Page 6 - 《广西植物》2024年第12期
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2 1 6 4 广 西 植 物 44 卷
Abstract: Bacterial wilt is a devastating soil ̄borne disease in tomato ( Solanum lycopersicum) production. The
pathogenic species are complex and tend to have a variationꎬ while mlo caused by the recessive mutation of MLO genes
has a broad ̄spectrum resistance. The previous study suggested that Slmlo1/ 6 may be involved in the resistance response
to bacterial wilt in tomato. In order to further study the gene function of Slmlo1/ 6 in tomato bacterial wilt resistanceꎬ the
genetic mutant plants were created by CRISPR/ Cas9 technology and their phenotypes were identified followed. The
results were as follows: (1) gRNA sequences of SlMLO1/ 6 were designed and assembled with the U6 promotersꎬ then
U6 ̄gRNA1/ 6 fragments containing highly effective targets were ligated to CRISPR vector of pBGK via restriction enzyme
Bsa I digestionꎬ to construct the two ̄gene fusion knockout vector of pBGK ̄SlMLO1/ 6. The recombinant plasmid of
pBGK ̄SlMLO1/ 6 was transformed into Escherichia coli DH5α competent cells and positive monoclonal clones were
selected via plate cultivation. Using Agrobacterium tumefaciens GV3101 strains ̄mediated genetic transformation and
resistance screening to hygromycinꎬ a total of nine edited tomato plants were obtained with sequencing validation. (2)
Target region sequencing showed that M2 and M8 plants had the 177 bp and 7 bp deletion of SlMLO1ꎬ respectivelyꎬ M7
had the 12 bp deletion of SlMLO6ꎬ and M9 had a single base T insertion of SlMLO6. Except for four single gene
homozygous mutants aboveꎬ the other mutations were heterozygous. (3) RT ̄qPCR showed that compared with the wild
type plantꎬ SlMLO1/ 6 gene expression of the mutants was significantly decreasedꎬ especially M2ꎬ M7ꎬ and M8
plants. (4) Phenotypic identification indicated that SlMLO1/ 6 might be tomato bacterial wilt susceptibility genes. In
conclusionꎬ the knockout vector is successfully constructed for resistance MLO genes and tomato transformation is also
achievedꎬ homozygous mutants acquire resistance to bacterial wilt. Amino acid deletion and frameshift mutation may be
the crucial reasons for the gene function change of Slmlo1/ 6 in resistance. The results provide a theoretical reference and
genetic engineering materials for the gene function study in resistance to bacterial wilt and disease resistance breeding
application of tomato.
Key words: tomatoꎬ Slmlo1/ 6ꎬ gene editingꎬ genetic transformationꎬ mutant
作为植物基因功能研究和作物遗传改良的有 annuum) Camlo1 / 2、 茄 子 ( Solanum melongena )
力工具ꎬ规律成簇间隔短回文重复序列及其相关 Smmlo1、烟 草 ( Nicotiana tabacum) Ntmlo1 和 黄 瓜
系 统 ( clustered regularly interspaced short (Cucumis sativus) Csmlo1 等 ( Zheng et al.ꎬ 2013ꎻ
palindromic repeat/ CRISPR ̄associated 9ꎬ CRISPR / Appiano et al.ꎬ 2015ꎻ Nie et al.ꎬ 2015ꎻ Bracuto et
Cas9)是目前应用最广的一项基因编辑技术ꎬ主要 al.ꎬ 2017)ꎮ 除白粉病以外ꎬmlo 也参与假单胞菌、
由单导 RNA ( single guide RNAꎬ sgRNA) 和 Cas9 黄单胞菌、卵菌、尖孢镰刀菌、炭疽菌和稻瘟病菌
组成(Mali et al.ꎬ 2013)ꎮ sgRNA 通过识别目的基 等 多 种 病 原 体 引 起 的 植 物 病 害 反 应 ( Kim &
因前间区邻近基序( protospacer aceradjacent motifꎬ Hwangꎬ 2012ꎻ Kim et al.ꎬ 2014ꎻ Acevedo ̄Garcia et
PAM)ꎬ引导 Cas9 对靶序列进行切割ꎬ产生双链断 al.ꎬ 2017)ꎮ
裂ꎬ修 复 过 程 中 引 起 靶 点 突 变ꎬ 进 而 产 生 表 型 番茄是全球范围内重要的蔬菜作物ꎬ也是一
变异ꎮ 种理想的基因编辑作物ꎬ但在生产中频繁遭受各
MLO (mildew resistance locus O) 是一类最早 种逆境胁迫ꎮ 作为最重要的植物病原细菌之一ꎬ
在大麦( Hordeum vulgare) 中发现并克隆的白粉病 青枯菌(Ralstonia solanacearum)引起的青枯病对番
易感基因(Buschges et al.ꎬ 1997)ꎬ其隐性突变 mlo 茄危害尤为严重ꎬ因此建立快速高效的抗病品种
对白粉菌的几乎所有生理小种都具有高效和持久 选育方法势在必行ꎮ 前期研究发现ꎬSlMLO1 / 6 均
抗性(Reinstädler et al.ꎬ 2010)ꎮ 此后ꎬ在多种高等 含有 7 个跨膜结构域ꎬ定位于原生质膜ꎬSlMLO1 含
植物中相继发现其同源基因ꎬ突变同样具有白粉 1 个 钙 调 素 结 合 区 ( calmodulin binding domainꎬ
病抗性( Kusch & Panstrugaꎬ 2017)ꎮ 利用基因编 CaMBD) (Shi et al.ꎬ 2020)ꎮ 同时ꎬSlMLO1 是已
辑技术ꎬ多个 mlo 被证明是白粉病抗性基因ꎬ如番 知的白粉病易感基因ꎬSlMLO6 与辣椒感青枯病基
茄( Solanum lycopersicum) Slmlo1、 辣 椒 ( Capsicum 因 CaMLO6 同 源 ( Bai et al.ꎬ 2008ꎻ Yang et al.ꎬ