非洲栽培稻抗稻瘟病新基因Pi67(t)的克隆

Oryza glaberrima, domesticated independently from wild specie O. barttii, is one of two cultivated species in Oryza genus. Because of its excellent traits for increased tolerance to biotic and abiotic stresses, O. glaberrima is considered to be one of the important reservoirs of useful genes transferring to Asican cultivated rice (O. sativa). In previous studies, we identified a novel gene Pi67(t) conferring broad spectrum resistance to rice blast from introgression line IL-106 of O. glaberrima. This gene was finely mapped to a region of about 76.1 kb.on the long arm of chromosome 6 of rice, flanked by 2 molecular markers STS67-15 and STS67-7. Although this gene was finely mapped, the structure, function and molecular mechanism for broad spectrum resistance to rice blast of Pi67(t) remain unknown. In order to clarify its structure and function, the strategies will be employed to clone Pi67(t) gene as follows, to amplify and assemble the target genomic region carrying Pi67(t) in introgression line IL-Og106 by PCR method based on the reference genomic sequences of O. sativa and O. glaberrima, and then annotate the candidate resistance genes in the target region through bioinformatic platform. After comparative analysis of allelic sequences amplified from resistant donor IL-Og106 and susceptible cultivars, the candidate resistant genes could be selected for transformation through Agrobacterium-mediated transformation method to further validate its function for resistance to Magnaporthe oryzae. The results will lay important foundation for further research on molecular mechanism for broad spectrum resistance to M. oryzae, evolution of Pi67(t), as well as interaction between resistance gene from rice and avirulence gene from M. oryzae. It will also provide important gene resources for disease-resistant rice breeding .

独立起源及驯化的非洲栽培稻是稻属的2个栽培种之一，具有许多抗生物胁迫和非生物胁迫的优异特性，是向亚洲栽培稻转移有利基因的优良基因库之一。项目组前期从非洲栽培稻渗入系IL-Og106中鉴定到1个广谱抗稻瘟病的新基因Pi67(t)，并将其精细定位到水稻第6染色体长臂上76.1kb的物理距离范围内。然而，对该基因的结构、功能和广谱抗性分子机制尚不清楚。本项目以Pi67(t)为研究对象，在基因精细定位的基础上，以已测序的亚洲栽培稻和非洲栽培稻的基因组序列为参考，采用PCR技术扩增IL-Og106的靶标区域、候选基因注释、抗病与感病品种间等位基因序列比较的方法确定候选抗病基因；通过农杆菌介导的遗传转化法开展候选抗病基因功能验证，完成Pi67(t)基因的克隆，阐明其结构与功能。研究结果将为开展广谱抗性机制、基因的演化以及抗病基因与病原菌无毒基因互作机制的研究奠定基础，并为抗病育种提供重要的基因源。

本项目利用非洲栽培稻渗入系IL106与亚洲栽培稻粳稻品种滇粳优1号杂交获得的19285个F2代个体组成的定位群体，在开展个体基因型及表型分析的基础上，从非洲栽培稻中鉴定到1个抗稻瘟病新基因Pi69(t)（原Pi67(t)），将其定位到水稻第6染色体长臂上76.1 kb的物理距离范围内，完成了该基因的精细定位；通过长距离PCR扩增技术及基于PCR扩增的染色体步移技术，完成渗入系IL106靶标区域的PCR扩增和分析；靶标区域的候选基因注释分析发现11个预测的编码基因，日本晴中的部分基因在IL106中缺失；通过RT-PCR分析发现IL106中仅2个候选基因P7和P10表达，这两个基因被确定为Pi69(t)的候选基因；在构建候选基因超表达遗传转化载体、基因编辑遗传转化载体和全长基因遗传转化载体的基础上，采用农杆菌介导的遗传转化法获得T0及T1代的转基因植株，通过表型鉴定及分子鉴定验证实验，证明了候选基因P7就是本项目要克隆的Pi69(t)基因，最终完成了Pi69(t)基因的克隆。Pi69(t)基因编码1个由1479个氨基酸组成的蛋白，该抗性基因编码具有核苷酸结合位点-亮氨酸重复结构域（nucleotide binding site and leucine-rich repeats, NBS-LRR）的蛋白；Pi69(t)基因的成功克隆，为进一步利用分子标记辅助选择和转基因技术开展抗稻瘟病育种提供了重要的基因源，同时也为进一步研究水稻抗病基因与稻瘟病菌无毒基因的互作，阐明Pi69(t)基因的抗病机理，寻找新的稻瘟病控制途径提供宝贵的研究素材。