柠檬酸分泌转运蛋白基因在山核桃抗铝毒中的功能解析及辅助选育

The hickory (Carya cathayensis Sarg.) fruit is in short supply due to its high nutritional values. However, severe acid aluminum (Al) stress was induced by increasing acidification of the soil, resulting in the decrease of its yield and quality, which hinders the development of hickory industry. Several mechanisms for external detoxification of aluminum have been proposed, but the most well-studied strategy is secretion of organic acid anions, including citrate, oxalate and malate, from the roots. These organic acid anions are able to chelate Al3+ externally, preventing aluminum from binding to root cells, and thereby detoxifying Al. A wide variety of plant species, including both dicots and monocots, have been reported to utilize this mechanism. However, it is unclear whether this mechanism is suitable for hickory. The purpose of this project is to parse the aluminum resistant mechanism of organic acids in hickory and rapidly breed aluminum resistance varieties. Firstly, this project intends to excavate the main organic acid secretion transporter encoding genes which involve in aluminum resistance using the completed genome sequencing data. Secondly, utilize the genetic transformation system of hickory to analyze the gene function and its regulatory elements. Finally, clone target genes and their promoter sequences of hickories collected from different districts and identify their SNP and InDel loci, then correlate the SNP and InDel loci with Al tolerance. The natural mutant hickory well-grown on acidic soils was selected. The results of this project will dispel people's worries about genetically modified foods, accelerate the breeding of acid-aluminum resistant hickory. It also provides theoretical basis and new ideas for future developing the more aluminum resistant forest germplasm by modifying target genes.

山核桃果实具有较高的营养价值，其市场供不应求。然而，由于其适宜生长的土壤日渐酸化引起了严重的铝（Al）胁迫，造成了其产量和品质的下降，给当前山核桃产业发展带来了难题。Al诱导的根系有机酸分泌来螯合质外体Al，被认为是大多数植物最主要的抗Al毒机制。然而，此机制是否适用于山核桃还不清楚。为了解析山核桃有机酸耐Al机制及快速选育出耐酸Al品种，本项目拟利用已完成的基因组测序数据和成熟的山核桃遗传转化体系，对主要参与山核桃耐Al胁迫的有机酸分泌转运蛋白进行挖掘，并将其在山核桃中进行功能验证和调控元件分析。通过克隆不同地区的山核桃目标基因和其启动子序列，找出SNP和InDel位点并将其与对应植株的耐Al性相关联，挑选出可在酸性土壤上良好生长的自然变异株系。研究结果将加速育种和应用；也为以后定向改造靶基因以培育更加耐Al的林木种质资源提供理论依据和全新思路。

山核桃适宜生长的微酸性土壤日渐酸化，造成土壤中的铝（Al）离子释放增加，Al离子胁迫造成山核桃产量和品质下降。植物分泌的有机酸能螯合Al离子达到解Al毒的目的，然而山核桃中Al诱导分泌有机酸模式及负责有机酸分泌的转运蛋白及其调控机制还不清楚。基于此，本项目首先确定了山核桃在Al诱导下的分泌模式，发现Al诱导下山核桃主要分泌的是柠檬酸，进一步对柠檬酸分泌转运蛋白MATE家族进行了生信分析，发现CcMATE4和CcMATE26两个基因受Al诱导表达上调最显著，且与已知其它植物具有解Al毒的MATE成员进化关系较近。对CcMATE4和CcMATE26的亚细胞定位及电生理特性进行分析，结果表明它们两个均定位于细胞膜，能够介导爪蟾卵母细胞的离子流变化，这符合柠檬酸转运蛋白的质子耦合转运机制。收集了不同酸性土壤上生长的山核桃并比较了它们的耐Al差异，克隆了它们的CcMATE4和CcMATE26编码区和启动子区序列、测定了cMATE4和CcMATE26的表达丰度，将表达丰度与耐Al程度进行了关联，发现表达丰度与耐Al程度之间无显著性关联，这可能是由于MATE4和MATE26的编码序列不同造成蛋白活性不同或者其它蛋白修饰而造成的。将带有不同SNP位点的CcMATE4和CcMATE26的CDS序列回复到拟南芥stop1突变体里来验证他们的解Al毒功能，结果表明不同的CcMATE4序列能够不同程度的提高stop1突变体对Al胁迫的耐受性，表明这些序列携带的SNP会影响CcMATE4的解Al毒能力。而CcMATE26则轻微提高stop1突变体对Al胁迫的耐受性。在山核桃中发现了5个C2H2 型锌指结构蛋白STOP1同源基因，利用双荧光素酶实验及酵母单杂交实验验证STOP1同源蛋白对CcMATE4启动子的调控作用，结果表明CcSTOP1a和CcSTOP1e能与CcMATE4起始密码子上游-936至-672 bp的DNA序列直接结合并正向调控CcMATE4的表达。以上研究结果为采用分子生物学技术结合自然变异，在一定范围内快速找出能在酸性土壤良好生长的优质非转基因的山核桃品系及为以后通过定点改造有机酸分泌转运蛋白编码基因以培育更加耐Al胁迫的山核桃种质资源提供理论依据和思路。