板栗菌根磷转运蛋白促磷吸收分子机制研究

Mycorrhizal fungi could establish mycorrhiza symbiosis with plant roots, and then increase plant phosphate uptake from soil. During mycorrhiza symbiosis, mycorrhizal phosphate transporter genes located on plant peri-membrane were specifically induced to transport inorganic phosphate into plant cytoplasm. The foundational data from our research group show that chestnut roots could form not only ecto-mycorrhiza (EM), but also vesiculer-arbuscular mycorrhiza (VA). These two types of mycorrhiza significantly increase phosphate uptake capacity of chestnut, respectively. Up to date, the molecular mechanism of mycorrhiza involved in pomology including chestnut hasn't been reported. .As known from literature reports, mycorrhizal phosphate transporter genes evolved into one subgroup based on phylogenic tree. Herewith the candidate mycorrhizal phosphate transporter genes of chestnut will be explored b, and then their full length of cDNA will be cloned to trace gene evolution via phylogeny. Meanwhile, the expression patterns of these candidate mycorrhizal phosphate transporter genes will be performed under different inoculation stages of EM fungi and VA fungi,respectively. Up-regulated mycorrhizal transporter gene in mycorrhizal roots will be anchored as an valid marker during chestnut mycorrhizal symbiosis. Therefore the expression characteristics of mycorrhizal phosphate transporter genes will be further revealed at different phosphate level. Finally combined with phosphate concentration in different organs, and morphologic and cellular observation of chestnut mycorrhiza, the mechanism increasing phosphate uptake capacity via mycorrhiza will be disclosed..Firstly, the conduction of this project will bring out more molecular proofs aimed to improving phosphate absorbance by mycorrhiza. Secondly, it will lay the foundation of mycorrhiza symbiosis between pomology and mycorrhizal fungi. Finally, it will provide scientific grounds for the production of green and organic fruits decreasing phosphate fertilizer in degree.

菌根真菌促进植物磷的吸收，与植物-菌根真菌共生过程植物菌根磷转运蛋白基因的特异性诱导表达密切相关。本课题组前期研究基础表明，板栗既可以形成外生（EM）菌根，也可以形成VA内生菌根，菌根真菌对板栗促磷吸收作用显著，但文献查新表明板栗等果树菌根共生分子生物学研究尚未报道。本项目通过发掘和克隆板栗菌根磷转运蛋白基因，追踪基因演化的历程；研究EM真菌和VA真菌侵染下菌根磷转运蛋白基因的表达特征，发掘稳定增强表达的菌根磷转运蛋白基因作为板栗-菌根真菌共生过程促进磷吸收的有效标记。同时研究不同磷供给的条件下菌根磷转运蛋白基因的表达谱，结合不同部位磷浓度的测定，以及菌根结构的形态学和细胞学观察，揭示板栗菌根促磷吸收的发生机理。本项目的实施将为板栗菌根促磷吸收提供分子证据，为进一步深入研究板栗等果树与菌根真菌的共生互作奠定基础，也为板栗等果树生产中不施或少施磷肥生产绿色、有机果品提供科学依据。

板栗是重要的生态经济林树种，可以与多种外生菌根真菌形成菌根。菌根可以扩大根系吸收面积，促进植物对营养元素的吸收，从而促进生长发育。本试验挖掘了北京地区优势菌根真菌并进行了分子鉴定，通过对病原菌的拮抗作用，挖掘板栗生防菌根真菌；开展了不同磷水平及外生和内生菌根真菌处理下，研究了板栗根系矿质元素和重金属元素的含量变化；探究了外生菌根形成后板栗根系H+流速以及板栗磷转运蛋白基因克隆表达、菌根磷转运蛋白基因的挖掘工作。同时，对板栗根毛转化体系的建立进行了探索，以期为板栗基因功能验证提供平台，为深入研究板栗菌根促磷吸收分析机理提供理论依据。主要研究结果如下：.1..研究了菌根真菌处理后板栗根系营养元素和重金属元素的水平变化。发现菌根真菌处理可以有效提高板栗根部对磷、钾、氮等植物必需矿质元素的吸收能力，且能够吸附土壤中铅等有害重金属，并阻碍其向地上部的运输。.2..分离和鉴定了北京地区板栗优势菌根真菌。采集田间板栗菌根，分离鉴定菌根真菌，鉴定得到螺旋木霉T1、钩状木霉T2、生赤壳属B1等15个属的21个菌种。 .3..研究了菌根真菌与病原菌的拮抗作用。与病原菌进行对峙培养，挖掘板栗生防菌根真菌。其中广谱性拮抗菌种有：螺旋木霉T1、生赤壳霉B1、钩状木霉T2等；专一性拮抗菌种有：白腐菌P1、拟茎点霉P2等。螺旋木霉T1、钩状木霉T2拮抗效果好，可作为候选菌种进行生防菌剂生产。生赤壳霉B1对板栗根腐病具有专一性抑菌效果，可做为板栗根腐病生防菌剂。.4..建立了板栗根毛转化体系。利用苗龄为3 d的板栗实生苗下胚轴为外植体，与发根农杆菌共培养5 d，头孢抗生素浓度为500 mg·L-1，转化效率可达40 %以上。.5..接种菌根真菌-马勃菌后板栗根系磷含量极显著提高，并且接种马勃真菌的板栗根系H+外排速率明显增加。.6..挖掘了一个菌根磷转运蛋白基因CmPT4。通过聚类分析及实时荧光定量PCR表明，5个磷转运蛋白基因在缺磷处理下均上调表达，其中CmPT4基因在板栗菌根中显著上调表达，结合聚类分析结果，证明CmPT4基因是板栗菌根磷转运蛋白基因。.7..克隆获得了板栗菌根磷转运蛋白基因CmPT4全长。CmPT4基因阅读框序列共1525bp，启动子序列722bp。启动子分析发现含有与植物菌根磷转运蛋白基因相关的顺式作用元件P1BS。