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多花黄精 PLT 基因家族的鉴定与表达分析

花匠小妙招
2025-03-12 17:05

摘要:

目的 PLT（Plethora）属于AP2/ERF型转录因子，在植物生长发育及抗胁迫等过程中发挥重要作用。本文旨在探究多花黄精PLT（Polygonatum cyrtonema Hua PLT，PcPLT）基因在根状茎器官生长发育中及盐胁迫下的调控作用，为深入研究PcPLT基因的功能提供理论依据。

方法基于多花黄精转录组数据库进行PcPLT基因家族鉴定及生物信息学分析，通过qRT-PCR技术检测其在多花黄精不同组织部位及不同浓度NaCl处理下的相对表达量，利用RT-PCR技术构建PcPLT2-2、PcPLT2-7基因融合表达载体，观察其荧光信号验证亚细胞定位。

结果共鉴定获得15个PcPLT家族成员，该家族无内含子结构，蛋白编码长度为159～601个氨基酸，均为亲水蛋白；进化树分析显示PcPLT与百合科石刁柏（Asparagus officinalis L.）亲缘关系最近；亚细胞定位预测及验证结果显示PcPLT2-2定位于细胞质、细胞核中，PcPLT2-7定位于细胞核中。qRT-PCR结果显示PcPLT2-3和PcPLT2-7在根状茎中表达最高，PcPLT1-3、PcPLT1-4、PcPLT2-7响应盐胁迫调控。

结论 PcPLT不仅具有组织特异性还能提高抗逆性，提示可能作为器官发育调节因子参与多花黄精根状茎膨大的形态建成，为深入研究PLT调控植物根状茎器官生长发育的生物学功能和多花黄精遗传改良奠定基础。

Abstract:

Objective Regulatory functions of Polygonatum cyrtonema Hua PLT (PcPLT) in the growth and development of rhizomes of the plant were investigated.

Methods Based on the P. cyrtonema transcriptome database, the identity and bioinformatics of PcPLT family were obtained. Relative expressions in tissues of the plant and that under NaCl stress were detected using the qRT-PCR technique. The fusion expression vectors of PcPLT2-2 and PcPLT2-7 were constructed, and their fluorescence signals examined to determine subcellular localization.

Results Fifteen PcPLT family members were identified. They were hydrophilic proteins, absent of intron structure, coded 159-601 amino acids, and evolutionarily closely related to Liliaceae Asparagus officinalis L. The predicted subcellular localization of PcPLT2-2 was in cytoplasm and nucleus, while PcPLT2-7 in nucleus only. PcPLT2-3 and PcPLT2-7 mostly in the rhizomes; and PcPLT1-3, PcPLT1-4, and PcPLT2-7 responsive to salt stress.

Conclusion PcPLT were tissue-specific and capable of enhancing the stress resistance of P. cyrtonema. They might act as an organ development regulator associated with the morphogenesis of rhizome expansion. If so, the result obtained in this study would be of value for the in-depth understanding of the biological functions of PLT.

图 1 PcPLT 蛋白保守基序Motif、保守结构域及基因结构

Figure 1. Conserved motif, conserved domains, and structure of PcPLT

图 2 多花黄精PLT 家族系统进化树

Pc：多花黄精， At：拟南芥， Os：水稻， Ao：石刁柏， Eg：油棕， Vv：葡萄。

Figure 2. Phylogenetic trees of P. cyrtonema PLT families

Pc: P. cyrtonema Hua; At: Arabidopsis thaliana L.; Os: Oryza sativa L.; Ao: A. officinalis L.; Eg: Elaeis guineensis; Vv: Vitis vinifera.

图 3 PcPLT在不同组织部位及盐胁迫下的表达模式分析

Figure 3. Expressions of PcPLTs in various tissues and under NaCl stress

图 4 PcPLT在不同组织部位及盐胁迫下的qRT-PCR表达分析

A：PcPLT在不同组织部位的表达；B：不同NaCl浓度处理下PcPLT的表达。

Figure 4. qRT-PCR expressions of PcPLTs in various tissues and under NaCl stress

A: Expressions of PcPLT in tissues; B: PcPLT expression under stresses of varied NaCl concentrations.

图 5 PcPLT蛋白在洋葱表皮中的亚细胞定位

A：pCAMBIA1302-GFP荧光信号；B：pCAMBIA1302-PcPLT2-2-GFP荧光信号；C：pCAMBIA1302-PcPLT2-7-GFP荧光信号。

Figure 5. Subcellular localization of PcPLT in onion epidermal cells

A: pCAMBIA1302-GFP fluorescence signal; B: pCAMBIA1302-PcPLT2-2-GFP fluorescence signal; C: pCAMBIA1302-PcPLT2-7-GFP fluorescence signal.

表 1 引物序列

Table 1 Sequences of primers used

引物名称
Primer name引物序列
Primer sequence (5′-3′)退火温度Tm/℃用途 Usage PLT2-2-Q-F
PLT2-2-Q-R CGTCATCTTATCGTGGAGTTAC
CATCTTTCCGCCTTGTTACT 55 qRT-PCR引物 PLT2-7-Q-F
PLT2-7-Q-R TCAAGTGTAATGGGAGGGA
AGAGAATAGCGGCAATGTTG 55 qRT-PCR引物 β-tubulin-Q-F
β-tubulin-Q-R TTGTCGAAAATGCTGACGAG
CAAGCTTCCGGAGATCAGAG 60 内参基因引物 PLT2-2-GFP-F
PLT2-2-GFP-R CATGCCATGGATGAAGAAGTTTGCGGTGCC
GGACTAGTTAACTCAAGTTTGCAGGGTT 60 载体构建、亚细胞定位 PLT2-7-GFP-F
PLT2-7-GFP-R CATGCCATGGATGGGGAACTTGACCAAG
GGACTAGTGCTCCTGGGGTAGAAATAGG 64 载体构建、亚细胞定位 PLT1-3-Q-F
PLT1-3-Q-R TACCTCCTCCACCACTTCTC
TCTTCAAATCAGAGTCCGC 60 qRT-PCR引物 PLT1-4-Q-F
PLT1-4-Q-R ATGGTGAGTGCTTGGCTT
TGCCTCCCTTTCCTTGTT 50 qRT-PCR引物

表 2 多花黄精PLT 家族成员信息

Table 2 Information on PcPLT family members

基因名称
Gene name基因ID
Gene ID注释到
拟南芥成员
Annotated
Arabidopsis氨基酸数量
Amino
acids/aa分子量
Molecular
weight/Da
等电点
pI脂肪系数
Aliphatic
index二级结构
Secondary structure/%亚细胞定位
Subcellular
localizationα-螺旋
Alpha
helixβ-转角
Beta
turn延伸链
Extended
strand无规卷曲
Random
coil PcPLT1-1＞Cluster-11683.110576.p1AtPLT131735195.858.9654.6432.185.3610.7351.74细胞核PcPLT1-2＞Cluster-11683.110575.p1AtPLT146951308.346.4753.4125.595.3312.1556.93细胞核PcPLT1-3＞Cluster-11683.173509.p1AtPLT146851236.276.4752.8825.215.1312.6157.05细胞核PcPLT1-4＞Cluster-11683.233671.p1AtPLT142147120.437.7158.2424.477.8413.7853.92细胞质、细胞核PcPLT2-1＞Cluster-11683.222146.p1AtPLT260166506.196.5159.6318.473.9912.4865.06细胞质、细胞核PcPLT2-2＞Cluster-11683.100274.p1AtPLT227730714.027.2561.6634.307.2215.5242.96细胞质、细胞核PcPLT2-3＞Cluster-11683.77849.p1AtPLT218020995.909.6470.9433.897.7826.1132.22细胞核PcPLT2-4＞Cluster-11683.163200.p1AtPLT215917827.936.4867.6142.775.6616.3535.22细胞核PcPLT2-5＞Cluster-11683.105355.p1AtPLT231234191.206.9473.9126.926.7313.1453.21细胞质、细胞核PcPLT2-6＞Cluster-11683.151415.p2AtPLT219922672.779.1174.0239.707.5422.6130.15细胞核PcPLT2-7＞Cluster-11683.204809.p1AtPLT233236898.449.1161.8425.005.1212.6557.23细胞核PcPLT2-8＞Cluster-11683.147730.p1AtPLT239544135.249.0763.0127.596.8415.9549.62细胞质、细胞核PcPLT2-9＞Cluster-11683.151416.p1AtPLT239143773.698.8959.6424.557.4213.8154.22细胞质、细胞核PcPLT2-10＞Cluster-11683.133871.p1AtPLT238042322.239.2761.8924.477.3713.6854.47细胞质、细胞核PcPLT2-11＞Cluster-11683.182808.p2AtPLT218420988.809.2669.4639.677.0718.4834.78细胞核 [1] 国家药典委员会. 中华人民共和国药典: 二部 (2020年版)[M]. 北京: 中国医药科技出版社, 2020. [2] 张娇, 王元忠, 杨维泽, 等. 黄精属植物化学成分及药理活性研究进展 [J]. 中国中药杂志, 2019, 44(10):1989−2008.

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