FaesPI基因参与调控甜荞lpls突变体雌雄蕊等长变异的分子机制

Common buckwheat (Fagopyrum esculentum, Polygonacea,), is a pseudo-cereal with healing benefits and dimorpic flower. Buckwheat possesses a dimorpic flowers, with populations being equally composed of plants with pin flowers (long pistil and short stamens) and plants with thrum flowers (short pistil and long stamens).Moreover, Buckwheat is strictly self-incompatible called heteromorphic self-incompatibility with obligate cross-pollination between pin and thrum flowers, which make it hard for hybrid breeding and result in low yield. We have developed lpls mutant line with long pistils and long stamens and cross-pollination freely. Our study revealed that FaesPI is involved only in stamen development in buckwheat, and encodes a MADS-box transcription factors. In addition, lpls mutant FaesPI promoter shows a 550 bp deletion of the －993/－384 region. In combination of various methods and techniques, such as Comparative Transcriptomics, molecular biology and morphology, key candidate genes involving stamen and pistil differentiation among pin, thrum and lpls mutant have been mined. Moreover, these upstream genes regulating the tancription of the FaesPI and they working together to specify the mutant of the stamen and pistil in lpls are discussed. The results should provide suitable candidate genes and excellent mutant line of self-compatibility for genetic improvement and cross breeding in buckwheat.

甜荞（Fagopyrum esculentum）是蓼科（Polygonaceae）食药兼用的杂粮作物，pin型（长雌短雄）和thrum型（短雌长雄）植株1:1分离。群体中，仅异型花间等长的雌雄蕊能正常授粉结实，同型花自交不亲和，成为其杂交育种瓶颈。申请人在前期育种工作中，筛选获得遗传性状稳定、同型花间能授粉结实的长雌蕊长雄蕊突变体lpls，进一步研究发现，突变体中雄蕊发育调控的MADS-box基因FaesPI启动子上游－993/－384区共550 bp的序列完全缺失。本项目拟从FaesPI基因启动子区的结构和功能变异入手，结合形态学、分子生物学和比较转录组学技术和方法，比较研究FaesPI基因参与调控甜荞pin型、thrum型和lpls突变体雌雄蕊形态差异的分子基础，以此解析甜荞lpls突变体雌雄蕊等长变异与自交亲和性形成的分子机制，为创制甜荞自交亲和性的新种质和杂交育种提供新的材料。

甜荞（Fagopyrum esculentum）是蓼科（Polygonaceae）食药兼用的杂粮作物，pin型（长花柱短雄蕊）和 thrum型（短花柱长雄蕊）植株1：1分离。群体中仅异型花间等高的柱头和花药正常授粉结实，同型花自交不亲和，成为限制其产量和杂交育种的瓶颈。长花柱长雄蕊lpls甜荞是课题组前其筛选获得的遗传性状稳定、自花、同型、异型花间均能正常授粉结实的新种质。结合基于二代和三代测序技术的甜荞花型差调控基因筛选、RACE技术、染色体步移等，分别从甜荞中分离出FaesPI_1、FaesPI_2、FaesAP3_1和FaesAP3_2基因及其启动子。蛋白序列同源比对和分子系统发育分析显示：FaesPI_1 和FaesPI_2属B类MADS-box家族中的PI/GLO进化系转录因子; FaesAP3_1和FaesAP3_2属B类MADS-box家族中的euAP3进化系转录因子。表达模式分析表明：4个B类MADS-box基因主要在甜荞雄蕊中表达；pFaesPI_1::GUS和pFaesAP3_2::GUS 转基因拟南芥雄蕊中GUS表达量高；而pFaesPI_2::GUS和pFaesAP3_1::GUS 转基因拟南芥雄蕊中GUS仅在花丝表达。功能分析表明：FaesPI_1 和 FaesPI_2仅能部分替代拟南芥内源PI基因的功能，将拟南芥pi-1突变体的雄蕊部分恢复，但不参与调控花瓣的发育；同样FaesAP3_1和FaesAP3_2也仅能部分替代拟南芥内源AP3基因的功能，将拟南芥ap3-3突变体的雄蕊部分恢复，而且TRV2-FaesAP3_1处理后甜荞lpls植株部分雄蕊转变为花被片；TRV2-FaesAP3_2处理后甜荞lpls植株仅部分雄蕊的花药瓣化；但均未对甜荞的花被片发育造成影响。上述结果表明：在甜荞花发育过程中，FaesPI_1和FaesAP3_2主要参与调控甜荞花药的发育；而 FaesPI_2和FaesAP3_1主要参与甜荞花丝的发育；4者协同参与调控正常雄蕊的发育，与模式植物中B类基因MADS-box基因参与调控花瓣和雄蕊发育的功能明显不同。该研究结果为理解lpls甜荞雌雄蕊等长变异调控的分子机制和荞麦属植物花型演化积累了理论基础，同时为优质高产同型长花柱甜荞新品种培育提供了新材料。受项目资助目前公开发表学术论文9篇，培养毕业硕士5人。