Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (23): 4727-4737.doi: 10.3864/j.issn.0578-1752.2020.23.001

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

Cloning, Expression and Functional Analysis of a Male Fertility Gene ThMs1 in Bread Wheat

HENG YanFang1(),LI Jian2,WANG Zheng2,CHEN Zhuo1,HE Hang2,DENG XingWang2,MA LiGeng1()   

  1. 1College of Life Sciences, Capital Normal University, Beijing 100048
    2Peking University Institute of Advanced Agricultural Sciences, Weifang 261325, Shandong
  • Received:2020-09-20 Accepted:2020-10-30 Online:2020-12-01 Published:2020-12-09
  • Contact: LiGeng MA E-mail:yanfangh@126.com;ligeng.ma@cnu.edu.cn

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Abstract:

【Objective】To clone a new male fertility gene, ThMs1, from the ms1g-4Ag wheat-Thinopyrum ponticum alien addition line, and characterize the expression pattern and biological function of ThMs1 in bread wheat, with the hope that ThMs1 will be useful in the application in the preparation of wheat hybrid breeding system. 【Method】4Ag chromosome from Thinopyrum ponticum in wheat-Thinopyrum ponticum alien addition line was identified by GISH. ThMs1 was cloned from the wheat-Thinopyrum ponticum alien addition line by homology-based cloning, and the analysis of the function of ThMs1 in the control of wheat male fertility was performed through stable genetic transformation. RT-PCR and qRT-PCR were performed to detect the expression pattern of ThMs1, further analyzing its tissue-specific expression by RNA in situ hybridization. The expression of ThMs1 protein in vivo was detected by Western blot using specific anti-Ms1 antibodies. ThMs1 protein structure were predicted by SignalP software. The detection of lipid-binding activity of ThMs1 was performed by dot blot. 【Result】The wheat-Thinopyrum ponticum alien addition line harbored 4Ag chromosome from Thinopyrum ponticum. ThMs1, the homolog of bread wheat male fertility gene Ms1, was cloned from the wheat-Thinopyrum ponticum alien addition line. The functional complementation analysis confirmed that the ThMs1 can completely restore the male fertility phenotype of the wheat ms1. The phylogenetic analysis suggested that the MS1 only exists in the Poaceae family. RT-PCR and qRT-PCR analysis confirmed that ThMs1 gene was expressed in anthers during meiosis. RNA in situ hybridization analysis revealed that ThMs1 was specifically expressed in microspore during wheat microsporogenesis. Western blot showed that ThMs1 was detected in wheat anther. The structure prediction analysis revealed that it has a predicted lipid binding domain, and protein lipid-binding experiments indicated that ThMs1 protein specifically binds PA and several phosphatidylinositols. 【Conclusion】A new male fertility gene, ThMs1, was cloned from wheat-Thinopyrum ponticum alien additiona line. ThMs1 exhibits similar expression pattern to that of Ms1 in bread wheat, and ThMs1 binds to the same phospholipid molecules as Ms1, thus ThMs1 is a structural and functional homolog of wheat Ms1. Therefore, the present work identified a new male fertility gene, and provided a foundation for the establishment of wheat hybrid breeding system through molecular design using wheat ms1.

Key words: bread wheat, wheat-Thinopyrum ponticum alien addition line, recessive nuclear male sterility, ThMs1, Ms1, wheat hybrid breeding system, molecular design

Table S1

The primers used in this study"

引物 Primer 序列 Sequence (5'→3') 目的 Intention
ThMs1-F AGATCCCGGCGCCTGCTGCTC ThMs1扩增引物
PCR primer of ThMs1
ThMs1-R CGCAGGAGCTGTAGAGCGTGAGGA
BF TAGCCATCTTTGATCAATGAGC 4Ag染色体鉴定
Analysis of 4Ag chromosome
BR TGATGAAAGAGCTAGGTGATAGTTG
TF1 CGCCAGGGTTTTCCCAGTCACGAC 转基因鉴定
Analysis of transgenic lines
TR1 TACAATGGCTAGTAGAGATTTC
TF2 ATACGTTTCCTGCTACAGATTTGAGG
TR2 AAGCAGTGCCGCCAGAGGATCAACGC
TF3 TTTGCGTTCTGCTGATGATGTG
TR3 CTCAATTGTCCTTTAGACCATGTCTAAC
TaACTIN-QF TTCCGTTGCCCTGAGGTCC RT-PCR和实时荧光定量PCR
RT-PCR and real time PCR
TaACTIN-QR TGATCTTCATGCTGCTTGGTGC
Ms1-QF ACATCATCCTCTGAGTCGCG
Ms1-QR GACCACGCAAACACGTACG
ThMs1-QF TCCCGGCGCCTGCTGCTC
ThMs1-QR CGGCAGAGGCAGGGGACG
Ms1-ISH-F aagcttGAGCGAGGGAGAGAGAGACC RNA原位杂交
In situ hybridization
Ms1-ISH-F gaattcATCACATAGCATCAGTGGTTC
ThMs1-ISH-F aagcttCTAGCGAGCGAGCGAGAGG
ThMs1-ISH-R gaattcCAACTGGACGGACCATGGC
ThMs1-SB-F1 TGAGATATACGGAGCGATTTAG Southern杂交
Southern blot
ThMs1-SB-R1 AGCACGGCAAGCTTTTGCTCTG
His-F aattcCATCATCATCATCATCACTGActgca MBP-His载体构建
Vector construction of MBP-His
His-R gTCAGTGATGATGATGATGATGg
Ms1s-His-F gagggaaggatttcagaattcCAGCCGGGGGCGCCGTGC MBP-Ms1-His载体构建Vector construction of MBP-Ms1-His
Ms1s-His-R cagtgccaagcttgcctgcagTCAGTGATGATGATGATGATGGGCCGCCTTGGACGGCG
ThMs1s-His-F gagggaaggatttcagaattcGCGTTCGGGCCGCAGC MBP-ThMs1-His载体构建
Vector construction of MBP-ThMs1-His
ThMs1s-His-R cagtgccaagcttgcctgcagtcaGTGATGATGATGATGATGGAAGAAGGCCGCCTTGGACG

Fig. S1

The structure of transgenic vector and analysis of transgenic lines A: Transgenic vector of pAHC20-ThMs1p::ThMs1; B: Analysis of transgenic lines by PCR"

Fig. 1

Phenotypic characterization and cytological analysis of bread wheat, ms1g and ms1g/addition line A: Spikes at flowering, Bar=1 cm; B: Anthers after filament elongation, Bar=1 mm; C: I2-KI staining of mature pollen, Bar=200 μm; D: Seeds at 15 d after pollination, Bar=1 mm, the numbers below the figure in B, C and D indicate the number of plants with the phenotypes and the number of all observed plants; E: Genomic in situ hybridization (GISH) of somatic metaphase chromosomes, Bar=10 μm; F: Microspores from DAPI-stained, Bar=50 μm"

Fig. S2

Paraffin sections of anthers from bread wheat, ms1g, ms1g/addition line at different stages E: Epidermis; Ar: Archesporial cell; Sp: Sporogenous cell; En: Endothecium; ML: Middle layer; T: Tapetum; MMC: Microspore mother cell; MC: Meiotic cell; Tds: Tetrads; BP: Bicellular pollen; MP: Mature pollen. Bar=50 μm"

Fig. S3

Sequence alignment of ThMs1 and its orthologues in bread wheat"

Fig. 2

Southern blot of ThMs1 in genomic DNA from bread wheat, ms1g, ms1g-4Ag addition line and Th. Ponticum Genomic DNA was digested with HindⅢ(A) and EcoRⅠ(B), respectively"

Fig. 3

Complementation of ms1g by transformation with ThMs1 Ms1, ms1g, T1 transgenic line containing ThMs1 in a ms1g background. A: Spikes at flowering, Bar=1 cm; B: Anthers before filament elongation, Bar=1 mm; C: Seeds at 15 d after pollination, Bar=1 mm; D: I2-KI staining of mature pollen grains, Bar=200 μm, the numbers below the figure in B, C and D indicate the number of plants with the phenotype and the number of observed plants"

Fig. 4

Expression analysis of ThMs1 and Ms1 in ms1g-4Ag addition line and bread wheat, respectively A, B: Expression analysis by RT-PCR; C, D: quantitative RT-PCR; E, F: RNA in situ hybridization analysis. Ms1 in bread wheat and ThMs1in 4Ag addition line anthers. Sp: Sporogenous cell; T: Tapetum; MMC: Microspore mother cell; MC: Meiotic cell; Tds: Tetrads; Le&pa: Lemma and palea; UM: Unicellular microspore; BP: Bicellular pollen; MP: Mature pollen; Bar = 50 μm"

Fig. S4

Analysis of expression of Ms1 and ThMs1 in developing anther during microspore meiosis from bread wheat and ms1g-4Ag addition line"

Fig. S5

Sequence alignment of ThMs1 and its orthologues in Poaceae plants Th：十倍体长穗偃麦草Thinopyrum ponticum;Aet：节节麦Aegilops tauschii;Ta：普通小麦Triticum aestivum;Tt：圆锥小麦Triticum turgidum;Tu：乌拉尔图小麦Triticum Urartu;Bradi：二穗短柄草Brachypodium distachyon;Brast：短柄藓Brachypodium stacei;Hordeum vulgare：大麦; Phyuostachys heterocycla：毛竹;Leersia perrieri：假稻属;Oryza sativa：水稻;Zoysia japonica：结缕草;Phragmites australis：芦苇;Lophtherum gracile：淡竹叶;Sb：高粱Sorghum bicolor;Si：谷子Setaria italica;Sevir：狗尾草Setaria viridis;Pavir：柳枝稷Panicum virgatum。下同The same as below"

Fig. S6

Phylogenetic tree of ThMs1 and its orthologs in Poaceae plants Sequences were used to produce the phylogenetic tree shown in Fig. S4"

Fig. S7

The predicted structures of ThMs1 A: The conserved domain with 8 Cysteine residues; B: Predicted lipid binding domain"

Fig. 5

Both Ms1 and ThMs1 bind phospholipids in vitro A, B: Purified MBP-His, MBP-Ms1-His and MBP-ThMs1-His; MBP-His, MBP-Ms1-His and MBP-ThMs1-His were overlaid in an Echelon P-6001 lipid strip (C) and Echelon P-6002 membrane strip (D). CL: Cardiolipin. Cs: Cholesterol. DAG: Diacylglycerol. LPA: Lysophosphatidic acid. LPC: Lysophos-phocholine; PC: Phosphatidylcholine; PE: Phosphatidylethanolamine; PG: Phosphatidylglycerol; PI: Phosphatidy-linositol; PI(3)P: Phosphatidylinositol-3-phosphate; PI(4)P: Phosphatidylinositol-4-phosphate; PI(5)P: Phosphatidylinositol-5-phosphate; PI(3,4)P2: Phosphatidylinositol-3,4-bisphosphate; PI(3,5)P2: Phospha- tidylinositol-3,5-bisphosphate; PI(4,5)P2: Phosphatidylinositol-4,5-bisphosphate; PI(3,4,5)P3: Phospha- tidylinositol-3,4,5-triphosphate; PS: Phosphatidylserine; SM: Sphingomyelin; S1P: Sphingosine-1 -phosphate; TAG: Triacylglycerol"

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