Cloning and Expression Analysis of TaSPP Gene in Wheat

doi:10.7668/hbnxb.20192653

Abstract

Abstract:

In order to clarify the expression characteristics and biological functions of the sucrose phosphate phosphatase gene(SPP),and further understand the regulatory mechanism of SPP involved in sucrose biosynthesis.Herein,three TaSPP homologs,TaSPP-5A,TaSPP-5B and TaSPP-5D,located on the fifth homologous group,were cloned using the cDNA of wheat variety Jinmai 47 as the template.The physical and chemical properties,gene structure,cis-acting elements,phylogenetic tree and protein conserved domains of TaSPP were analyzed by the method of bioinformatics.The expression pattern of TaSPP was analyzed by qRT-PCR.The results showed that TaSPP-5A,TaSPP-5B and TaSPP-5D contained eight exons and seven introns.TaSPP-5A and TaSPP-5D encoded 422 amino acids,while TaSPP-5B encoded 413 amino acids.Phylogenetic analysis showed that TaSPP in wheat and its related species belonged to the same evolutionary branch with highly genetic similarity.The specific expression analysis showed that TaSPP genes were expressed in roots,stems,flag leaves,leaf sheaths,flower and seeds,whereas the higher expression levels were identified in flag leaves and stems.The expressions of TaSPP gene could be induced by ABA,PEG-6000,NaCl and IAA,indicating that TaSPP genes could play an essential role in abiotic stress tolerance in wheat.

Key words: Wheat, SPP, Cloning, Abiotic stress, Expression analysis

JING Fanli, ZHANG Peipei, MIAO Yongping, CHEN Tao, LIU Yuan, YANG Delong. Cloning and Expression Analysis of TaSPP Gene in Wheat[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(2): 28-34. doi: 10.7668/hbnxb.20192653.

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Figures/Tables 12

Tab.1 Primers for cDNA amplification and fluorescence quantification

基因 Genes	引物序列(5'—3') Sequence of primers(5'—3')
TaSPP-cDNA-F/R	Forward:ATGGATAAGCTCAAGGGCTCT
	Reverse:CTAGAGTTTGGATGACTGCTCG
TaSPP	Forward:TGAGAGGTGTGCTGCTGGTATT
	Reverse:CTGTAGGCTTGAAAGGGTCCTCC
TaACTIN	Forward:GACCCAGACAACTCGCAAC
	Reverse:GGAATCCATGACCACCTAC

Fig.1 The PCR amplification of TaSPP gene

Fig.2 Sequence alignment of TaSPP proteins

Fig.3 Gene structure of wheat TaSPP

Fig.4 The cis-acting element of the TaSPP promoter

Tab.2 Cis-acting elements and functional annotations of TaSPP gene

顺式元件 Cis-acting element	功能 Function
ABRE	脱落酸诱导涉及的顺式作用元件
ARE	厌氧诱导的顺式作用元件
CGTCA-motif	茉莉酸响应的顺式作用元件
G-box	光响应顺式作用元件
LTR	低温诱导的顺式作用元件
MBS	参与干旱诱导的MYB结合位点
Sp1	光响应元件
TCA-element	水杨酸反应涉及的顺式作用元件
TGACG motif	MeJA反应的顺式作用元件

Tab.3 Predicted secondary structure of TaSPP protein%

蛋白名称 Protein name	α-螺旋 Alpha helix	β-折叠 Beta turn	无规则卷曲 Random coil	延伸链 Extended strand
TaSPP-5A	36.26	7.11	37.91	18.72
TaSPP-5D	36.73	7.58	36.97	18.72
TaSPP-5B	36.26	6.87	37.91	18.96

Fig.5 Prediction of the tertiary structure of TaSPP

Fig.6 Phylogenetic analysis of the SPP proteins from wheat and other plants(A)and TaSPP protein clustering interaction network diagram(B)
Zm.Zea mays;Os.Oryza sativa;At.Arabidopsis thaliana;Md.Malus domestica;Sc.Solanum lycopersicum;Tu.Triticum urartu;As.Aegilops speltoides;Aet.Aegilops tauschii;Nt.Nicotiana tabacum;Gm.Glycine max;Pt.Populus trichocarpa;St.Solanum tuberosum;Rc.Ricinus communis;Sc.Secale cereale.

Fig.7 The conserved function domain of amino acid sequence of sucrose-phosphate synthase and sucrose-phosphate phosphatase in the wheat

Fig.8 Expression patterns of TaSPP in wheat tissue
Reference gene.β-actin;Different lowercase letters indicate significant differences(P<0.05).The same as Fig.9.

Fig.9 Expression analysis of TaSPP gene under ABA,PEG-6000,NaCl and IAA treatments

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