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作物学报 ›› 2021, Vol. 47 ›› Issue (7): 1332-1341.doi: 10.3724/SP.J.1006.2021.02049

• 耕作栽培·生理生化 • 上一篇    下一篇

多肽配体R18促进水稻弱势籽粒灌浆的初步研究

张志兴1,2, 陈花1, 敏秀梅1, 许海龙1, 宋果1, 林文雄1,2,*()   

  1. 1福建农林大学生命科学学院, 福建福州, 350002
    2福建农林大学 / 福建省农业生态过程与安全监控重点实验室, 福建福州, 350002
  • 收稿日期:2020-07-18 接受日期:2020-12-01 出版日期:2021-07-12 网络出版日期:2021-01-09
  • 通讯作者: 林文雄
  • 作者简介:E-mail: zhangzhixingfz@163.com
  • 基金资助:
    本研究由国家自然科学基金项目(31871542);国家重点研发计划项目资助(2018YFD0301105)

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 Published:2021-07-12 Published online:2021-01-09
  • Contact: LIN Wen-Xiong
  • 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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摘要：

14-3-3蛋白在植物生长发育中具有重要的调控作用, 其亚型GF14f在水稻弱势籽粒中高表达是其灌浆结实差的一个重要的原因。本研究首先通过分子对接的方式, 发现多肽R18与GF14f蛋白具有潜在的结合位点, 进而采用体外竞争性实验, 发现R18能够竞争性的与GF14f蛋白结合, 从而导致GF14f与SuS2、SS和AGPS三个互作靶蛋白间的结合力减弱, 与此同时, 体外酶活实验表明, 外源添加R18能够显著提高淀粉合成酶(StSase)、蔗糖合成酶(SuSase)和ADPG焦磷酸化酶(AGPase) 3个酶的活性。免疫共沉淀实验表明, R18在水稻籽粒中除了能和GF14f结合外, 还能与GF14b、GF14c、GF14d及GF14e结合, BiFC结果也证实了上述结果。籽粒灌浆期, 外源喷施60 mg L -1浓度的R18于弱势籽粒上, StSase、SuSase及AGPase酶活性显著提高, 弱势籽粒千粒重及结实率也显著提高。本研究结果表明, 多肽配体R18能够与14-3-3互作靶蛋白竞争性结合, 减弱14-3-3与靶蛋白间的结合力, 进而释放籽粒灌浆过程中蔗糖转化及淀粉合成途径中关键酶的活性, 从而有利于弱势籽粒灌浆充实。

关键词: 水稻, 弱势籽粒, 灌浆, 14-3-3蛋白, 多肽配体

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

表1

引物序列及用途"

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

图1

GF14f蛋白与R18多肽分子对接示意图"

图2

多肽配体R18与GF14f互作靶蛋白竞争性结合 A: 随着His-R18浓度的增加GST-GF14f与His-SUS2之间的结合力逐渐减弱; B: 60 mg L-1 His-R18作用下GF14f与AGPS间的结合力显著下降; C: 60 mg L-1 His-R18作用下GF14f与SS间的结合力显著下降。"

图3

体外添加R18多肽对蔗糖降解及淀粉合成相关酶活的影响 A: 随着R18浓度的增加蔗糖合成酶的活性逐渐提高。B: 60 mg L-1 R18作用下ADPG焦磷酸化酶活性显著提高。C: 60 mg L-1 R18作用下淀粉合成酶活性显著提高。"

图4

外源涂抹R18体内 Western Blot验证 DAF: 开花后天数; CBB: 考马斯亮蓝。"

表2

R18与水稻籽粒14-3-3蛋白亚型互作列表"

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

图5

R18与14-3-3蛋白家族成员GF14b和GF14f能够在烟草叶表皮细胞中相互作用 A: R18与GF14b相互作用; B: R18与GF14f相互作用。"

图6

外源喷施R18溶液对籽粒灌浆过程中蔗糖降解及淀粉合成相关酶活性的影响 A: 60 mg L-1 R18溶液作用下ADPG焦磷酸化酶活性显著提高; B: 60 mg L-1 R18溶液作用下蔗糖合成酶活性显著提高; C: 60 mg L-1 R18溶液作用下淀粉合成酶活性显著提高。不同小写字母表示P < 0.05差异显著。"

图7

外源喷施R18溶液对弱势籽粒千粒重及结实率的影响 A: 弱势籽粒千粒重变化; B: 弱势籽粒结实率变化。不同小写字母表示P < 0.05差异显著。"

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