易变山羊草色氨酸脱羧酶基因克隆及对禾谷孢囊线虫抗性研究

Cereal cyst nematode (CCN, causal agent Heterodera avanae) has been documented as one of major restrictive factors to cereal production in China and around the world. However, few CCN resistance genes were cloned and characterized in wheat and its relatives up to date, and neither of them had confirmed function in CCN resistance. Based on generation of a root transcriptome of Ae. variabilis accession No.1 during CCN infection, the phenylalanin and isoquinoline metabolisms were deduced to have central roles in CCN resistance of Ae. variabilis No.1. By investigation of gene expression levels involved in these metabolism pathway, a cross-hub enzyme, tryptophan decarboxylase (TDC), was found to be significantly up-regulated by CCN infection. In this study, we will clone the genes encoding tryptophan decarboxylase from Ae. variabilis No.1, and characterize them by means of gene structure analysis, subcellular localization, and gene expression. Furthermore, genetic transformation approach will be utilized into tobacco, Arabidopsis thaliana, and wheat to validate its function. Meanwhile, the gene silencing will be also performed by RNAi technology. According to the secondary metabolites change, we will explore the association among the secondary metabolism, the CCN resistance and morphology of root of Ae. variabilis No.1 during the CCN infection. Overall, a critical role for phenylalanine and isoquinoline metabolism in the resistance of Ae. variabilis No.1 to the CCN, even the RKN will be elucidated via our research, which will provide new insights into the molecular mechanisms related to CCN resistance.

小麦禾谷孢囊线虫(H. avenae, CCN)已成为我国麦类作物高产、稳产的重要限制因素之一。但小麦种内抗性资源匮乏，其相关抗性机理尚不清楚。本研究组通过禾谷孢囊线虫侵染易变山羊草的深度转录组测序，发现苯丙氨酸代谢及异喹啉生物碱代谢在整个抗性应答中具有重要作用，其中色氨酸脱羧酶为两代谢途径共有关键酶。因此，本项目拟通过对色氨酸脱羧酶基因（TDC）全序列克隆，揭示基因结构、亚细胞定位、基因表达特性；通过构建该基因植物根部特异表达载体对烟草及小麦遗传转化，评价其对禾谷孢囊线虫及根结线虫的抗性作用。通过对色氨酸脱羧酶（TDC）基因过表达或抑制表达（沉默），探索根部在CCN的侵染诱导下次生代谢产物的变化及与对根线虫抗性和表型性状发育的关系，揭示苯丙氨酸代谢及异喹啉生物碱代谢途径在小麦抗禾谷孢囊线虫，乃至植物根线虫方面的作用，为进一步揭示植物抗根线虫生化和分子机理及生防新途径研究奠定基础。

禾谷孢囊线虫（Cereal cyst nematode, CCN）是小麦生产上重要的病原，引起的经济损失日益增长。然而，小麦种内抗CCN的基因资源少，因此从其近缘物种中分离抗性相关基因是重要且有效的途径。易变山羊草1号是小麦近缘物种，对CCN具有高抗性。但至今从该材料中分离到的CCN抗性相关基因很少。色氨酸脱羧酶（TDC）催化色氨酸转变成色胺，该反应是很多与抗病相关次生代谢产物的共同骨架。本研究中，我们克隆了两条易变山羊草TDC基因，AeVTDC1 和AeVTDC2。功能验证发现，在CCN 幼虫侵染后的30小时，AeVTDC1和AeVTDC2的表达受其诱导显著上调，其中AeVTDC1比AeVTDC2对CCN侵染更为敏感。病毒介导的AeVTDC1沉默导致植株根部对CCN抗性降低，CCN侵染的数目显著增加；在普通烟草中过量表达AeVTDC1 会显著提高植株对根结线虫（root-knot nematode, RKN）的抗性，导致根部形成的根结数目显著减少。HPLC MS对AeVTDC1转基因烟草/非转基因烟草和AeVTDC1沉默植株/对照植株的代谢产物比较分析发现，一些具有病原抗性活性的次生代谢产物的含量受AeVTDC1 表达水平变化呈现出一致的变化。植物生长激素IAA的含量并不受AeVTDC1 表达水平影响。另外，我们已获得了两个AeVTDC1 过表达的转基因小麦纯系。CCN抗性鉴定结果发现，与对照相比，AeVTDC1 高表达的株系对CCN的抗性显著增强，而AeVTDC1 表达较低的株系对CCN的抗性与对照并无显著差异。以上结果表明，我们克隆的AeVTDC1 是一个有价值、可应用且不影响IAA合成和植株生长的基因资源，可用于CCN和RKN抗性育种。.本项目还创制了对植物根线虫有抗性的转色氨酸脱羧酶基因烟草及小麦材料各2个株系；发表了SCI研究论文3篇，申请专利3件，培养研究生3名。