Os水稻。
Figure 1. Phylogenetic analysis on DoHCT family of D. officinale
Note: At: A. thaliana;Do: D. officinale; Gm: G. max; Td: T. dicoccoides; Os: O. sativa.
图 2 铁皮石斛DoHCT 1~8多重蛋白序列比对
注：红色背景表示的是完全相同的氨基酸序列，蓝框标出的序列为相似度较高的序列。
Figure 2. Sequence alignment of multiple proteins of D. officinale DoHCT1-8
Note: Red background indicates identical amino acid sequence; blue box, sequences with high similarity.
图 3 铁皮石斛DoHCT保守基序及分布图
注：A. 左为DoHCTs保守基序图，不同颜色区域表示不同保守基序；右为DoHCTs保守结构域，绿色表示转移酶超家族，黄色表示PLN02481超家族。B. DoHCTs保守基序序列。
Figure 3. Distribution of DoHCT conserved motifs of D. officinale
Note: A: DoHCT conserved motif map on the left, and different conserved motifs in differently colored regions; DoHCT conserved domain on the right, transferase superfamily colored in green, and PLN02481 superfamily colored in yellow. B: DoHCT conserved motif sequence.
图 4 铁皮石斛DoHCT基因结构
注：绿色表示UTR序列，黄色表示CDS序列，灰色线条表示内含子。
Figure 4. Structure of D. officinale DoHCT
Note: Green represents UTR sequence; yellow, CDS sequence; gray line, intron.
图 5 DoHCTs蛋白二级结构预测
注：蓝色表示α螺旋；红色表示β折叠；绿色表示β转角；紫色表示无规卷曲。
Figure 5. Predicted DoHCTs protein secondary structure
Note: Blue indicates alpha helix; red, beta sheet; green, beta turn; purple, random coil.
图 6 铁皮石斛DoHCTs 蛋白三级结构预测
注：蛋白三级结构模型根据彩虹色由N端向C端着色。
Figure 6. Predicted 3-D structure of D. officinale DoHCT
s protein Note: Protein 3-D structure colored from N to C end as in a rainbow spectrum.
图 7 DoHCTs在根茎叶中的相对表达水平
注：不同小写字母表示表达水平在 95 %置信区间水平上差异显著；EF1-α为内参。
Figure 7. Relative expressions of DoHCTs in roots, stems, and leaves
Note: Data with different lowercase letters indicate significant difference on expression level at 95% confidence interval level; EF1-α: internal reference.
图 8 DoHCTs在不同胁迫处理下的相对表达水平
注：CK：对照，1/2 MS液体培养基中培养6 h；ABA：含5 μmol·L−1的ABA的1/2 MS液体培养基中培养6 h；NaCl：含100 mmol·L−1 NaCl的1/2 MS液体培养基中培养6 h；PEG：含10% PEG的1/2 MS液体培养基中培养6 h；HT：40 ℃1/2 MS液体培养基中培养6 h；LT：4 ℃1/2 MS液体培养基中培养6 h。EF1-α为内参。其中*表示P＜0.05，**表示P＜0.01，***表示P＜0.001。
Figure 8. Relative expression levels of DoHCTs under different stresses
Note: CK: control, cultured in 1/2MS liquid medium for 6 h; ABA: cultured in 1/2 MS liquid medium containing 5 μmol·L−1 ABA for 6 h; NaCl: cultured in 1/2 MS liquid medium containing 100 mmol·L−1 NaCl for 6 h; PEG: cultured in 1/2 MS liquid medium containing 10% PEG for 6 h; HT: cultivated in 1/2 MS liquid medium at 40 ℃ for 6 h; LT: cultivated in 1/2 MS liquid medium at 4 ℃ for 6h. EF1-α: internal reference. * at P＜0.05, ** at P＜0.01, and *** at P＜0.001.
表 1 铁皮石斛DoHCT家族PCR引物
Table 1. RT-PCR primers for DoHCT family of D. officinale
基因名称
Gene name引物序列 (5′-3′)
Primer sequenceDoHCT1 F: GACGATTTTGGGGACTTCGC；
R: GAGGTACTCGTCGTCCATGCDoHCT2 F: TCAGCCCTTGCTTCCAGATG；
R: ACGAATTACCGCTGCTCGATDoHCT3 F: ACCGACCCGACTCTACATGA；
R: ATCAACACCGACAACCCTCCDoHCT4 F: TGGGCCAGAATTACCCACAC；
R: ACCACCCACAAATCCGTCTCDoHCT5 F: AGAATCACCCACACCGACAC；
R: AACCGAAACGCCTCCTTCTTDoHCT6 F: GAGTGGCGATAGCATTCCGA；
R: CGAGGGTAGATGAAGGTCGCDoHCT7 F: GACGATTTTGGGGACTTCGC；
R: GAGGTACTCGTCGTCCATGCDoHCT8 F: TCAGCCCTTGCTTCCAGATG；
R: ACGAATTACCGCTGCTCGATEF1-α F: CCACCACCCCCAAATACTCC；
R: TCCCTAACAGCGAAACGTCC
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表 2 铁皮石斛DoHCT家族成员基因组信息和蛋白特征
Table 2. Genomic information and protein characteristics of DoHCTs of D. officinale
基因名
Gene name氨基酸长度
Amino acid
length/aa相对分子质量
Relative molecular
mass/kDa等电点
Isoelectric
point不稳定系数
Instability
index脂溶指数
Aliphatic
index亲水指数
Grand average of
hydropathicityDoHCT1 430 47.43 6.46 51.54 83.77 −0.148 DoHCT2 426 46.80 7.24 36.51 85.23 −0.071 DoHCT3 454 49.17 6.18 56.00 90.51 −0.046 DoHCT4 445 48.92 5.78 57.93 94.52 0.076 DoHCT5 447 48.99 5.64 53.67 83.38 −0.116 DoHCT6 433 47.71 7.56 52.25 88.78 0.014 DoHCT7 430 47.018 6.47 33.62 92.09 0.026 DoHCT8 443 49.29 6.39 47.99 91.76 −0.023
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表 3 铁皮石斛DoHCTs蛋白二级结构信息
Table 3. Information on secondary structure of D. officinale DoHCTs protein
蛋白名称
Protein nameα螺旋
Alpha helix/%β折叠
Beta sheet/%β转角
Beta turn/%无规卷曲
Random coil/%DoHCT1 31.63 18.60 3.95 45.81 DoHCT2 31.92 18.08 4.46 45.54 DoHCT3 32.16 17.40 4.63 45.81 DoHCT4 32.36 17.75 4.27 45.62 DoHCT5 32.89 17.45 4.25 45.41 DoHCT6 31.18 18.94 4.16 45.73 DoHCT7 31.16 18.84 4.88 45.12 DoHCT8 31.16 18.51 4.74 38.37
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