大蒜遗传转化体系建立与CRISPR-Cas9敲除蒜氨酸酶基因研究  

Establishment of the Genetic Transformation System and Studies on CRISPR-Cas9 Knockout Allinase Gene

作　　者：牛忠露 杨彩霞 李宁阳[1] 李祥[2] NIU Zhong-lu;YANG Cai-xia;LI Ning-yang;LI Xiang(College of Food Science and Engineering,Shandong Agricultural University,Tai’an 271018,China;State Key Laboratory of Crop Biology,College of Life Sciences,Shandong Agricultural University,Tai’an 271018,China)

机构地区：[1]山东农业大学食品科学与工程学院,山东泰安271018 [2]山东农业大学生命科学学院,作物生物学国家重点实验室,山东泰安271018

出　　处：《山东农业大学学报（自然科学版）》2023年第3期319-343,共25页Journal of Shandong Agricultural University：Natural Science Edition

基　　金：国家自然科学基金面上项目(31972000);山东省重点研发计划项目(2021TZXD001)。

摘　　要：为了为大蒜转基因研究和蒜氨酸酶基因研究提供技术支撑和理论依据,本文首先建立了愈伤组织再生培养体系,在基因型、生长发育时期、植物激素配比和外植体来源方面对比MS、B5、N6和SH基础培养基,明确大蒜愈伤诱导和培养的最适基础培养基为MS和SH,其中以苍山大蒜根尖为外植体、培养基为MS+1 mg/L 2,4-D+0.1 mg/L 6-BA是大蒜愈伤组织诱导的最佳组合;分化阶段为MS+3 mg/L 6-BA;生根培养和鳞茎诱导阶段则使用不添加任何激素的培养基。然后,通过对影响农杆菌介导遗传转化因素的筛选,得到最佳转化条件:农杆菌菌株选用LBA4404、侵染液组成MS/LB+100μM As+1%葡萄糖+1%蔗糖为宜,侵染时间10 min,共培养3 d,草铵膦筛选浓度为250 mg/L,在此条件下该方法的遗传转化率为1.68%。利用CRISPR-Cas9敲除技术,成功实现了对鳞茎蒜氨酸酶关键基因的敲除,突变体中目的基因mRNA的表达量和蒜氨酸酶活均明显下降。To provide technical support and theoretical basis for garlic transgenic research and allinase gene research.In this paper,a callus regeneration culture system was established,and MS,B5,N6 and SH were compared in terms of genotype,growth and development stage,plant hormone ratio and the source of explants.The optimal medium for callus induction and culture of garlic was MS and SH.The root tip of Cangshan garlic as explants and medium MS+1 mg/L 2,4-D+0.1 mg/L 6-BA were the best combinations for callus induction.The differentiation stage was MS+3 mg/L 6-BA.In root culture and bulb induction phase,medium without any hormone was used.Then,by screening factors affecting Agrobacterium-mediated genetic transformation,the optimal transformation conditions were obtained:The optimal solution was MS/LB+100μM As+1%glucose+1%sucrose with LBA4404 and infecting liquid.The infection time was 10min,the co-culture was conducted for 3 days,and the screening concentration of phosphine oxalammonium was 250 mg/L.Under these conditions,the genetic conversion rate of this method was 1.68%.Using CRISPR-Cas9 knockout technique,the key gene of bulb allinase was successfully knocked out,and the mRNA expression of target gene and allinase activity in the mutant were significantly decreased.

关 键 词：大蒜 遗传转化 愈伤组织 蒜酶 

分 类 号：S633.4[农业科学—蔬菜学]