类受体蛋白激酶基因LENG参与水稻耐冷的分子机制研究

Damage caused by low temperature is a major factor affecting rice production. During our previous research, we have identified a cold-sensitive mutant （named leng）from an EMS mutant library derived from the indica variety Huanghuazhan (HHZ). The mutation site was mapped to a gene encoding a receptor-like kinase. The CRISPR knockout mutant of LENG gene in japonica variety Wuyungeng (WYG) was more sensitive to cold-damage than the wild-type plant, while over-expression of LENG gene in WYG increased the cold-tolerance of the plant. The LENG gene in HHZ has a 3.2 kb DNA insertion in the promoter region that is absent in WYG. Cold treatment rapidly induced the LENG gene in WYG but not in HHZ. This proposal will determine whether the difference in the promoter region confers differential functions of the two genotypes in cold-tolerance. We will also confirm the function of LENG gene in cold-tolerance by investigating the phenotypic, physiological, and biochemical changes of leng mutant plant, LENG over-expressing plant, and the wild-type plant in response to cold treatment. Further, we will conduct the transcriptomics and phosphoproteomics comparisons among the leng mutant plants, LENG over-expressing plants, and the wild-type plants. Protein interacting with the LENG kinase will be isolated by screening the yeast two-hybrid library and/or co-immuno-precipitation. Through these experiments, we expect to illustrate the signal transduction pathway operated by the LENG gene for cold-tolerance. This project will not only reveal the molecular mechanism underlying LENG-mediated cold-tolerance, but also provide the theoretical guidance and source of genes for molecular breeding of cold-tolerant rice varieties.

低温冷害是影响水稻安全生产的主要气象灾害之一。项目前期在黄华占EMS突变体库中获得一个苗期冷敏感突变体leng，经基因克隆发现候选基因LENG编码一个类受体蛋白激酶。LENG基因敲除植株对低温敏感，而超表达植株具有更强的耐受性。在籼稻黄华占和粳稻武运粳中，LENG基因对冷害的应答模式完全不同，前者不受低温诱导，后者受低温快速诱导；序列比对发现两者启动子序列相差3.2kb。本项目将通过分析野生型、突变体和超表达转化体在低温处理后的表型差异及生理生化变化，明确该基因在低温耐受中的功能；通过比较野生型、突变体和超表达植株转录组与磷酸化蛋白质组的差异、鉴定分析互作蛋白、以及比较籼粳稻LENG基因序列差异对水稻低温耐受性的影响，从而初步阐明LENG基因参与水稻耐冷调控的作用机理。本项目不仅有助于阐明LENG基因介导低温耐受性的分子机制，同时也为耐低温水稻新品种的分子设计育种提供基因资源和理论基础。

低温冷害是影响水稻安全生产的主要气象灾害之一。本研究发现一个细胞膜定位的类受体假激酶蛋白LENG受低温诱导表达，其基因敲除植株对冷胁迫敏感，而超表达植株具有更强的耐受性。LENG基因可能通过ICE-CBF-COR途径参与水稻苗期耐冷性的调控，目前已鉴定到LENG蛋白的互作蛋白LIP1，LENG可能通过LIP1蛋白对冷信号进行转导。在籼稻黄华占和粳稻武运粳中，LENG基因对低温处理的应答模式不同，前者不受低温诱导，后者受低温快速诱导；序列比对发现籼稻黄华占的LENG基因在启动子区存在3.2 kb的大片段插入，并且在被检的108份水稻核心种质材料中，这种大片段插入只存在于部分籼稻中。不含大片段插入的武运粳启动子无论驱动武运粳基因区还是黄华占基因区，互补LENG基因敲除材料所得的幼苗在冷处理后植株存活率都显著高于含大片段插入的黄华占启动子驱动的互补材料，表明这个3.2kb的大片段插入确实影响LENG基因对水稻苗期耐冷性的调控。