基于CRISPR/Cas9技术创制耐盐香稻

Development of Aromatic Salt-tolerant Rice Based on CRISPR/Cas9 Technology

【目的】为了促进耐盐香稻育种,利用CRISPR/Cas9系统对粳稻品种连粳11的Badh2和OsRR22基因进行编辑,以期快速获得一批不含有转基因成分且具有耐盐性和香味的纯合水稻材料。【方法】根据Badh2和OsRR22基因序列中编辑位点的敲除效率设计靶位点,构建pH-Ubi-Cas9-Badh2-OsRR22敲除载体,利用农杆菌介导法转入受体品种连粳11中。对转基因后代进行潮霉素和Cas9标记PCR检测以及靶基因测序,获得无外源基因插入的badh2-osrr22纯合株系,并对后代种子性状和苗期耐盐性进行分析。【结果】T2代成熟种子中2-AP含量较背景材料连粳11显著增加,千粒重、粒长、粒宽无明显变化;128 mmol/L氯化钠处理14 d后株系21-30较连粳11苗高增加15.2%,苗鲜质量增加45.2%,苗干质量增加13.2%。【结论】利用CRISPR/Cas9技术对Badh2和OsRR22基因编辑获得能稳定遗传且无外源基因插入的耐盐香稻材料,加快了水稻多性状聚合的选育进程。

【Objective】It is of importance to promote salt-tolerant and fragrant rice breeding.We obtained transgene free homozygous rice materials with salt-tolerance and fragrance by CRISPR/Cas9-mediated editing of Badh2 and OsRR22 in japonica variety Lianjing 11.【Method】Target sites were designed according to knockout efficiency of editing sites in Badh2 and OsRR22 gene sequence.The pH-Ubi-Badh2-OsRR22 knockout vector was constructed and transferred into the recipient variety Lianjing 11 by the Agrobacterium-mediated technology.PCR detection for hygromycin,Cas9 marker and target gene sequencing were performed in the transgenic progenies to obtain badh2-osrr22 homozygous lines without exogenous gene insertion.The seed traits and salt tolerance at seedling stage were analyzed.【Result】Compared with the background material Lianjing 11,the 2-AP content of T2 significantly increased.After treated with 128 mmol/L NaCl solution for 14 days,the seedling height,the fresh and the dry weight of T2 line 21-30 increased by 15.2%,45.2%and 13.2%,respectively.【Conclusion】The successful editing of Badh2 and OsRR22 by CRISPR/Cas9 technology developed aromatic salt-tolerant homozygous lines without exogenous gene insertion,speeding up the breeding process of pyramiding multiple traits in rice.