玉米单向杂交不亲和基因Ga1-S的分子克隆与功能验证

Cross incompatibility genes known as gametophyte factors (ga) are numerous in maize. Many popcorn strains carry these genes and cannot be fertilized by pollen of dent and flint maize strains although the reciprocal crosses are successful. The Chinese popcorn strain SDGa25 carries the strongest allele of Ga1 (Ga1-S) and the majority of Chinese dent and flint maize germplasm are incompatible to SDGa25. The incompatibilty is due to pollen tube growth obstruction 2h after pollination. Pollen tube is arrested in the silk segment 5.5cm distal to pollination area and never reach ovule. The Ga1-S carried by SDGa25 behaves as a single dominant gene. We have mapped Ga1-S on chromosome 4 between markers SD3 on BAC AC200747 0.827cM apart on the telomere side and SD12 on BAC AC204382 0.709cM apart on the centromere side. The genetic region mapped spanning the Ga1-S locus was estimated 1.5cM in length and the physical distance is 2,056,343bp on ctg156 based on B73 RefGen_v2 sequence. Gametophyte factors influence gene flow direction and the strongest Ga1-S allele is useful for isolating one category of commercial varieties from another. Ga1-S also is a great candiadte for pollen-silk interaction and signal transduction of maize.The objectives of this project are to 1) further fine mapping Ga1-S, 2)cloning of Ga1-S, 3)functional analysis of its function through maize transgenic approach, 4)its expression profile, and 5)its subcellular localization.

玉米配子体基因(Ga)是指影响单倍体配子有性传递的遗传因子，来自于爆裂玉米的配子体基因Ga1-S，对绝大多数普通玉米(ga1)具有100%的单向杂交不亲和性，即ga1/ga1基因型玉米给Ga1-S/Ga1-S基因型玉米授粉不能结实。Ga1-S可作为不同类型玉米间生物学隔离的工具，也是研究玉米传粉授精信号识别传导的珍贵材料。在国家自然基金(30971788, 2010-2012)的资助下，我们将Ga1-S定位在BAC AC200747和AC204382之间，距Ga1-S分别为0.827cM和0.709cM，相距2,056,343bp。同时，我们还利用SDGa25(Ga1-S的供体)构建了具有30X基因组覆盖率的BAC文库。本研究将对Ga1-S进一步精细定位，继而克隆该基因，并进行转基因功能验证，同时开展该基因的时空表达和亚细胞定位等研究。

玉米单向杂交不亲合是由玉米配子体基因(Gametophyte factor, Ga)控制，主要存在三个等位基因，ga1、Ga1-M和 Ga1-S。ga1花粉不能给Ga1-S/Ga1-S母本授粉结实，反之，Ga1-S花粉却能给ga1/ga1母本授粉结实；和Ga1-S相比，Ga1-M失去了对ga1花粉不亲和的功能，但仍具有给Ga1-S/Ga1-S母本授粉的功能，这就是Ga1基因位点所控制的单向杂交不亲和性。至于Ga1基因单向杂交不亲和的机理，目前仍是个谜。.我们通过多年的杂交及遗传分析证明，Ga1位点是由遗传上紧密连锁的显性配子体性质基因male factor（PP）和隐性孢子体性质基因female factor（ss）共同控制。通过对国内外的1299个玉米自交系进行不亲和性测定，筛选出21份Ga1-M基因型材料，18份Ga-S基因型材料。通过构建精细定位群体，将male和female基因分别定位于玉米Chr.4s物理距离为1.7 Mb和2.2 Mb的区间内。结合转录组和BAC测序分析，得到位于花粉和花丝中特异表达的male和female候选基因。虽然female基因理论上只在Ga1-S基因型材料的花丝中表达，但依然有2份Ga1-M材料的female基因在花丝中表达量较高。测序表明，上述材料的female基因的编码区序列存在碱基插入，造成编码氨基酸序列的移码突变。最后，构建了玉米ubiquitin启动子驱动的male和female候选基因的过载体转化ga1基因型材料Hi II进行功能验证。授粉实验表明，转male候选基因的阳性植株够能突破Ga1-S基因型材料花丝的阻碍，使之结实；而female作为孢子体性质的基因，只有在纯合基因型条件下才能阻碍ga1花粉，但考虑到过表达载体能够显著提高基因的表达量，我们对转female基因的阳性植株分别授以ga1和Ga1-M花粉，Ga1-M花粉能够使之结实，但授ga1花粉的材料结实状态差。据此，我们初步证明female基因的真实性，进一步的研究正在进行。Ga1基因的研究对深入了解杂交不亲和机理和育种实践具有重要意义。受课题资助，在《Crop science》、《Plant Physiology》、《The Plant Journal》上共发表论文三篇，相关发明专利一项，培养硕士研究生一名，博士研究生两名，预算结余0.877805万元。