Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (13): 2710-2723.doi: 10.3864/j.issn.0578-1752.2021.13.002

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Cloning of TaBG and Analysis of Its Function in Anther Dehiscence in Wheat

TAN ZhaoGuo1,2(),LI YanMei2(),BAI JianFang2,GUO HaoYu2,LI TingTing2,DUAN WenJing2,LIU ZiHan2,YUAN ShaoHua2,ZHANG TianBao2,ZHANG FengTing2,CHEN ZhaoBo2,ZHAO FuYong1(),ZHAO ChangPing2(),ZHANG LiPing1,2()   

  1. 1College of Life Sciences, Yangtze University, Jingzhou 434025, Hubei
    2Beijing Engineering and Technique Research Center for Hybrid Wheat, Beijing Academy of Agriculture and Forestry Sciences/The Municipal Key Laboratory of the Molecular Genetics of Hybrid Wheat, Beijing 100097
  • Received:2020-11-25 Revised:2021-02-05 Online:2021-07-01 Published:2021-07-12
  • Contact: FuYong ZHAO,ChangPing ZHAO,LiPing ZHANG E-mail:tanzhaoguo@foxmail.com;lymei1013@163.com;fyzhao@yangtzeu.edu.cn;cp_zhao@vip.sohu.com;lpzhang8@126.com

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Abstract:

【Objective】 β-glucosidase (BG, 4-β-D-glueosidase) is a kind of hydrolase which hydrolyzes glycosidic bond from glycopolymer or oligosaccharide to release non reducing sugar group, which plays an important role in controlling anther dehiscence. The anthers of the photoperiod-temperature sensitive genic male sterile (PTGMS) wheat line BS366 did not dehiscence in sterile environment, but completely or partially cracked in fertile environment. In order to study the regulatory function of BG gene on anther dehiscence in wheat, the TaBG was cloned from BS366 and its potential function in anther dehiscence was analyzed, which provided theoretical basis for further analysis of molecular mechanism of abnormal anther dehiscence in photoperiod thermo sensitive male sterile line of wheat.【Method】TaBG was cloned from the anther of PTGMS line BS366, and the primary structure, secondary structure, tertiary structure of TaBG protein were predicted. In addition, the cis-acting elements in promoter region prediction, phylogenetic tree construction with other species, interaction between miRNA and TaBG were performed using bioinformatics software. The expression levels of TaBG and the interacted tae-miR395a in anthers and glumes under MeJA and SA treatment were determined. 【Result】 The total length of TaBG is 1 473 bp, which encoded 490 amino acids, and the theoretical pI is 8.12, belonging to the glycosylhydrolase superfamily. The results showed that TaBG may be regulated by stress resistance-related miRNAs, such as miR169and miR395a. Based on the prediction of protein interaction, it was found that TaBG might interact with glucose-methanol-choline oxidoreductase (GMC) and endoglucanase (EG). TaBG is located in the liquid bubble of the wheat proton. The qPCR results showed that the expression of TaBG in Bilocular stage (stage 13), Dehiscence (stage14) and Senescence (stage15) showed an upward and then downward trend. TaBG was the highest expression in Dehiscence, and the expression of TaBG in no dehiscence of anther was 2.8 times higher than that of complete dehiscence of anther. After treatment of MeJA, the expression ofTaBG in anther and glume showed downward. The tae-miR395a expression pattern was the opposite.【Conclusion】 TaBGmight be involved in anther dehiscence with stress tolerance related miRNA such as miR169andmiR395a. High expression of TaBG could increase the content of soluble sugar in the anther under the sterile environment, and then increase the osmotic potential in the anther, so as to slow down the dehydration of anther dehiscence. This study will lay a foundation for elucidating the biological function of BG in regulating anther dehiscence.

Key words: wheat, 4-β-D-glueosidase, miRNA, anther dehiscence

Table 1

Primer sequences used in this study"

引物名称 Primer name 引物序列 Primer sequence (5'-3') 用途 Usage
TaBG-F CTCCACTATGGAGCTCCTCCG 基因扩增Gene amplification
TaBG-R TATTCACCCATCTGGCACCACT
TaBG-CF ATGGAGCTCCTCCGCG 蛋白编码区克隆 CDS cloning
TaBG-CR TCAGTACTCCTCGCTCTT
TaBG-DF TATCTCTAGAGGATCCATGGAGCTCCTCCGCG 亚细胞定位 Subcellular localization
TaBG-DR TGCTCACCATGGATCCGTACTCCTCGCTCTTGATCAC
TaBG-QF CCTCACGCGCTCTACGACACC 实时荧光定量PCR qPCR
TaBG-QR CCATGCGAAGTAGCCCGTCACC
β-Actin-QF TACTCCCTCACAACAACCG 内参基因 Reference gene
β-Actin-QR AGAACCTCCACTGAGAACAA
tae-miR395a-QF GTGAAGTGTTTGGGGGAACTC 实时荧光定量PCR qPCR
tae-miR395a-QR AGAACTTCACAGTGGTCTTA
U6snRNA-QF CAAGGATGACACGCAAATTCG 内参基因 Reference gene
U6snRNA-QR GTGCAGGGTCCGAGGT

Fig. 1

Anther dehiscence morphology of BS366 at senescence A: Complete dehiscence (Beijing); B: Incomplete dehiscence (Beijing); C: No dehiscence (Dengzhou, Henan)"

Fig. 2

PCR amplification and sequence analysis of TaBG A: Cloning of TaBG, M: DL 2000 maker; 1: TaBG PCR product. B: Sequence analysis of TaBG "

Fig. 3

Prediction of signal peptide and conserved domain of TaBG A: Signal peptide; B: Conservative domain"

Table 2

Analysis of promoter element"

上游启动子元件 Upstream promoter elements 功能 Function
CAAT-box、TATA-box 核心启动子元件 Core promoter element
ABRE 脱落酸响应元件 Abscisic acid responsiveness element
CGTCA-motif、TGACG-motif 茉莉酸甲酯响应元件 MeJA-responsiveness responsiveness element
TCA-element 水杨酸响应元件 Salicylic acid responsiveness element
G-box、TCT-motif 光响应元件 Light responsiveness element
CAT-box 分生组织表达相关元件 Related to meristem expression element

Fig. 4

Amino acid sequence alignment of TaBG with other species homologous proteins"

Fig. 5

Phylogenetic tree analysis of TaBG and BG in other species and their corresponding motif prediction Different color parts in the evolutionary tree represents different groups. XP_020155850.1: Aegilops tauschiiCoss.; XP_020155849.1: Aegilops tauschiiCoss.; XP_020166122.1: Aegilops tauschiiCoss.; VAI78197.1: Triticum turgidumL.var.durumDesf. Yan. ex P. C.; EMS63424.1: Triticum aestivum L.; VAI90324.1: Triticum turgidum L. var. durum Desf. Yan. ex P. C.; KAE8767126.1: Hordeum vulgare L.; XP_010237292.1: Brachypodium distachyon L. Beauv.; XP_006664007.1: Oryza rufipogon Griff.; KAF0922613.1:Oryza rufipogonGriff.; EEC69164.1: Oryza rufipogonGriff.; XP_015620634.1: Oryza sativa L.; KAB8117340.1: Oryza sativa L.; TVU50559.1: Eragrostis curvula Schrad Nees.; PWZ23263.1: Zea mays L.; XP_020393252.1: Zea maysL.; XP_021304604.1: Sorghum bicolor L.; RLN3116: Panicum miliaceumvar.Compactrm; RLM98073.1: Panicum miliaceumvar. Compactrm; PUZ66950.1: Panicum miliaceumvar. Compactrm; XP_025809448.1: Panicum miliaceum var. Compactrm; RLN31163.1: Panicum miliaceumvar. Compactrm; TVU50559.1: Eragrostis curvulaSchrad. Nees; RLM98073.1: Panicum miliaceumvar.Compactrm; XP_021304604.1: Sorghum bicolorL.; PWZ23263.1: Zea maysL.; XP_004963948.1: Setaria italica L. Beauv. var. germanicaMill. Schrad.; XP_020393252.1:Zea mays L.; XP_006490742.1: Citrus sinensis "

Table 3

Motif sequences of TaBG"

基序 Motif 基序序列 Motif sequence
Motif 1 DFCFKTYGDRVKNWFTINEPRMMAWHGYGDGFFPPGRCTGC
Motif 2 CIWDTFLKYPGATPDNATANVTVDEYHRYMDDVDNMVRVGFDAYRFSISW
Motif 3 DGARVTGYFAWSLLDNFEWRLGFTSKFGJVYVDRKTFTRYPKDSTRWFRK
Motif 4 GKIGILLDFVWYEPLTYSVEDEYAAHRAREFTLGWFLHPITYGHYPETMZ
Motif 5 SRIFPSGIGRINKDGVDYYHRLIBYMLANNITPYVVLYHYDLPZVLNBQY
Motif 6 ERNGVPIGKQAYSDWLYVVPWGFYKAVMHVKEKFNBPVILIGENGIDQSG
Motif 7 KJVGGRLPNFTFEQSKMVKGSADYIAINHYTTYYVSHHVNLTHMSYANDW
Motif 8 LLANGEHTBLTRDSFPPGFVFGTASSAYQVEGNALKYGRGP
Motif 9 NSATEPYIAGHHLLLAHAAAVKVYRDKYQ
Motif 10 NDTLPHALYDTFRIDYFDQYLHELKRAIA

Fig. 6

Interaction analysis ofTaBG with miRNA and protein interaction prediction of TaBG A: TaBGand miRNA interaction; B: miRNA hairpin of tae-miR395a; C: Network diagram of TaBG protein interaction "

Fig. 7

Subcellular localization of TaBG-GFP fusion in wheat protoplasts "

Fig. 8

Relative expression pattern of TaBG gene in different anther development stages and tissues of wheat "

Fig. 9

Expression pattern of TaBG and tae-miR395a in anther and glumes under the stresses of MeJA and SA Expression profiling of TaBG and tae-miR395a in anthers (A and B) and glumes (C and D) treated with MeJA (0, 0.5, 2, and 4 mmol·L-1) and SA (10 mmol·L-1). MeJA: Spikelets were treated with MeJA (0, 0.5, 2, and 4 mmol·L-1). MeJA+SA: Treatment of spikelets with different concentration of MeJA (0, 0.5, 2, and 4 mmol·L-1) and than application of SA (10 mmol·L-1) "

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