滇水金凤花距发育相关基因<i>TCP4</i>的克隆及表达分析

doi:10.3785/j.issn.1008-9209.2022.04.064

浙江大学学报（农业与生命科学版）

2023, Vol. 49

Issue (3): 341-348 DOI: 10.3785/j.issn.1008-9209.2022.04.064

园艺科学

滇水金凤花距发育相关基因TCP4的克隆及表达分析

李洋(

),李凡,孟丹晨,李林菊,魏春梅,黄美娟(

),黄海泉(

)

西南林业大学园林园艺学院/国家林业和草原局西南风景园林工程技术研究中心/云南省功能性花卉资源及产业化技术工程研究中心/西南林业大学园林园艺花卉研发中心，云南昆明 650224

Cloning and expression analysis of petal spur development related gene TCP4 in Impatiens uliginosa

Yang LI(

),Fan LI,Danchen MENG,Linju LI,Chunmei WEI,Meijuan HUANG(

),Haiquan HUANG(

)

College of Landscape Architecture and Horticulture Sciences, Southwest Forestry University/Southwest Research Center for Engineering Technology of Landscape Architecture (State Forestry and Grassland Administration)/Yunnan Engineering Research Center for Functional Flower Resources and Industrialization/Research and Development Center of Landscape Plants and Horticulture Flowers, Southwest Forestry University, Kunming 650224, Yunnan, China

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摘要：

为探究滇水金凤（Impatiens uliginosa）TCP4基因（IuTCP4）对花距发育的调控作用，通过反转录聚合酶链反应技术克隆其cDNA全长，对其序列进行生物信息学分析，并采用实时荧光定量聚合酶链反应（real-time fluorescent quantitative polymerase chain reaction, qRT-PCR）技术探讨IuTCP4基因在花距的不同发育阶段及不同组织部位的时空表达模式。结果表明：滇水金凤IuTCP4基因2个拷贝（IuTCP4.1和IuTCP4.2）的cDNA全长分别为1 194、1 173 bp，各编码397个和390个氨基酸，且均不包含内含子；IuTCP4.1和IuTCP4.2所编码的蛋白均为不稳定的亲水性蛋白，且均不存在信号肽和跨膜结构域，两者与茶（Camellia sinensis）、苦瓜（Momordica charantia）等15个物种TCP4蛋白的同源性达到66.11%。在系统发育树中这2个拷贝聚为一支，推测它们为旁系同源基因。qRT-PCR结果显示：IuTCP4.1在初期、开花期的花距和檐部存在差异性表达，且在初期的檐部表达量最高；IuTCP4.2在3个发育时期的花距和檐部均存在差异性表达，且在初期的花距中表达量最高。综上所述，IuTCP4基因在花距发育过程中具有一定的调控作用，且主要在花距发育初期发挥作用。上述结果为凤仙花花距发育机制、花形改良和新品种培育提供了一定的理论依据。

关键词： 滇水金凤; 花距; TCP4基因; 基因克隆; 表达分析

Abstract:

To explore the regulation mechanism of TCP4 gene on the development of petal spur of Impatiens uliginosa, the full length cDNA of TCP4 gene in I. uliginosa (named as IuTCP4) was cloned by reverse transcription polymerase chain reaction, and its sequence was analyzed by bioinformatics. Then, real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to investigate the spatiotemporal expression pattern of IuTCP4 gene at different developmental stages and different tissue locations of petal spur. The results showed that the full-length cDNA of two copies (IuTCP4.1 and IuTCP4.2) of TCP4 gene in I. uliginosa were 1 194 bp and 1 173 bp, encoding 397 and 390 amino acids, respectively, and neither contained introns. Both of IuTCP4.1 and IuTCP4.2 encoded proteins were unstable and hydrophilic without signal peptide and transmembrane domain. The homology of amino acid sequences of TCP4 protein between I. uliginosa and other 15 species, such as tea (Camellia sinensis) and balsam pear (Momordica charantia), reached 66.11%. In the phylogenetic tree, IuTCP4.1 and IuTCP4.2 clustered into one branch, suggesting that the two copies of IuTCP4 are paralogous genes. The results of qRT-PCR showed that IuTCP4.1 was differentially expressed between petal spur and limb at both the early stage and blooming stage, with the highest expression quantity in the limb at the early stage; IuTCP4.2 was differentially expressed between petal spur and limb at all three developmental stages, and the highest expression quantity was observed in the petal spur at the early stage. In conclusion, IuTCP4 plays a certain regulatory role in the development of petal spur and mainly functions at the early stage of petal spur development. It is provided a theoretical basis for the development mechanism of petal spur, flower shape improvement and new variety cultivation of Impatiens.

Key words: Impatiens uliginosa petal spur TCP4 gene gene cloning expression analysis

收稿日期: 2022-04-06 出版日期: 2023-06-25

CLC:

Q78

基金资助: 国家自然科学基金项目(32060364);云南省重大科技专项计划项目(202102AE090052);云南省重点研发计划项目(2018BB013);云南省教育厅科学研究基金项目(2020Y0410);云南省中青年学术和技术带头人培养项目(2018HB024);云南省高校园林植物与观赏园艺科技创新团队建设项目(51700204);云南省园林植物遗传改良与高效繁育博士生导师团队建设项目(503210103)

通讯作者: 黄美娟,黄海泉 E-mail: 1050502487@qq.com;xmhhq2001@163.com;haiquanl@163.com

作者简介: 李洋（https://orcid.org/0000-0001-9453-473X），E-mail：1050502487@qq.com

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引用本文:

李洋,李凡,孟丹晨,李林菊,魏春梅,黄美娟,黄海泉. 滇水金凤花距发育相关基因TCP4的克隆及表达分析[J]. 浙江大学学报（农业与生命科学版）, 2023, 49(3): 341-348.

Yang LI,Fan LI,Danchen MENG,Linju LI,Chunmei WEI,Meijuan HUANG,Haiquan HUANG. Cloning and expression analysis of petal spur development related gene TCP4 in Impatiens uliginosa. Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(3): 341-348.

链接本文:

https://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2022.04.064 或 https://www.zjujournals.com/agr/CN/Y2023/V49/I3/341

基因

Gene

引物序列（5 $′$ →3 $′$ ）

Primer sequence (5 $′$ →3 $′$ )

IuTCP4.1

F: ATGGGAGGAGATACTCATAATACC

R: TTAATGGCGAGAATCGGAG

IuTCP4.2

F: ATGGGAAGAGGAGCTAACTC

R: TCAATCGATAATATTATTATGTTGAGAGTCGG

表1 IuTCP全长扩增引物序列

图1 滇水金凤花距的3个发育时期（A）及其组织部位（B）

基因

Gene

引物序列（5 $′$ →3 $′$ ）

Primer sequence (5 $′$ →3 $′$ )

IuTCP4.1

F: AGATTGTCGAGGTTCAGGGC

R: TCACTCTGTTTCCTGCGGTC

IuTCP4.2

F: GGAGGAGTTCGTGGGACAAG

R: GCACCTACCGGGAAGAACA

IuActin

F: TGAATGTCCCTGCTGTTTG

R: ACCTTCCGCATAACTTTACC

表2 IuTCP qRT-PCR引物序列

图2 IuTCP4.1 和 IuTCP4.2 的PCR扩增结果M：DL2000+ DNA标志物（marker）；1：IuTCP4.1 cDNA；2：IuTCP4.1 gDNA；3：IuTCP4.2 cDNA；4：IuTCP4.2 gDNA。

表3 滇水金凤IuTCP4蛋白生物信息学分析

图3 滇水金凤IuTCP4蛋白序列同源性比对

图4 TCP4蛋白同源序列的系统进化树

图5 IuTCP4 基因在滇水金凤3个时期花距和檐部的相对表达量短栅上不同小写字母表示在P＜0.05水平差异有统计学意义，n=3。

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