拟南芥agl16突变体的创制及功能鉴定

Creation and Functional Identification of Arabidopsis agl16 Mutant

AGL16是调控拟南芥气孔密度和ABA含量的重要负调控转录因子,在拟南芥抗旱反应过程中发挥重要的作用。为了获取拟南芥agl16突变体材料,采用棉花叶皱缩病毒(CLCrV)介导的VIGE系统筛选靶向敲除拟南芥AGL 16的sgRNA,同时利用农杆菌介导的浸花法将完整编辑载体转化野生型拟南芥,创制了拟南芥agl16突变体并进行抗旱性鉴定。结果表明:(1)利用CLCrV介导的VIGE系统筛选获得2个能靶向敲除AGL 16基因的sgRNA,同时构建AtU6-26∷AtAGL 16-sgRNA1-35S∷Cas9-Ter-p1300编辑载体转化野生型拟南芥Col-0,筛选获得靶位点缺失4 bp的agl16纯合突变体(AGL 16:-4)。(2)抗旱表型性鉴定结果显示,干旱处理18 d后,大部分野生型植株干枯死亡,而突变体植株受胁迫的表型相对较轻;复水后,野生型和突变体植株的存活率分别为34.5%(10/29)和75%(27/36)。(3)与野生型相比,干旱胁迫下AGL 16:-4纯合突变体植株叶片单位面积气孔数量减少、离体叶片失水率显著降低,但二者的单株种子量并没有发生明显的改变。研究认为,AGL 16:-4纯合突变体的抗旱性较野生型明显增强,且种子量与野生型基本一致,表明AGL 16基因可作为作物抗旱育种理想的候选靶基因;所获得的agl16突变体为后期从农作物中克隆的AGL 16同源基因进行功能回补验证提供了有利的转基因受体材料。

AGL16 is an important negative regulatory transcription factor that regulates stomatal density and ABA content in Arabidopsis,and plays an important role in drought resistance in Arabidopsis.In order to obtain Arabidopsis agl16 mutant material,we used cotton leaf crumple virus(CLCrV)-mediated VIGE system to screen the target and knockout Arabidopsis AGL 16 sgRNAs.At the same time,the intact editing vector was transformed into wild-type Arabidopsis by Agrobacterium-mediated flower dip method,and Arabidopsis agl16 mutant was created and identified for drought resistance.The results showed that:(1)two sgRNAs that can target and knockout Arabidopsis AGL 16 gene were obtained by CLCrV-mediated VIGE system,while AtU6-26∷AtAGL 16-sgRNA1-35S∷Cas9-Ter-p1300 editing vector was constructed to transform wild-type Arabidopsis Col-0,and agl16 homozygous mutant(AGL 16:-4)with 4 bp deletion of the target site was screened.(2)The results of drought resistance phenotypic identification showed that after 18 days of drought treatment,most of the wild-type plants dried up and died,while the mutant plants had a relatively lightly stressed;After rehydration,the survival rates of wild-type and mutant plants were 34.5%(10/29)and 75%(27/36),respectively.(3)Compared with the wild-type,the number of stomata per unit area in leaves and the water loss rate of isolated leaves of AGL 16:-4 homozygous mutant plants were reduced under drought stress,but the seed amount per plant did not change significantly.The results showed that the drought resistance of AGL 16:-4 homozygous mutant was significantly enhanced compared with the wild-type,and the seed quantity was basically the same as that of the wild-type,indicating that AGL 16 gene could be an ideal candidate gene for crop drought resistance breeding;The obtained agl16 mutant provides a favorable transgenic receptor material for the functional complementation verification of the AGL 16 homologous gene cloned from crops in the future.