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作 者 ：李强 刘华伟 王渭玲

期 刊 ：植物营养与肥料学报 2014年 20卷 4期 页码：930-937

关键词：田菁茎瘤固氮根瘤菌；小麦；定殖；营养元素；miRNA；

Keywords:Azorhizobium caulinodans, wheat, colonization, nutrient element, miRNAs,

摘 要 ：【目的】随着化肥过度使用引起的环境问题的出现，无公害农业的推广，以及新型肥料研究领域不断拓宽，微生物肥料，尤其是植物根际促生菌的研究成为近年来的热点。然而微生物肥料的增产机理还基本停留在作物农学性状的表观调查上，没有从分子水平进行研究。因此，本文用田菁茎瘤固氮根瘤菌（Azorhizobium caulinodans ORS571）侵染小麦，探索GFP-A.caulinodans在小麦幼苗组织中的分布与定殖规律以及营养元素相关miRNAs在小麦与A.caulinodans互作中的作用机制。【方法】使用A.caulinodans侵染小麦种子（品种为小偃22），将接菌6 d后的小麦幼苗的根和接菌12 d后的叶制作玻片，利用激光共聚焦显微镜对样品进行逐层扫描，检测GFP-A.caulinodans在幼苗不同组织中的分布与定殖情况。同时，采集接菌后0 h、 6 h、 12 h、 24 h、 48 h、 72 h、 96 h的小麦幼根取样，用Trizol法提取总RNA。利用试剂盒进行加尾和反转录反应，将样品总RNA中的miRNA合成为cDNA，使用β-tubulin作为内参基因，进行实时定量PCR反应，使用2-ΔΔCT方法计算相对表达量。利用psRNATarget 在线软件，采用默认参数，对miRNA的靶基因进行预测。【结果】 1）激光共聚焦结果显示，GFP-A.caulinodans可定殖于根的表皮细胞、 细胞间隙、 根尖破损处和根毛，在根维管组织和叶片气孔部位，也发现有GFP-A.caulinodans存在。2）实时定量PCR分析结果表明，6条与营养元素代谢有关的miRNA表达发生变化，其中miR164、 miR167和miR827相对表达量呈现出先上调后下调的趋势，miR169 和miR398相对表达量也基本呈现出这一趋势。miR164、 miR167、 miR169和miR398的相对表达量在接菌12 h时上调至最高点，分别为对照的4.13、 2.84、 2.46和3.99倍； miR827相对表达量在接菌24 h时达到最高点，为对照的2.17倍。miR399相对表达量呈现出先下调后上调的趋势，在接菌24 h时降至最低点，为对照的0~21倍。3）通过靶基因预测，6条miRNA的靶基因分别编码了NAC1转录因子、 生长素响应因子、 HAP转录因子、 Cu/Zn 超氧化物歧化酶、 蛋白缀合酶PHO2和SPX-MFS亚家族蛋白。【结论】GFP-A.caulinodans能够从根毛和根尖破损处等部位进入小麦幼苗根部定殖，并可通过向上迁移到达叶片，在气孔处定殖。接种A.caulinodans可不同程度增加小麦根中响应氮素、 磷素、 微量元素的miRNAs相对表达量,增强小麦幼苗对营养元素的吸收和利用，促进小麦根的形态建成。

Abstract:【Objectives】Microbial fertilizer, especially organic materials inoculated with plant growth-promoting bacteria, is becoming a popular type of fertilizers as the environmental concernes caused by over use of chemical ferlitizers, the extension of pollution-free agriculture production. In this paper, wheat seeds were inoculated with Azorhizobium caulinodans ORS571 (A.caulinodans), and the mechanism of nutrient-related miRNAs and colonization of green fluorescence protein (GFP)-labeled A.caulinodans in wheat seedlings were investigated.【Methods】Wheat seeds were infected with A.caulinodans, the root samples of 6 d and leaf samples of 12 d after inoculation were made into slides, scanned using laser confocal microscope. The distribution and colonization of GFP-A.caulinodans in wheat seedlings were detected. Trizol method was used to extract RNA in wheat seedling roots of 6 h, 12 h, 24 h, 48 h, 72 h and 96 h after inoculation. Through the tail adding and reverse transcription reaction, the miRNA within total RNA of samples was synthesized to cDNA. Using β-tubulin as reference gene, the expression patterns of miRNAs response to nutrient element were validated by real-time quantitative PCR. The relative expressions of miRNA were calculated with 2^-ΔΔCT method. Using default parameters, the target genes of 6 miRNAs were predicted on psRNATarget online software. 【Results】 1) The laser scanning result shows that GFP-A. caulinodans can colonize in epidermal cells, intercellular spaces of lateral roots, root tip breakages and root hairs. GFP-A.caulinodans can also be detected in root vascular tissue and leaf stoma. 2) The expression of 6 nutrient-related miRNAs varies with inoculation time. The expression of miR164, miR167 and miR827 are upregulated and then down-regulated, and those of miR169 and miR398 are almost consistent. The relative peak expression of miR164, miR167, miR169 and miR398 appear at 12 h after inoculation and the expression are 4-13, 2-84, 2-46 and 3-99 fold of control. The relative peak expression of miR827 occurs at 24 h after inoculation, 2.17 fold of the control. The expression of miR399 shows a trend of down-regulated first and then up-regulated. The relative expression of miR399 reaches the lowest point at 24 h after inoculation, equaling 0.21 fold of the control. 3) Target prediction shows that the target genes of 6 miRNAs code NAC1 transcription factor, Auxin response factor, HAP transcription factor, Cu/Zn-superoxide dismutase, Ubiquitin-conjugating E2 enzyme PHO2 and SPX-MFS Subfamily protein respectively.【Conclusions】GFP-A.caulinodans can colonize in roots of wheat seedlings via root hairs and root tip breakages and ascend into the leaves around stomas. The relative expressions of N, P and microelements related miRNAs can be up-regulated through the inoculation of A.caulinodans. Healthy root morphology is constructed for more efficient uptake of nutrients.

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