烟草NtSKOR1基因的克隆及功能分析

该研究利用拟南芥AtSKOR蛋白序列，通过NCBI的Blast搜索获得烟草NtSKOR1基因CDS序列。设计全长CDS扩增引物，通过RT PCR方法从栽培烟草中克隆得到NtSKOR1基因，对其进行生物信息学、表达特性等分析，并利用CRISPR/Cas9技术获得NtSKOR1的敲除材料。结果表明：(1)NtSKOR1基因CDS由2 466 bp核苷酸组成，编码821个氨基酸，推测NtSKOR1蛋白等电点为6.36，分子量为94.21 kD。(2)NtSKOR1定位于细胞膜，有6个跨膜区，无信号肽序列；NtSKOR1蛋白含有孔形成区(P)及锚定蛋白区(ANK)等典型SKOR类蛋白功能域。(3)进化树分析表明，烟草NtSKOR1蛋白与番茄和马铃薯的SKOR蛋白遗传距离最近，与禾本科植物的SKOR蛋白遗传距离较远。(4)组织特异性表达分析表明，NtSKOR1基因主要在烟草根中表达，其表达模式与拟南芥一致；钾胁迫处理后，NtSKOR1基因呈先降低后升高再降低的表达模式。(5)CRISPR/Cas9敲除NtSKOR1基因，烟草叶片钾含量显著降低，表明NtSKOR1基因是控制烟叶钾离子的关键基因之一。该研究结果为解析烟草钾离子吸收转运的分子机制提供重要依据。

Cloning and Function Analysis of NtSKOR1 in Tobacco

In this study, the tobacco NtSKOR1 gene that is homologous to AtSKOR gene in Arabidopsis was identified from tobacco via NCBI Blast and RT PCR. The bioinformatics and expression characteristics of NtSKOR1 were analyzed, and the knock out materials of NtSKOR1 was obtained by CRISPR/Cas9 technology. The results showed that: (1) full length CDS of NtSKOR1 was found to be 2 466 bp, encoding a sequence of 821 amino acid residues, and successfully cloned with gene specific primer pair by RT PCR. The isoelectric point of this protein is 6.36 and the molecular weight is 94.21 kD. (2) The subcellular localization result of NtSKOR1 showed that this gene was localized in cell membrane. Amino acid sequence analysis showed that this protein contains 6 trans membrane structures, but with no signal peptide sequence. As a typical SKOR family protein, NtSKOR1 is featured by pore forming domain and ankyrin structural domains. (3) Phylogenetic analysis showed that NtSKOR1 protein is close to SKOR proteins identified from tomato and potato, while is distant from the SKOR proteins of Gramineae species. (4) Tissue specific expression analysis showed that NtSKOR1 was preferentially expressed in roots, which was in line with AtSKOR1 expression pattern in Arabidopsis. After potassium stress treatment, the expression pattern of NtSKOR1 showed a fluctuant trend. (5) CRISPR/Cas9 based genome editing of NtSKOR1 significantly decreased the potassium content in the leave of transgenic lines, suggesting NtSKOR1 gene is one of the key genes in controlling potassium content in tobacco leaves. Our results provided significant evidence of the molecular mechanism of potassium intake and transport in tobacco.