利用CRISPR/Cas9技术创制番茄粉果和无绿肩材料  

Creating Pink Fruit and Fruit Without Green Shoulder Using CRISPR/Cas9 Technology in Tomato

作　　者：李国斌 蔡梁玉 肖立成 王家发 张得迪 张俊红[1] LI Guobin;CAI Liangyu;XIAO Licheng;WANG Jiafa;ZHANG Dedi;ZHANG Junhong(National Key Laboratory for Germplasm Innovation and Utilization for Fruit and Vegetable Horticultural Crops,College of Horticulture and Forestry Sciences,Huazhong Agricultural University,Wuhan 430070,China)

机构地区：[1]华中农业大学园艺林学学院,果蔬园艺作物种质创新与利用全国重点实验室,武汉430070

出　　处：《园艺学报》2023年第5期985-999,共15页Acta Horticulturae Sinica

基　　金：国家自然科学基金项目(31772317);国家现代农业产业技术体系建设专项资金项目(CARS-23-A13)。

摘　　要：以栽培番茄品种(系)Ailsa Craig(AC)、B5和B9为试材,利用CRISPR/Cas9双元表达载体系统,对番茄品质调控关键基因SlGLK2和SlMYB12进行定点敲除。获得AC背景下SlGLK2和SlMYB12的基因编辑植株各9株,B5背景下SlGLK2的基因编辑植株3株,B9背景下SlMYB12的基因编辑植株4株。通过对靶点测序发现,在靶位点的PAM上游3~4 bp处发生了不同形式的碱基缺失、插入或替换,主要以1~10 bp的缺失和单碱基G和T的插入为主;也产生了两靶点间的大片段缺失,最大片段为334 bp。AC背景下敲除SlMYB12产生粉色果实,敲除SlGLK2基因,果实肩部的绿色明显变浅,B5背景下的3株基因编辑番茄均无“绿肩”。本研究中利用CRISPR/Cas9技术在不同番茄种质中成功创制了敲除SlGLK2或SlMYB12的基因编辑材料,以期为番茄品质育种提供优良种质。The key quality-regulating genes SlGLK2 and SlMYB12 were knocked out in tomato cultivars(lines)Ailsa Craig(AC),B5 and B9 using CRISPR/Cas9 binary expression vector system. Nine CRISPR/Cas9 transgenic plants for each of SlGLK2 and SlMYB12 in the AC,three CRISPR/Cas9transgenic plants of SlGLK2 in the B5,and four CRISPR/Cas9 transgenic plants of SlMYB12 in the B9were obtained. By sequencing the target sites,it was found that different forms of base deletion,insertion and replacement occurred at 3–4 bp upstream of PAM,mainly 1–10 bp deletion as well as single base G and T insertion. In addition,a large deletions produced between the two targets is 334 bp. Knockout of SlMYB12 in AC showed pink fruits. Knockout of SlGLK2 gene in AC showed the green shoulders of fruits were obviously lighter,and knockout of SlGLK2 gene in B5 showed no‘Green Shoulder'in tomato fruits comparing to the corresponding wild type lines. In this study,SlGLK2 and SlMYB12 gene editing plants were successfully generated in different tomato materials using CRISPR/Cas9 technology,which would provide excellent germplasm for tomato quality breeding.

关 键 词：番茄 基因编辑 SlGLK2 SlMYB12 粉果 绿肩果 

分 类 号：S641.2[农业科学—蔬菜学]