利用CRISPR/Cas9技术突变BnaMPK6基因降低甘蓝型油菜的耐盐性  

Genome editing of BnaMPK6 gene by CRISPR/Cas9 for loss of salt tolerance in Brassica napus L.

作　　者：张文宣 梁晓梅[1] 戴成 文静[1] 易斌[1] 涂金星[1] 沈金雄[1] 傅廷栋[1] 马朝芝[1] ZHANG Wen-Xuan;LIANG Xiao-Mei;DAI Cheng;WEN Jing;YI Bin;TU Jin-Xing;SHEN Jin-Xiong;FU Ting-Dong;MA Chao-Zhi(National Key Laboratory of Crop Genetic Improvement,Huazhong Agricultural University/National Engineering Research Center of Rapeseed/Hongshan Laboratory,Wuhan 430070,Hubei,China)

机构地区：[1]华中农业大学作物遗传改良全国重点实验室/国家油菜工程技术研究中心/洪山实验室,湖北武汉430070

出　　处：《作物学报》2023年第2期321-331,共11页Acta Agronomica Sinica

基　　金：中央高校基本科研业务费专项基金(2662020ZKPY020);国家重点研发计划项目(2020BBB061)资助。

摘　　要：甘蓝型油菜是我国一种重要的油料作物。MPK6是一种丝裂原活化蛋白激酶,MAPK级联途径可以被各种胁迫激活,进而调节植物对胁迫的响应,在植物响应非生物胁迫过程中发挥着重要的作用,但在甘蓝型油菜盐胁迫响应过程中的功能还尚不清楚。基因结构和蛋白序列分析表明,MAPK6在十字花科芸薹属植物种间分化相对保守,基因结构相似,蛋白之间均有相同的STKc_TEY_MAPK结构域,与胁迫和生长发育相关。为探究其功能,本研究利用CRISPR/Cas9技术对BnaMPK6基因进行编辑,通过农杆菌介导的方法将BnaMPK6基因转入到甘蓝型油菜中,获得了BnaMPK6基因4个同源拷贝同时突变的材料:cr-bnampk6-13-1和cr-bnampk6-49-1。cr-bnampk6突变体表现出明显的盐敏感性:在100 mmol L^(-1)和150 mmol L^(-1)浓度的NaCl溶液处理下,功能缺失突变体长势明显缓慢,并且株高和鲜重显著低于野生型植株,但根长无明显差异。此外,在盐胁迫下,功能缺失突变体体内的活性氧和丙二醛含量的积累显著高于野生型对照,脯氨酸含量也明显高于野生型植株。本研究结果表明,BnaMPK6基因在盐水胁迫下正向调控植株的生长和发育,影响甘蓝型油菜的耐盐性。本研究不仅为BnaMPK6基因调控甘蓝型油菜盐胁迫的研究提供了理论基础,而且为甘蓝型油菜耐盐性遗传改良提供了一定的技术支持。Brassica napus L.is an important oil crop.MPK6(Mitogen-Activated Protein Kinases 6)is activated by various stresses to control plant stress tolerance,which is the key gene in response to abiotic stresses.However,the function of MAPK6 in B.napus stress tolerance is still unclear.Gene structure and protein sequence analysis showed that MPK6 gene structures were similar,and all proteins had the same STKc_TEY_MAPK domain in Brassica.To explore the potential role of BnaMPK6,the BnaMPK6 mutants were generated by CRISPR/Cas9 genome editing technology.Two quadruple mutants,cr-bnampk6-13-1 and cr-bnampk6-49-1,were obtained.cr-bnampk6 mutant lines were hypersensitive to salt treatment(100 mmol L^(-1) and 150 mmol L^(-1)),the growth of the mutant was strongly inhibited after salt treatment,and the plant height and fresh weight were significantly lower than those of wild-type plants,but there was no significant difference in root length.In addition,reactive oxygen species(ROS),malondialdehyde(MDA),and free proline were more accumulated in cr-bnampk6 mutant lines.In conclusion,these results revealed that BnaMPK6 gene positively regulated salt tolerance in B.napus,providing theoretical and technical supports for genetic improvement of salt tolerance in Brassica napus.

关 键 词：甘蓝型油菜 BnaMPK6 CRISPR/Cas9 盐胁迫 

分 类 号：S565.4[农业科学—作物学]