Cloning and Expression Analysis of the Flowering Related Gene UrAP1 in Uncaria rhynchophylla

doi:10.7668/hbnxb.20195829

Abstract

Abstract:

During the reproductive phase of Uncaria rhynchophylla,stem hooks undergo conversion into peduncles,consequently diminishing stem hook yield.To identify key regulatory factors governing this organ homology shift between stem hooks and peduncles in U.rhynchophylla,tissues from stem hooks and flowers were utilized to isolate its floral meristem gene,UrAP1(APETALA1).Comprehensive bioinformatic characterization was subsequently performed to elucidate the physicochemical properties of its encoded product.Concurrently,qRT-PCR analysis was conducted to quantifiy the relative transcript abundance of UrAP1 across distinct tissues of vegetative and reproductive branches,aiming to clarify its functional role and regulatory network in floral organogenesis.Results demonstrated that the UrAP1 coding sequence (CDS) spanned 729 bp,encoding a 242 amino acid polypeptide.This sequence exhibited substantial homology(94% identity) with the AP1 gene from Cephalanthus occidentalis and harbored conserved MADS-box and K-box domains.Furthermore,the UrAP1 protein lacked transmembrane domains,with primary localization predicted within the nucleus.UrAP1 expression was detectable and relatively stable across leaves,stems,stem nodes,and stem hooks of vegetative shoots.In contrast,its transcript levels varied significantly in reproductive shoots,showing distinct abundances in leaves,stems,stem nodes,peduncles,and flower buds.Expression peaked in young buds during early flowering stages and subsequently declined as buds matured.Comparison of the expression profiling between the two growth phases suggests a crucial role of UrAP1 in regulating floral bud differentiation in U.rhynchophylla.

Key words: Uncaria rhynchophylla, UrAP1 gene, Bioinformatics, Gene expression, Cloning

CLC Number:

LI Yuqiong, QIN Ya, HUANG Qiulan, SHEN Jie, YANG Cuihong, LIANG Wenjing, WEI Shugen, PAN Limei, GUI Lingjian, FU Jine, LI Linxuan, WAN Lingyun. Cloning and Expression Analysis of the Flowering Related Gene UrAP1 in Uncaria rhynchophylla[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(6): 92-99. doi: 10.7668/hbnxb.20195829.

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Figures/Tables 14

Tab.1 Primers sequences

引物 Primer	引物序列(5'—3') Sequence (5'—3')
UrAP1-F	GGATCTTCCAGAGATATGGGGAGAGGAAAGGTACAGTT
UrAP1-R	CTGCCGTTCGACGATTCAGGCCGCAAAGCATCC
SAM-F	CACAATCTGGCATACGAAA
SAM-R	AACTCACTTGGCTGGAAAC
qRTPCR-F	GCACGGAAACAGTGAGTCTA
qRTPCR-R	CCAATCCTTCCTTGCCTAAATG

Tab.2 Bioinformatics analysis softwares of UrAP1

名称 Name	作用 Function	网址URL
ProtParam	蛋白质理化性质分析	https://web.expasy.org/protparam/
ProtScale	亲/疏水性评估	https://web.expasy.org/protscale/
WOLF PSORT	亚细胞定位分析	https://www.genscript.com/wolf-psort.html/
SMART	保守结构域鉴定	https://smart.embl-heidelberg.de/
TMHMM	跨膜拓扑预测	https://services.healthtech.dtu.dk/service.php?TMHMM-2.0
PSIPRED	二级结构推断	https://bioinf.cs.ucl.ac.uk/psipred/
SWISS-MODEL	三级结构建模	https://swissmodel.expasy.org/
DNAMAN 6.0	蛋白同源性比对	—
MEGA 11	系统进化树构建	—

Fig.1 Agarose gel electrophoresis of total RNA 1—8.The leaf,young bud,middle-stage bud,old bud,stem,peduncle,stem node,and peduncle node of the reproductive branches of Uncaria rhynchophylla;9—12.The vegetative leaf,stem hook,stem,and stem node of the vegetative plants.

Fig.2 PCR amplification of the UrAP1 gene cDNA M.DNA Marker (DL5000);1—6.Biological replicates.

Fig.3 Hydrophily analysis on UrAP1 coded protein Score>0.Hydrophobicity;score<0.Hydrophilicity.

Fig.4 Conserved domains of UrAP1 protein

Fig.5 Prediction of transmembrane structure of UrAP1 protein

Fig.6 Prediction of secondary structure of UrAP1 protein

Fig.7 Prediction of tertiary structure of UrAP1 protein

Fig.8 Regulatory elements on the UrAP1 promoter

Fig.9 Multiple sequence alignment of the protein encoded by the UrAP1 gene with AP1 amino acids from other species

Fig.10 Construction of phylogenetic tree of UrAP1 protein and AP1 homologous proteins from other plants

Fig.11 Relative expression levels of UrAP1 gene in different tissues of nutritional branches Different lowercase letters indicate significant difference at P<0.05.The same as Fig.12.

Fig.12 Relative expression levels of UrAP1 gene in different tissues of reproductive branches

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