番茄匍柄霉SlCmr1基因克隆及其敲除载体的构建

【目的】克隆番茄匍柄霉（Stemphylium lycopersici）SlCmr1基因，并构建SlCmr1基因敲除载体，为研究该基因功能打下基础。【方法】根据NCBI数据库提供的番茄匍柄霉基因组信息设计引物，PCR扩增得到SlCmr1基因的DNA全长和CDS序列，并对SlCmr1基因编码的蛋白进行生物信息学分析。利用SlCmr1基因上、下游1100 bp左右的序列设计引物，分别PCR扩增SlCmr1基因的上、下游片段，通过酶切连接的方法将SlCmr1基因的上、下游序列分别插入载体pCX62，构建SlCmr1基因敲除载体。【结果】SlCmr1基因的DNA全长为3275 bp，CDS长度为3018 bp，有3个内含子，编码蛋白序列全长1005 aa，分子式为C₄₈₈₂H₇₆₄₂N₁₄₀₄O₁₄₉₉S₆₁，分子量为111.94 kD，理论等电点为（pI）6.64，半衰期30 h，不稳定指数58.72，亲/疏水性平均值为-0.424，预测亚细胞定位于细胞核，存在2个相邻的ZnF_C2H2结构域和1个GAL4结构域，推测SlCMR1为不含跨膜区域的非分泌、不稳定可溶性蛋白。分别将SlCmr1基因上、下游序列插入pCX62载体，成功构建了基因敲除载体pCX62-SlCmr1。【结论】SlCmr1可能是真菌调控色素合成的关键基因，在真菌的次级代谢产物合成中发挥重要作用。

Cloning and knockout vector construction of SlCmr1 gene in Stemphylium lycopersici

【Objective】In order to study the function of SlCmr1 gene in Stemphylium lycopersici, cloning of SlCmr1 gene was carried out, and gene knockout vector was constructed.【Method】The primers of SlCmr1 gene were designed according to the S. lycopersici genome sequence provided by NCBI database. The full length DNA and CDS sequence of SlCmr1 gene were obtained by PCR amplification and the protein coded by SlCmr1 gene was analyzed through bioin formatics methods. Sequences around 1100 bp upstream and downstream of SlCmr1 gene were used to design primers. The upstream and downstream sequences of SlCmr1 were amplified by PCR and inserted into pCX62 vector by restriction enzyme digestion and SlCmr1 gene knockout vector was constructed.【Result】SlCmr1 gene was 3275 bp in DNA sequence and 3018 bp in CDS sequence, including 3 introns, encoded 1005 amino acid protein sequence with molecular formula C₄₈₈₂H₇₆₄₂N₁₄₀₄O₁₄₉₉S₆₁ and molecular weight 111.94 kD, predicted isoelectric point(pI) 6.64, halflife 30 h, instability index 58.72, mean value of hydrophilicity/hydrophobicity-0.424 and subcellular localization in nucleus. It was speculated that SlCMR1 was a non-secretory, unstable soluble protein without transmembrane region, and there were 2 adjacent ZnF_C2H2 domains and 1 GAL4 domain. SlCmr1 gene knockout vector pCX62-SlCmr1 was successfully constructed by inserting the upstream and downstream fragments of SlCmr1 gene into pCX62 vector.【Conclusion】Slcmr1 may be a key gene regulating pigment synthesis and play an important role in the secondary metabolite synthesis in fungi.