基于CRISPR/Cas9技术创制高维生素C番茄材料  

Enriching Vitamin C in Tomato Fruits by CRISPR/Cas9-Mediated SlCSN5B Gene Editing

作　　者：安煊森 王雁伟 田文超 任亚娟 艾鹏飞[1] AN Xuan-Sen;WANG Yan-Wei;TIAN Wen-Chao;REN Ya-Juan;AI Peng-Fei(Collage of Food And Biological Science,University of Science and technology,Shijiazhuang 050018,China)

机构地区：[1]河北科技大学食品与生物学院,石家庄050018

出　　处：《中国生物化学与分子生物学报》2025年第3期460-469,共10页Chinese Journal of Biochemistry and Molecular Biology

基　　金：河北省重点研发计划项目(No.22326312D,No.21326309D-3)资助。

摘　　要：组成型光形态建成9信号复合体的5B亚基(subunit 5B of constitutively photomorphogenic 9 signalosome,CSN5B)是植物中L-半乳糖途径合成维生素C的抑制因子。为了获得高维生素C番茄突变体,本文构建双靶点载体pKSE402-SlCSN5B并对番茄育种亲本“1912”进行了遗传转化。通过分子生物学鉴定和DNA序列分析,17株转基因阳性植株中6株发生编辑,编辑效率为35.3%;其中csn5b-6和csn5b-8为纯合突变体,分别缺失了180 bp和3 bp。对这2种缺失纯合体T 1代中无外源T-DNA插入的株系分别进行生物学观测,在植株和果实的表型上无明显差异;在果实红熟期,对其进行生理指标测定,csn5b-6的T 1代株系在GMPase活力、维生素C和过氧化氢含量变化显著,分别比野生型提高43%、37.8%和降低25.9%,而可溶性固形物含量无明显差异;csn5b-8的T 1代株系与野生型一致。采用基因表达分析和蛋白质结构预测发现,csn5b-6-11中SlCSN5B基因表达量正常,但其编码的蛋白质由于较大的肽段丢失引起结构的改变,从而影响其功能,这可能是引起维生素C含量提高的原因。以上结果表明,利用CRISPR/Cas9技术编辑SlCSN5B基因可以提高番茄果实中维生素C的含量,为后续培育优质番茄新品种提供材料。Subunit 5B of constitutively photomorphogenic 9 signalosome(CSN5B)is an inhibitory factor for the biosynthesis of vitamin C in the L-galactose synthesis pathway in plants.To create mutants with richer vitamin C in tomato fruits,a dual-target vector of pKSE402-SlCSN5B was constructed and transformed into the breeding parent of 1912.Based on the molecular biological assay and DNA sequencing,17 transgenic positive lines were determined,and 6 lines of them were genetically mutated at the target site of SlCSN5B,with an editing efficiency of 35.3%.Among the mutants,2 lines were homozygous mutants,csn5b-6 with 180 bp deletion and csn5b-8 with 3 bp deletion.The biological traits of two types of deficiency homozygotes without exogenous T-DNA insertion derived from the T 1 generation were observed,and there were no significant differences in phenotypes of the plant and fruit.Through physiological testing of red-ripe fruits from T 1 generation lines of csn5b-6,the GMPase activity and the vitamin C content significantly increased by 43%and 37.8%,respectively,the content of hydrogen peroxide significantly decreased by 25.9%,and the content of soluble solids did not obviously change,compared with the wild type.There were no significant differences in plant traits and physiological characteristics between T 1 generation lines of csn5b-8 and the wild type.Based on gene expression analysis and protein structure prediction,the results showed that the gene of SlCSN5B normally transcribed,and the loss of large peptide segments of CSN5B encoded by SlCSN5B caused changes in its structure to affect functioning,which led to an increase of vitamin C content in the line of csn5b-6-11.The findings suggest that the vitamin C content of tomato fruit can be improved by CRISPR/Cas9-mediated SlCSN5B gene editing,which provides the valuable resources for high-quality breeding in tomato.

关 键 词：CRISPR/Cas9 番茄 SlCSN5B 维生素C 

分 类 号：Q812[生物学—生物工程] S64[农业科学—蔬菜学]