小麦<i>TaSPP</i>基因的克隆及表达分析

doi:10.7668/hbnxb.20192653

摘要/Abstract

摘要：

为明确磷酸蔗糖磷酸酶基因(SPP)的表达特征和生物学功能,进一步了解SPP参与蔗糖生物合成的调控机制。以小麦抗旱品种晋麦47的cDNA为模板克隆出3个位于第5同源群上的TaSPP同源基因,分别命名为TaSPP-5A、TaSPP-5B和TaSPP-5D。并利用生物信息学对小麦TaSPP的理化性质、基因结构、顺式作用元件、系统进化树和蛋白保守结构域等进行分析,通过qRT-PCR分析基因TaSPP的表达模式。结果表明,TaSPP-5A、TaSPP-5B、TaSPP-5D都包含8个外显子和7个内含子,TaSPP-5A和TaSPP-5D编码422个氨基酸,TaSPP-5B编码413个氨基酸。系统进化分析显示,小麦TaSPP基因与小麦的近缘物种在进化上处于同一分支,相似度较高。qRT-PCR表达分析结果显示,TaSPP基因在根、茎秆、旗叶、叶鞘、颖花、种子中均表达,其中在旗叶和茎秆中表达量较高;ABA、PEG-6000、NaCl和IAA胁迫均能诱导TaSPP基因的表达,表明小麦TaSPP基因可能在逆境胁迫过程中发挥重要作用。

关键词: 小麦, 磷酸蔗糖磷酸酶基因, 克隆, 非生物胁迫, 表达分析

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

景凡丽, 张沛沛, 苗永平, 陈涛, 刘媛, 杨德龙. 小麦TaSPP基因的克隆及表达分析[J]. 华北农学报, 2022, 37(2): 28-34. doi: 10.7668/hbnxb.20192653.

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.

http://www.hbnxb.net/CN/Y2022/V37/I2/28

图/表 12

表1 扩增cDNA、荧光定量的引物

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

图1 TaSPP基因的PCR扩增

Fig.1 The PCR amplification of TaSPP gene

图2 TaSPP蛋白序列比对

Fig.2 Sequence alignment of TaSPP proteins

图3 小麦TaSPP的基因结构

Fig.3 Gene structure of wheat TaSPP

图4 TaSPP启动子顺式作用元件

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

表2 TaSPP基因的顺式作用元件及功能注释

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

表3 TaSPP蛋白的二级结构预测

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

图5 TaSPP蛋白的三级结构

Fig.5 Prediction of the tertiary structure of TaSPP

图6 小麦与其他植物SPP蛋白的进化分析(A)和TaSPP蛋白聚类互作网络图(B)
Zm.玉米;Os.水稻;At.拟南芥;Md.苹果;Sc.番茄;Tu.乌拉尔图小麦;As.拟斯卑尔托山羊草;Aet.粗山羊草;Nt.烟草;Gm.大豆;Pt.杨树;St.马铃薯;Rc.蓖麻;Sc.黑麦。

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.

图7 TaSPS与TaSPP氨基酸序列功能域分析

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

图8 小麦不同组织中TaSPP的表达模式
内参基因.β-actin;不同小写字母表示差异显著(P<0.05)。图9同。

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.

图9 TaSPP基因在ABA、PEG-6000、NaCl、IAA胁迫下的表达分析

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

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