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Acta Agronomica Sinica ›› 2021, Vol. 47 ›› Issue (7): 1332-1341.doi: 10.3724/SP.J.1006.2021.02049

• TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY • Previous Articles     Next Articles

Preliminary study of the peptide aptamer R18 promotes grain filling of rice inferior spikelets

ZHANG Zhi-Xing1,2, CHEN Hua1, MIN Xiu-Mei1, XU Hai-Long1, SONG Guo1, LIN Wen-Xiong1,2,*()   

  1. 1College of Life Sciences, Fujian Agricultural and Forestry University, Fuzhou 350002, Fujiang, China
    2Fujian Provincial Key Laboratory of Agroecological Processing and Safety Monitoring, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujiang, China
  • Received:2020-07-18 Accepted:2020-12-01 Online:2021-07-12 Published:2021-01-09
  • Contact: LIN Wen-Xiong E-mail:wenxiong181@163.com
  • Supported by:
    This work was supported by the National Natural Science Foundation of China(31871542);the National Key Research and Development Program of China(2018YFD0301105)

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Abstract:

14-3-3 protein plays an important role in plant growth and development. The high expression abundant of 14-3-3 isoform (GF14f) was an important reason for the poor grain-filling of rice inferior spikelets. In the present study, firstly we used molecular docking to study the most possible binding sites between GF14f protein and peptide aptamer R18. Furthermore, in vitro competitive experiment showed that the R18 could block GF14f binding to its interaction proteins, in turn, lead to a weakened interaction of GF14f with the SuS2, SS, and AGPS. Meanwhile, in vitro enzymatic assays showed that exogenous added R18 significantly increased the activity of sucrose synthase (SuSase), adenosine diphosphate-glucose pyrophosphorylase (AGPase), and starch synthase (StSase). Coimmunoprecipitation (Co-IP) experiments revealed that, in addition to GF14f, R18 could interacted with the other 14-3-3 isoforms, such as GF14b, GF14c, GF14d, and GF14e, which were further confirmed by Bimolecular Fluorescent Complimentary (BiFC) assay. These results also showed that the exogenous application (60 mg L -1) of R18 at grain-filling stage could significantly increase the activity of StSase, SuSase, and AGPase, resulting in the improvement of 1000-grain weight and seed-setting rate of rice inferior spikelets. In the present study, R18 had been investigated to competitively interfere with the interaction of rice 14-3-3 protein with multiple client proteins, weak the binding force, which increased the activity of sucrose conversion and starch synthesis related enzyme, resulting in the improvement of grain filling of rice inferior spikelets.

Key words: rice, inferior spikelets, grain filling, 14-3-3 protein, peptide aptamer

Table 1

Primers used in this study"

引物名称
Primer name		 引物序列
Primer sequence (5′-3′)		 用途
Usage
pet32a_His_R18-F GCTGATATCGGATCCGAATTCCCCCACTGCGTGCCCAGG 载体构建
Vector construct
pet32a_His_R18-R GTGGTGGTGGTGGTGCTCGAGGGGGGGCAGGCACATGTT
YFPN-GF14b-F TATACTAGTATGTCGGCACAGGCGGAGC 双分子荧光互补实验载体
Vector for BiFC
YFPN-GF14b-R ATATACTAGTCTGCCCCTCGCTGGAGTCG
YFPN-GF14f-F TTTTGCCACTAGTATGTCGCCTGCTGAGGCATCGCGT 双分子荧光互补实验载体
Vector for BiFC
YFPN-GF14f-R TTTTCCACTAGTGTGGCCCTCTCCTTCAGGCTTCGCT
YFPN- R18-F CTAGAATGCCCCACTGCGTGCCCAGGGACCTGAGCTGGCTGGACCTGGAGGCCAACATGTGCCTGCCCCCCT 双分子荧光互补实验载体
Vector for BiFC
YFPN- R18-R CTAGAGGGGGGCAGGCACATGTTGGCCTCCAGGTCCAGCCAGCTCAGGTCCCTGGGCACGCAGTGGGGCATT

Fig. 1

Molecular docking between GF14f protein and R18 polypeptide"

Fig. 2

Peptide aptamer R18 competes with client proteins for binding to rice GF14f proteins A: the in vitro interaction between GST-GF14f and His-SUS2 is gradually weakened with an increase in the concentration of His-R18; B: the in vitro interaction between GST-GF14f and His-AGPS is weakened at 60 mg L-1 of His-R18; C: the in vitro interaction between GST-GF14f and His-SS is weakened at 60 mg L-1 of His-R18."

Fig. 3

Effects of in vitro with the exogenous R18 on the activities of sucrose breakdown and starch synthesis-related enzyme A: the enzyme activity of SuSase is gradually increased with an increase in the concentration of R18. B: the enzyme activity of AGPase is significantly increase at R18 concentrations of 60 mg L-1. C: the enzyme activity of StSase is significantly increase at R18 concentrations of 60 mg L-1."

Fig. 4

Western blot verification of R18 in vivo DAF: days after flowering; CBB: coomassie brilliant blue."

Table 2

List of interaction isoforms between R18 and rice 14-3-3 proteins"

蛋白ID
Protein IDs		 蛋白名称
Protein name		 独特肽数量
Unique peptides
LOC_Os04g38870.5 GF14b 2
LOC_Os08g33370.2 GF14c 4
LOC_Os11g34450.1 GF14d 3
LOC_Os02g36974.4 GF14e 2
LOC_Os03g50290.3 GF14f 5

Fig. 5

Interaction between R18 with 14-3-3 family members GF14b and GF14f in pavement cells of Nicotiana benthamiana A: the interaction between R18 with GF14b; B: the interaction between R18 with GF14f."

Fig. 6

Effects of the exogenous application R18 on the activities of sucrose breakdown and starch synthesis-related enzyme at grain-filling stage A: the enzyme activity of AGPase is significantly increase with the 60 mg L-1 R18 solution; B: the enzyme activity of SuSase is significantly increase with the 60 mg L-1 R18 solution; C: the enzyme activity of StSase is significantly increase with the 60 mg L-1 R18 solution. Different lowercase letters indicate significant difference at P < 0.05 among the treatments."

Fig. 7

Effects of the exogenous application R18 on 1000-grain weight and seed setting rate of rice inferior spikelets A: the change in the 1000-grain weight of rice inferior spikelets; B: the change in the seed setting rate of rice inferior spikelets. Different lowercase letters indicate significant difference at P < 0.05 among the treatments."

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