利用CRISPR/Cas技术研究拟南芥LEA蛋白家族在环境胁迫中的功能

胚胎发育晚期丰富(LEA)蛋白最早在胚胎发育晚期的棉花种子中被发现, 该家族具有亲水性高、序列复杂性低和缺乏明显高级结构等特点，在种子自然失水过程中发挥保护作用。LEA基因广泛分布于植物界， 目前大量的LEA基因已在植物或部分动物中被分离鉴定， 该家族基因受非生物胁迫诱导表达。拟南芥中共有51个LEA基因，被分为LEA_1、LEA_2、LEA_3、LEA_4、LEA_5、dehydrin、SMP、AtM和PvLEA18，共9个亚家族。我们利用CRISPR/Cas9基因编辑技术对拟南芥中的LEA基因上设计了sgRNA并进行了靶向敲除。根据亚家族分类、非生物胁迫诱导表达基因的特异性和亚细胞定位的特异性，我们构建了不同组合的转化载体，通过转基因浸染，获得了不同组合的突变体材料库，通过进一步表型筛选和突变基因型鉴定，最终解析LEA蛋白家族在非生物胁迫上的功能和分子机制。

The late embryogenesis abundant (LEA) proteins were first found in cotton seeds in the late period of embryonic development. They play protective roles during the natural dehydration process of the seeds with the characteristics of high hydrophilicity, low sequence complexity and lack of obvious high-level structure. LEA genes are widely distributed in the plant kingdom. Many LEA genes have been isolated and identified in plants and some animals. The expression of LEA genes can be induced by different abiotic stress. There are 51 LEA genes in Arabidopsis, which can be divided into nine subfamilies: LEA_1, LEA_2, LEA_3, LEA_4, LEA_5, dehydrin and SMP. We designed sgRNAs for targeted knockout of all LEA genes in Arabidopsis by CRISPR/Cas9 gene editing system. According to the subfamily classification, the specificity of abiotic stress-induced expression genes, and the specificity of subcellular localization, we constructed different combinations of transformation vectors. The different CRISPR binary vectors were used to transform Arabidopsis Col-0 plants using the Agrobacterium-mediated floral dip method and T1 transgenic lines were obtained for each construct. Phenotypic screening of the transgenic lines on abiotic stress, and then we identified the mutation type of by Hi-TOM sequencing. we aim to reveal the function and molecular mechanism of LEA protein family on abiotic stress.