铁皮石斛<i>DcbHLH</i>14基因克隆及表达分析

理雅; 刘博婷; 赖思慧; 罗伟红; 于白音; 刘羽佳

doi:10.19303/j.issn.1008-0384.2022.009.005

铁皮石斛DcbHLH14基因克隆及表达分析

doi: 10.19303/j.issn.1008-0384.2022.009.005

理雅^{1, 2,},
刘博婷¹,
赖思慧¹,
罗伟红¹,
于白音^{1, 2},
刘羽佳^{1, 2, ,}

1.
广东省粤北食药资源利用与保护重点实验室/韶关学院，广东　韶关　512005
2.
河南农业大学农学院，河南　郑州　450046

基金项目: 国家自然科学基金项目（32000266）；广东省基础与应用基础研究基金项目（2020A1515011438）；广东省普通高校创新团队项目（2020KCXTD037）；韶关市科技计划项目（200810224537583、210731084530203）；韶关学院科研重点项目（SZ2019ZK04）

详细信息

作者简介:
理雅（1997−），女，硕士研究生，研究方向：铁皮石斛分子分析与基因改良（E-mail：1498391781@qq.com）

通讯作者:
刘羽佳（1984−），女，博士，讲师，研究方向：植物逆境生理与分子生物学（E-mail：liuyj1713@dingtalk.com）

中图分类号: Q 78
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出版历程
- 收稿日期: 2022-04-01
- 修回日期: 2022-07-28
- 刊出日期: 2022-09-30

Cloning and Expression of DcbHLH14 from Dendrobium catenatum Lindl.

LI Ya^{1, 2
,},
LIU Boting¹,
LAI Sihui¹,
LUO Weihong¹,
YU Baiyin^{1, 2},
LIU Yujia^{1, 2
, ,}

1.
Guangdong Provincial key Laboratory of Utilization and Conservation of Food and Medicinal Resources in Northern Region/Shaoguan University, Shaoguan, Guangdong　512005, China
2.
College of Agriculture, Henan Agricultural University, Zhengzhou, Henan　450046, China

摘要

摘要: 目的克隆铁皮石斛转录因子DcbHLH14基因并分析其在非生物胁迫响应中的表达情况，为研究DcbHLH14基因的功能提供理论参考。方法通过同源克隆从铁皮石斛叶组织中得到DcbHLH14基因，对其编码的蛋白序列特征和组织表达特性进行分析，并采用qRT-PCR对DcbHLH14基因在低温、干旱和ABA处理过程中的表达量进行分析。结果 DcbHLH14基因开放阅读框（Open reading frame, ORF）为1269 bp，与参考序列存在7个碱基差异，仅含有1个外显子且无内含子，编码422个氨基酸。DcbHLH14蛋白的分子式为C₂₀₁₁H₃₁₉₂N₅₈₆O₆₁₃S₁₃，理论分子量为45.8 kD，理论等电点（pI）为5.98，含有bHLH家族保守结构域bHLH-MYC-N和HLH，属于bHLH家族，与鼓槌石斛（Dendrobium chrysotoxum）和春兰（Cymbidium goeringii）bHLH蛋白的氨基酸序列同源性较高，分别为97.16%和86.90%。转录组分析结果显示，DcbHLH14基因在云南产地野生铁皮石斛花蕾中的表达量最高，在叶中的表达量最低。进一步qRT-PCR分析结果表明，该基因在广东丹霞铁皮石斛叶中的表达量最高，而在茎中的表达量最低。DcbHLH14基因启动子富含多种与水分胁迫、低温、脱水以及ABA响应等相关的顺式作用元件。DcbHLH14基因明显受到低温、干旱和ABA诱导，低温和ABA处理6 h后DcbHLH14表达量被显著提高并达到峰值，分别是处理前的12.6倍和3.7倍；干旱处理9 h后，DcbHLH14表达量最高，是处理前的6.5倍，达到极显著差异水平。结论 DcbHLH14基因可能在转录水平上通过依赖于ABA信号通路途径响应低温和干旱胁迫，从而调控下游功能基因表达，提高铁皮石斛抗逆性。
- 铁皮石斛 /
- DcbHLH14基因 /
- 非生物胁迫 /
- 表达分析
Abstract: Objective Functions of DcbHLH14, a basic helix-loop-helix transcription factor of Dendrobium catenatum Lindl., in response to abiotic stresses were studied. Method DcbHLH14 was cloned from D. catenatum leaves using homologous cloning method for a bioinformatic analysis on the gene and expression in tissues. Gene expressions under low temperature, drought, and abscisic acid (ABA) stresses were determined. Result The ORF of DcbHLH14 was 1 269 bp and encoded 422 amino acids. It contained one exon, no intron, and 7 bases that were different from the reference sequences. The theoretical molecular weight was 45.8 kD, isoelectric point pH 5.98, and molecular formula C₂₀₁₁H₃₁₉₂N₅₈₆O₆₁₃S₁₃. Its conserved domains contained bHLH-MYC-N and HLH proteins that had high similarities with the bHLH proteins in D. chrysotoxum at 97.16% and in Cymbidium goeringii at 86.90%. The transcriptome analysis revealed high expressions in the flower buds and columns but low in the leaves of the wild D. catenatum from Yunnan, whereas the qRT-PCR analysis showed high expressions in the leaves and low in the stems of the sample from Danxia, Guangdong. The promoters of DcbHLH14 contained numerous cis-acting elements associated with the responses to water-depletion, low temperature, dehydration, and ABA stresses, which significantly affected expression of the gene. For instance, DcbHLH14 was upregulated to peak in 6 h after a low temperature or ABA treatment reaching 12.6 or 3.7 times, respectively, as well as by a 9 h drought stress to become as high as 6.5 times of control. Conclusion It was postulated that DcbHLH14 responded to low temperature or drought stress through the ABA signaling pathway at transcription level. Hence, the tolerance of D. catenatum to the abiotic stresses could be manipulated by regulating the expression of the downstream functional gene.
- Dendrobium catenatum /
- DcbHLH14 /
- abiotic stress /
- expression analysis

HTML全文

图 1 DcbHLH14基因克隆（A）及染色体定位（B）结果

M：Marker；1：DcbHLH14基因 cDNA 电泳。

Figure 1. DcbHLH14 cloning (A) and chromosome locating (B)

M: Marker; 1: cDNA electrophoresis of DcbHLH14.

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图 2 DcbHLH14基因cDNA序列及其推导氨基酸序列分析

红色箭头表示DcbHLH14基因与参考序列间存在的碱基差异。

Figure 2. cDNA and deduced amino acid sequences of DcbHLH14

Red arrow represents difference on base between DcbHLH14 and reference sequences.

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图 3 DcbHLH14 蛋白结构域预测

Figure 3. Domains of DcbHLH14 protein

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图 4 DcbHLH14蛋白亲疏水分析（A）、二级结构预测（B）及三级结构预测（C）

Figure 4. Hydrophilicity and hydrophobicity of DcbHLH14 protein (A), predicted secondary structure (B), and tertiary structure (C)

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图 5 DcbHLH14蛋白与其他物种bHLH蛋白的多重序列比对分析结果

实线表示bHLH-MYC-N结构域；虚线表示HLH结构域。

Figure 5. Multiple sequence alignment between DcbHLH14 and bHLH proteins from other plants

Solid line represents bHLH-MYC-N domain; dashed line, HLH domain.

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图 6 基于不同物种bHLH蛋白氨基酸序列构建的系统发育进化树

Figure 6. Phylogenetic tree based on amino acid sequences in bHLH protein of different species

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图 7 DcbHLH14基因在野生铁皮石斛不同组织中的表达

小写字母表示不同组织间差异显著（P＜0.05）。图8同。

Figure 7. Expressions of DcbHLH14 in tissues of wild D. catenatum　　　

Letters represent significant difference between different organizations at P＜0.05. Same for Fig. 8.

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图 8 DcbHLH14基因组织表达特性的qRT-PCR分析

Figure 8. qRT-PCR analysis on DcbHLH14 expressions in tissues

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图 9 DcbHLH14基因在低温（A）、干旱（B）和ABA（C）处理下的表达情况

*表示与0 h差异显著（P＜0.05）；***表示与0 h极显著差异（P＜0.001）；

Figure 9. Expressions of DcbHLH14 under cold stress (A), drought stress (B), and ABA treatment (C)

* represents significant difference from control (0 h) at P＜0.05; *** represents extremely significant difference from control (0 h) at P＜0.001.

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图 10 DcbHLH14基因在低温、干旱和ABA处理下表达水平的qRT-PCR分析结果

不同小写字母表示同一处理不同时间之间差异显著（P<0.05）。

Figure 10. qRT-PCR analysis on expressions of DcbHLH14 under low temperature, drought, and ABA treatment

Different lowercase letters indicant significant difference among different treatment times of the same treatment (P<0.05).

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表 1 DcbHLH14基因启动子顺式作用元件分析结果

Table 1. Cis-acting elements in DcbHLH14 promoter

序号 No.	元件名称 Element	元件序列 Element sequence	数目 Number	功能预测 Predicted function
1	MYCCONSENSUSAT	CANNTG	18	低温响应 Low temperature responsive
2	MYBCORE	CNGTTR	4	水分胁迫调控 Regulation of water stress
3	MYB2CONSENSUSAT	YAACKG	3	脱水响应 Dehydration responsive
4	LTRECOREATCOR15	CCGAC	3	低温响应 Low temperature responsive
5	MYBATRD22	CTAACCA	2	脱水响应 Dehydration responsive
6	MYB1AT	WAACCA	1	脱水响应 Dehydration responsive
7	ABRELATERD1	ACGTG	1	ABA响应 Abscisic acid responsive

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