遗传 ›› 2018, Vol. 40 ›› Issue (6): 496-507.doi: 10.16288/j.yczz.17-424

• 研究报告 • 上一篇    下一篇

利用CRISPR/Cas9双基因敲除系统初步解析大豆GmSnRK1.1和GmSnRK1.2对ABA及碱胁迫的响应

李慧卿,陈超,陈冉冉,宋雪薇,李佶娜,朱延明(),丁晓东()   

  1. 东北农业大学农业生物功能基因重点实验室,哈尔滨 150030
  • 收稿日期:2017-12-27 修回日期:2018-05-16 出版日期:2018-06-20 发布日期:2018-05-21
  • 通讯作者: 朱延明,丁晓东 E-mail:ymzhu2001@yahoo.com.cn;xiaodong.ding@neau.edu.cn
  • 作者简介:李慧卿,硕士研究生,专业方向：植物基因工程与分子生物学。E-mail: 632651739@qq.com
  • 基金资助:
    国家自然科学基金项目(31670272);黑龙江省自然科学基金项目(C2017014);东北农业大学人才项目资助(C2017014)

Preliminary analysis of the role of GmSnRK1.1 and GmSnRK1.2 in the ABA and alkaline stress response of the soybean using the CRISPR/Cas9-based gene double-knockout system

Huiqing Li,Chao Chen,Ranran Chen,Xuewei Song,Jina Li,Yanming Zhu(),Xiaodong Ding()   

  1. Key Laboratory of Agricultural Biological Functional Genes, Northeast Agricultural University, Harbin 150030, China
  • Received:2017-12-27 Revised:2018-05-16 Online:2018-06-20 Published:2018-05-21
  • Contact: Zhu Yanming,Ding Xiaodong E-mail:ymzhu2001@yahoo.com.cn;xiaodong.ding@neau.edu.cn
  • Supported by:
    Supported by the National Natural Science Foundation of China(31670272);Heilongjiang Province Natural Science Foundation(C2017014);the Northeast Agricultural University Talent Project(C2017014)

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摘要：

蔗糖非发酵相关激酶(sucrose non-fermenting related protein kinases, SnRKs)是广泛存在于植物中的一类Ser/Thr蛋白激酶,在植物的生长、发育、代谢和抗逆等方面具有重要调节作用。大豆(Glycine max L.)基因组中含有4个SnRK1同源基因,其中GmSnRK1.1和GmSnRK1.2为两个主要表达基因,可能参与大豆多种抗逆途径。为解析大豆GmSnRK1.1和GmSnRK1.2对ABA及碱胁迫的响应,本研究构建了双靶点CRISPR载体定向敲除GmSnRK1.1和GmSnRK1.2基因,利用发根农杆菌(Agrobacterium rhizogenes)介导大豆遗传转化,获得双基因敲除突变体毛状根,经测序鉴定双基因突变率为48.6%;同时,利用实验室前期构建的植物超量表达载体获得超量表达GmSnRK1基因大豆毛状根。经25 μmol/L ABA处理15 d,对照组和超量表达毛状根的生长受到明显抑制,其根长与根鲜重均显著低于双基因敲除突变体毛状根;经50 mmol/L NaHCO3处理15 d,对照组和双基因敲除突变体毛状根的生长受到明显抑制,其根长与根鲜重均显著低于超量表达毛状根。本研究建立的CRISPR/Cas9系统能够有效地对大豆进行GmSnRK1.1和GmSnRK1.2双基因敲除,基因敲除突变降低了植物对ABA的敏感性及对碱胁迫的耐性,研究结果初步说明SnRK1激酶在植物响应非生物胁迫中具有重要作用。

关键词: 大豆, CRISPR/Cas9, GmSnRK1.1, GmSnRK1.2, ABA信号, 碱胁迫

Abstract:

Sucrose non-fermenting related protein kinases (SnRKs) are a ubiquitous Ser/Thr protein kinase in the plant kingdom. These kinases play important roles in plant growth, development, metabolism and resistance to environmental stresses. The soybean (Glycine max L.) genome has four SnRK1 genes, of which GmSnRK1.1 and GmSnRK1.2 are predominant and participate in multiple stress response pathways. To dissect the mechanism of the role of GmSnRK1.1 and GmSnRK1.2 proteins in response to ABA and alkaline stresses, we constructed a dual-gRNA CRISPR vector to specifically knock out GmSnRK1.1 and GmSnRK1.2. The resultant constructs were transformed into soybean cotyledon nodes to induce hairy roots by agrobacteria (Agrobacterium rhizogenes). The soybean hairy roots obtained were genotyped, and the results showed that GmSnRK1.1 and GmSnRK1.2 were efficiently doubly knocked out in 48.6% hairy roots. We also generated control hairy roots that over-expressed GmSnRK1. The materials were treated with 25 μmol/L ABA for 15 days and the results showed that the growths of wild-type and GmSnRK1 over-expressed roots were significantly inhibited than GmSnRK1.1 GmSnRK1.2 double-knockout roots, as the controls displayed less root lengths and fresh weights. However, after treating with 50 mmol/L NaHCO3 for 15 days, we found that the growths of GmSnRK1.1 GmSnRK1.2 double-knockout roots were significantly inhibited than the wild-type and GmSnRK1 over-expressed control roots, as the knockout groups contained less root lengths and fresh weights. These results implied that the GmSnRK1.1 GmSnRK1.2 double knockout mitigated hairy root sensitivity to ABA and resistance to alkaline stress. Taken together, we established the CRISPR/Cas9 system to perform gene double knockout in the soybean and by using this technique, we determined the roles of GmSnRK1.1 and GmSnRK1.2 in response of abiotic stresses.

Key words: soybean, CRISPR/Cas9, GmSnRK1.1, GmSnRK1.2, ABA signal, alkaline resistance