摘要： 为探究CRISPR/Cas9基因编辑技术在花生中的应用，利用花粉管注射浸花法和农杆菌介导法转化花生，通过在侵染液中添加合适浓度的AS、MES以及MgCl₂ · 6H₂O促进了转化事件的发生，并通过注射液每日现用现配最大程度地保持农杆菌的活力，提高了转化效率。本试验首次将该转化法用于基于CRISPR/Cas9技术的花生基因编辑，根据花生FatB基因序列设计编辑靶位点，成功构建CRISPR/Cas9基因编辑载体PX458-Cas9-FatB并成功转化农杆菌菌株GV3101；利用1 mL无菌注射器将当日离心配置的新鲜农杆菌侵染液注射到花生龙骨瓣中至花瓣浸透，每日8：00以前完成注射，连续注射15日，待注射花朵下针后用尼龙绳进行捆绑标记；待荚果成熟后，收获捆绑标记的花生荚果，正常晾晒干燥后剥取花生籽粒，提取籽粒基因组DNA，进行PCR扩增，筛选转化阳性籽粒。结果显示，在被检测的274粒籽粒中，有108粒扩增出了相应目的条带，转基因阳性率为39.42%；经测序验证，108粒阳性籽粒中有1粒在靶位点外发生了基因编辑，在部分阳性籽粒的自交后代中，发现了靶位点发生编辑的籽粒。因此，本研究初步证实利用注射浸花以及农杆菌介导法对基于CRISPR/Cas9技术的花生基因编辑是有效的，但编辑效率有待于进一步提高。

关键词: 花生, CRISPR/Cas9, 注射浸花法, FatB, 基因编辑

Abstract: In order to explore the possible application of CRISPR/Cas9 gene editing technology in peanut, the genetic transformation by pollen tube injection pathway and Agrobacterium tumefaciens mediated method was conducted firstly in peanut. The transformation efficiency was improved dramatically by adding appropriate concentration of AS, MES and MgCl₂ · 6H₂O to the infection solution, and using the fresh infection solution produced daily which could maximize the activity of A. tumefaciens.In this experiment, the transformation method was applied to peanut gene editing based on CRISPR/Cas9 technology for the first time. The editing target site was designed according to the peanut FatB gene sequence, and the CRISPR/Cas9 gene editing vector PX458-Cas9-FatB was successfully constructed and transformed into the A. tumefaciens strain GV3101. The fresh A. tumefaciens infection solution was injected into the carina of peanut plants by 1 mL aseptic syringe until the petals were soaked. The injection was conducted before 8:00 everyday, with 15 d continuous injection. Those pod needles from injected flowers were then marked by tied with nylon cord. Those pods tied by nylon cord from injected flowers were harvested, and then dried by normal sunlight. The genomic DNAs from those seeds from marked pods were extracted,and the positive transformed seeds were screened by PCR amplification.The results showed that among the 274 seeds tested, the corresponding target bands were obtained from 108 seeds, and the positive rate of transformation was 39.42%.The sequencing results showed that one of the 108 positive kernels had gene editing outside the target site, the kernels with edited target sites were found in the inbred progenies of some positive kernels. Therefore, this study preliminarily showed that the injection dipping flower method was effective for peanut gene editing based on CRISPR/Cas9 technology, but the editing efficiency needs to be further improved.

Key words: Peanut, CRISPR/Cas9, Infection and soaking flower, FatB, Gene editing

中图分类号: 

• Q78

• S565.03