Cloning and Functional Prediction of Tappc3A Gene in Wheat

doi:10.7668/hbnxb.20193798

Abstract

Abstract:

PEPC catalyzes phosphoenolpyruvate(PEP)to generate oxaloacetate(OAA)to participate the tricarboxylic acid(TCA),which plays an important role in development and stress adaptation of plant.However,there have been no reports of PEPC involvement in plant organ development.Exploration and study on biological function of Tappc3A gene in wheat flower development,and provides new clues to explore the molecular mechanism of homologous conversion of stamens into pistillody in wheat.The Tappc3A gene was cloned from CM28TP and HTS-1 by PCR,and the sequence and phylogenetic tree were analyzed by bioinformatics tools,and the expression level of Tappc3A gene in different developmental stages and different reproductive organs of wheat young spikes was analyzed by using qRT-PCR,and whether the protein function encoded by Tappc3A gene was analyzed by prokaryotic expression.The wheat RNA-Seq database was used to analyze the co-expression of Tappc3A gene and other genes that regulate flower organ development.The ORF of Tappc3A gene was 2 901 bp in length,encoding 966 amino acid residues,with a typical phosphoenolpyruvate carboxylase(PEPase)conserved domain,a conserved serine(Ser,S)reversible phosphorylation site(SIDAQLR)at the N-terminal,a plant-type PEPC protein signature sequence(QNTG)at the C-terminal,and the 774th amino acid was the typical alanine of C3 plant PEPC.Cluster analysis also showed that Tappc3A belonged to the C3 type PEPC family.qRT-PCR analysis,in three stages of wheat young spike development,showed that the expression of Tappc3A gene in HTS-1 was higher than that in CM28TP at the dichotomous stage to the florescence differentiation stage and the pharmacophore period,and the expression of Tappc3A gene was significantly higher in pistils(P)and pistillody stamens(PS)than stamens(S).The prokaryotic expression results showed that the protein encoded by Tappc3A gene,which catalyzes the production of OAA from PEP,and its activity was significantly enhanced after IPTG induction.The gene co-expression analysis showed that Tappc3A gene might be involved in the morphogenesis of wheat floral organs.Tappc3A gene might be involved in wheat pistil development,and its overexpression in stamens might be associated with the homologous transformation of stamens into pistillody trait formation.

Key words: Wheat, PEPC gene, Gene expression, Pistil development

XIANG Guili, WU Rina, YAMAMOTO Naoki, WU Yichao, JIANG Jin, LIAO Mingli, WEI Shuhong, PENG Zhengsong, YANG Zaijun. Cloning and Functional Prediction of Tappc3A Gene in Wheat[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(4): 28-37. doi: 10.7668/hbnxb.20193798.

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Figures/Tables 8

Tab.1 Primers of Tappc3A gene used for gene cloning,qRT-PCR and prokaryotic expression

引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')	退火温度/℃ Annealing temperature	引物用途 Primer purpose
Tappc3A-F	GGGGGAAGAGGGTTTTCTCG	56.0	克隆扩增
Tappc3A-R	CACACCATCTCACATAACATAACAGT
Tappc3A-qF	CAACATTTGGACTATCCCTTG	56.0	qRT-PCR
Tappc3A-qR	TTGGCGTTTCTCCTCTGAC
Tappc3A-edF	CCGGAATTCATGGCGCGCAA	61.5	原核表达
Tappc3A-edR	CCCAAGCTTTCAGCCGGTGT

Fig.1 PCR amplification and CDS sequence of Tappc3A gene A.Cloning of Tappc3A gene:M.DL5000 Marker,1.HTS-1 PCR product,2.CM28TP PCR product;B.CDS sequence of Tappc3A gene.

Fig.2 Amino acid sequence alignment of Tappc3A and PEPC in other plants Boxes are the reversible phosphorylationsite at the N terminus and plant-type PEPC motifs at the C terminus; Underlines.Conserved subdomains essential for PEPC catalysisis;Arrow.Amino acid residue in 774.

Fig.3 Analusis of the Tappc3A protein sequence A.Domain of the Tappc3A protein;B.Secondary structure prediction of the Tappc3A protein; C.Prediction of the hydrogen.The red part.Alpha helix;The blue part.β-extended strand;The black part.Random coil.

Fig.4 Phylogenetic tree of Tappc3A and PEPC in other species

Fig.5 Relative expression level of Tappc3A gene in CM28TP,HTS-1 and P,PS,S 1.Double ridge stage to floret differentiation stage 0.2—0.5 cm;2.Pistil and stamen primordiuxn formation stage 0.5—0.7 cm;3.Anther seperation stage 0.7—1.0 cm;P.Pistil;PS.Pistilloid-stamen;S.Stamen.Means with different letters are significant differences at the 0.05 probability level.The same as Fig.6.

Fig.6 Prokaryotic expression analysis A.Identification of recombinant plasmid pET-28a-Tappc3A by double enzyme digestion:M.Marker 15000;1.pET-28a-Tappc3A digested by double enzyme EcoRⅠand Hind Ⅲ;2.pET-28a-Tappc3A linear plasmid.B.Enzymatic activity assay of PEPC:VC.Control group;3A.Experience group.

Fig.7 GO enrichment analysis of Tappc3A co-expression genes Color indicates enrichment degree;Size indicates number of gene.

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