【摘要】 为研究半胱氨酸转移酶(CYS)基因对烟草生长发育及花青素合成的影响，在烟草中建立CRISPR/Cas9基因编辑体系，构建双靶点CRISPR/Cas9基因编辑载体p35S-H-CYS，利用农杆菌介导的遗传转化体系获得CYS烟草突变体，通过碱基测序和RT-qPCR检验CYS基因敲除效果，分析靶位点序列突变类型。结果表明，3个突变体株系在靶位点发生了基因突变，突变类型包括碱基插入、碱基替换，其中，靶位点1可以更有效地与靶位点结合，靶位点2处产生2个碱基替换，均为氨基酸同义突变。RT-qPCR结果表明，突变体株系中CYS基因的相对表达量均显著低于野生型烟草(P<0.01)。与野生型对比，CYS-1、CYS-2和CYS-3突变体株系中CYS基因转录水平倍数分别为0.31、0.45和0.34。敲除CYS基因后对烟草株高、叶形等表型未见明显影响，而突变体烟草中花青素含量均有不同程度的提高，其中，CYS-1突变体株系中花青素含量为野生型的1.3倍，显著高于野生型烟草(P<0.05)，说明CYS基因会抑制花青素的合成，该结果与先前转录组结果相符。本研究通过CRISPR/Cas9基因编辑技术对CYS基因设计2个靶位点进行定点编辑，以烟草为转化受体，为进一步研究CYS基因功能提供了重要的材料基础。更多还原

【Abstract】 In order to study the effects of cysteine transferase(CYS) gene on the growth, development and anthocyanin synthesis in tobacco, we established CRISPR/Cas9 gene editing system in tobacco and constructed a double target CRISPR/Cas9 gene editing vector P35S-H-CYS. CYS tobacco mutant lines were obtained by Agrobacteriummediated genetic system, the CYS gene knockout effect was tested by base sequencing and RT-qPCR, the mutation types of target genes were analyzed. Sequencing results showed that 3 mutant lines had gene mutations at target sites, including base insertion and base replacement. Target site 1 could bind to target site more effectively, and target site 2 produced two bases mutations, both synonymous mutations of amino acids. RT-qPCR results showed that the relative expression level of CYS gene in mutants were significantly lower than the wild type tobacco(P<0.01). Compared to wild type, the multiple of gene transcription levels were 0.31, 0.45 and 0.34. The knockout of CYS gene had no significant effect on tobacco plant height and leaf shape, but the anthocyanin content in the mutant tobacco were increased in different degrees. The content of anthocyanin in CYS-1 mutant was 1.3-fold,which was significantly higher than that of wild type(P<0.05). These results indicated that CYS gene can inhibit anthocyanin synthesis, which was consistent with the previous transcriptome analysis results. In this study, CRISPR/Cas9 gene editing technology was used to design two target sites for targeted editing of CYS gene. Using tobacco as the transformation receptor provided an important material basis for further study of the function of CYS.更多还原