转录因子MYB14调控玉米淀粉合成的分子途径解析

The starch synthesis rate in grain determined the grain filling rate and grain weight during crop grain filling period. Expanding the whole synthesis pathway of starch is an ideal way to increase the starch synthesis rate. In maize kernel, the starch synthesis rate-limiting enzyme is AGPase, which is a heterotetramer and encoded by Sh2 and Bt2 genes. Synchronize improve the expression of Sh2, Bt2 genes and other kernel starch content affecting genes such as Bt1, SSIV and other key enzyme coding genes of starch synthesis is an effective method to expand the whole starch synthesis pathway. The transcription factor MYB14 can significantly enhance the expression of Sh2, Bt2, Bt1 genes and other key enzyme coding genes in starch synthesis, which implied it has great potential to improve the starch synthesis rate in maize grain. In this project, the effect of MYB14 on the promoters’ activities of the maize starch synthesis key enzyme coding genes and the active regulatory sites of MYB14 on the promoters will analyze by endosperm transient expression system; The binding properties and binding sites of MYB14 and key enzyme coding gene promoters were analyzed by yeast one hybrid and electrophoretic mobility shift assay (EMSA); then, the effect of MYB14 on the expression of the starch synthesis key enzyme coding genes, the key enzyme activities, the starch content and the grain filling rate of maize kernel will analyze by over-expressed and knock out of MYB14 gene in maize elite inbred line. The carrying out of this project will reveal the role and the molecular regulation mechanism of MYB14 on the starch synthesis of maize kernel; the result of this project will supply an excellent gene resource for high grain filling rate of maize molecular breeding.

作物灌浆期籽粒淀粉合成速率决定灌浆速率和粒重。扩增整个淀粉合成通路是提高淀粉合成速率的理想途径。玉米籽粒淀粉合成限速酶AGPase是由Sh2、Bt2编码的异源四聚体，同时提高Sh2、Bt2以及影响籽粒淀粉含量的Bt1、SSIV和其它关键酶基因表达是扩增整个淀粉合成通路的有效方法。转录因子MYB14能极显著提高Sh2、Bt2、Bt1和其它淀粉合成关键酶基因的表达，在提高淀粉合成速率方面有较大潜能。本项目拟通过胚乳瞬时表达系统分析MYB14对玉米淀粉合成关键酶基因启动子活性的影响及其调控位点；通过酵母单杂交和凝胶阻滞实验（EMSA）分析MYB14与关键酶基因启动子的结合特性和结合位点；通过玉米骨干自交系中过表达和敲除MYB14分析对籽粒淀粉合成关键酶基因表达、关键酶活性、淀粉含量及灌浆速率的影响，解析MYB14在调控玉米淀粉合成中的作用和调控途径，为高效灌浆速率玉米分子育种提供优异基因资源。

淀粉是玉米籽粒的主要贮藏成分，籽粒淀粉含量和组成结构对玉米产量和品质有重要影响。转录因子ZmMYB14在灌浆期玉米胚乳中特异高表达，胚乳瞬时过表达ZmMYB14同时提高Sh2、Bt2等多个淀粉合成关键酶编码基因启动子活性，胚乳特异启动子Pzein27驱动ZmMYB14过表达玉米自交系胚乳中Sh2、SSI、SSIIIa（Du1）、SSIV 、ISA1（Su1）等基因的表达量极显著上调，而GBSSI（Waxy）的表达量则极显著下调，对Bt1、Bt2、SSIIa（Su2）、SBEIIb、ISA2等基因的表达调控效果不显著。酵母单杂交实验显示，ZmMYB14与Bt1、SsI、SsIIa、SsIV、Wx、SbeI、Isa1、Isa2等启动子能直接相互作用，而与Sh2、Bt2、SsIII启动子不能相互作用。淀粉合成相关酶活性测定显示，ZmMYB14过表达胚乳中AGPase、SSS酶活性极显著提高，而GBSS酶活性极显著降低。胚乳淀粉含量测定显示，过表达ZmMYB14使胚乳总淀粉含量极显著增加，而直链淀粉含量有所降低，抗性淀粉含量极显著降低。研究结果说明ZmMYB14在调控玉米淀粉含量特别是抗性淀粉含量方面具有利用价值。