多肽配体R18促进水稻弱势籽粒灌浆的初步研究

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

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。

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.