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2021 , Vol. 42 >Issue 11: 3120 - 3125

DOI: https://doi.org/10.3969/j.issn.1000-2561.2021.11.010

Omics & Biotechnology

Construction and Verification of Editing Vector for EBE Region of MeSWEET10a Promoter in Cassava

  • ZHANG Tong 1 ,
  • WANG Yajie 1 ,
  • GUO Wenya 1 ,
  • LI Ruimei 2 ,
  • LIU Jiao 2 ,
  • GUO Jianchun 2 ,
  • HU Xinwen 1 ,
  • YAO Yuan , 2, * ,
  • GENG Mengting , 1, *
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  • 1. Hainan University, Haikou, Hainan 570228, China
  • 2. Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences / Hainan Institute for Tropical Agricultural Resources, Haikou, Hainan 571101, China

Received date: 2021-02-19

  Request revised date: 2021-04-09

  Online published: 2021-12-24

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Copyright reserved © 2021.
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Abstract

Cassava bacterial blight (CBB) is an important cassava disease caused by Xanthomonas axonopodis pv. manihotis (Xam), which affects the yield of cassava. The main cassava varieties in China are not resistant to Xam. The TAL20 effector protein secreted by Xam, could bind to the EBE region of the cassava MeSWEET10a promoter, activate the gene expression, promote the transport of sugars to the site of Xam infection, and provide carbohydrates for the reproduction of the pathogen. In this study, the gene editing target of EBE region was designed by using online software CRISPR-Pv2.0, and the CRISPR/Cas9 gene editing plasmid pCAMBIA1301-Cas9-EBE-sgRNA was constructed. The recombinant plasmid was transformed into Agrobacterium tumefaciens LBA4404, and the fragile embryogenic calli of cassava was infected by the A. tumefaciens-mediated method. DNA was extracted from the fragile embryogenic calli of infected and uninfected cassava. The target EBE region and potential miss sites were amplified by PCR, and the editing effect was analyzed by Sanger sequencing. It was found that the EBE region was edited successfully without off-target phenomenon. This study is helpful to further obtain cassava mutants in the EBE region of MeSWEET10a promoter and enhance the resistance of cassava to bacterial blight.

Key words： cassava; bacterial blight; MeSWEET10a; CRISPR/Cas9

Cite this article

ZHANG Tong , WANG Yajie , GUO Wenya , LI Ruimei , LIU Jiao , GUO Jianchun , HU Xinwen , YAO Yuan , GENG Mengting . Construction and Verification of Editing Vector for EBE Region of MeSWEET10a Promoter in Cassava[J]. Chinese Journal of Tropical Crops, 2021 , 42(11) : 3120 -3125 . DOI: 10.3969/j.issn.1000-2561.2021.11.010

木薯是热带地区重要的粮食作物,而病害是木薯产量提高的重要制约因素之一[1,2]。木薯细菌性枯萎病（cassava bacterial blight, CBB）是我国木薯面临的主要病害,该病是由地毯草黄单胞菌木薯萎蔫致病变种（Xanthomonas axonopodis pv. Manihotis, Xam）引起的细菌性病害[3]。我国木薯主栽品种均不抗Xam病原菌的侵染,CBB曾导致我国木薯大面积减产。因此,培育抗CBB木薯新品种有益于我国木薯产业持续健康发展。
Xam病原菌侵染木薯时,可通过Ⅲ型分泌系统将转录激活因子样效应物蛋白（transcription activator-like effectors,TAL）分泌到宿主细胞中[4],TAL效应蛋白的可变重复区（repeat variable diresidues,RVD）识别宿主感病相关基因启动子上的效应蛋白结合元件（effector-binding element,EBE),并激活下游感病基因表达,以促进Xam病原菌的侵染[5]。不同的Xam病原菌菌株包含的TAL效应蛋白数目存在差异,一般为1~5个。其中TAL20的RVD区可识别木薯糖转运蛋白编码基因MeSWEET10a启动子的EBE区,激活MeSWEET10a基因的表达,以利于糖类物质转运至Xam病原菌侵染部位,以促进病原菌的繁殖[6]。因此,MeSWEET10a基因启动子的EBE区是利用基因编辑技术创制抗CBB木薯种质的重要靶标。类似的抗病育种策略已应用水稻抗白叶枯病育种。利用基因编辑技术,编辑水稻SWEET基因启动子EBE区,阻止水稻白叶枯病病原菌水稻黄单胞菌水稻致病变种（Xanthomonas oryzae pv. oryzae,Xoo）的TAL效应蛋白激活水稻SWEET基因,可获得广谱抗白叶枯病的水稻种质[7]。本研究利用CRISPR/Cas9技术构建木薯糖转运蛋白基因MeSWEET10a启动子EBE区域的编辑载体,验证了载体的编辑效果,及分析了脱靶的可能性,为进一步获得MeSWEET10a启动子突变体,创制抗细菌性枯萎病木薯新种质奠定了基础。

1 材料与方法

1.1 材料

植物材料：‘华南8号’（SC8）木薯品种来源于中国热带农业科学院热带作物品种资源研究所。
菌株及质粒：大肠杆菌菌株DH5α、根癌农杆菌菌株LBA4404、基因编辑质粒pCAMBIA1301- Cas9-sgRNA为本实验室构建。
试剂：T4 DNA ligase、DNA聚合酶Ex Taq采购于Takara公司,BsaⅠ限制性内切酶购于NEB公司,质粒提取试剂盒购生工生物工程（上海）股份有限公司,植物DNA提取试剂盒购于成都福际生物技术有限公司。

1.2 方法

1.2.1 靶点引物的设计及基因编辑载体构建 根据MeSWEET10a（Manes.06G123400）基因的启动子序列,利用CRISPR-P v2.0在线软件分析MeSWEET10a启动子EBE区的靶点及潜在脱靶位点。合成靶点退火引物（表1）,参照李崭等[8]的方法进行退火及克隆至基因编辑载体。菌液PCR筛选阳性克隆后送生工生物工程有限公司测序,将正确的质粒命名为pCAMBIA1301-Cas9- EBE-sgRNA,并将该质粒转化LBA4404农杆菌,菌液PCR验证正确后备用。
表1 本研究用的引物

Tab.1 Primers for this study

引物
Primer		 上游序列（5°‒3°）
Forward sequence (5°‒3°)		 下游序列（5°‒3°）
Reverse sequence (5°‒3°)		 产物大小
Product size/bp		 用途
Use
靶点退火 GATTGTGGGCGAGAAGCGTTTATAT AAACATATAAACGCTTCTCGCCCAC 25 载体构建
阳性克隆筛选 GCAAGCTGCTCTAGCCAATACGC AAACATATAAACGCTTCTCGCCCAC 351 PCR检测
EBE-HT ATGCAACTTGACTTCATTCCGTTC TTCGTTTTTGGCGTTTTAGGTACTA 200 编辑效果分析
Off-target-1 TTATGCATGCAACTTGACCTGA TGCAAGGCCATCTCGAAATCT 169 脱靶分析
Off-target-2 TCCTTGTGGTTCAGGACACAT GTCCCCAATCCGACATCGTT 226 脱靶分析
Off-target-3 TTAACATGGTATCAAGCCTCCCC TCAGCCATCCACAGCAAAACG 178 脱靶分析
Off-target-4 GTCTGCTCCATAAAGTGGCTC ACAGGTCTCCGTGGTTGTACT 231 脱靶分析
1.2.2 木薯脆性胚性愈伤组织的诱导及侵染 参照李崭等[8]的方法诱导‘SC8’木薯脆性胚性愈伤组织,将携带基因编辑质粒的LBA4404农杆菌侵染脆性胚性愈伤组织,经过共培养3 d,洗菌继续培养21 d后用于检测EBE区的编辑效果。
1.2.3 EBE区编辑效果及脱靶分析 设计MeSWEET10a启动子EBE区编辑靶点和4个潜在脱靶位点区域的PCR扩增引物（表1）。提取农杆菌侵染的木薯脆性胚性愈伤组织DNA,PCR扩增获得目的片段并测序。基于Sanger测序峰值图,分析启动子EBE区编辑效果,以及是否存在脱靶情况。

2 结果与分析

2.1 启动子编辑靶点的设计及潜在脱靶位点分析

通过在线设计软件CRISPR-P v2.0,根据木薯MeSWEET10a基因启动子EBE区序列,设计获得编辑MeSWEET10a基因启动子EBE区域的靶点序列,该靶点CG含量值为45%,PAM序列为CCT（图1）。脱靶位点预测分析结果显示（表2）,该靶点在木薯基因组存在4个潜在脱靶位点。其中,Off-target-1的脱靶分值为0.587,有3个错配的碱基数,有可能脱靶到14号染色体;Off-target-2的脱靶分值为0.142,有4个错配的碱基数,位于15号染色体;Off-target-3的脱靶分值为0.102,有3个错配的碱基数,位于10号染色体;Off-target-4的脱靶分值为0.098,位于10号染色体。全部潜在脱靶位点均不属于基因的编码区,表明即使脱靶也不会造成基因功能的缺失。sgRNA的二级结构预测分析结果显示（图2）,靶点序列二级结构较为松散,有利于结合靶标位置。
图1 基因编辑靶点位置

Fig. 1 Location of gene editing target

表2 EBE区编辑靶点和潜在脱靶位点

Tab. 2 Editing target and potential off-target sites of EBE region

位点
Site		 序列
Sequence		 sgRNA编辑或潜在脱靶得分Score of sgRNA editingor off-target potential 错配数量
Number of mismatch		 基因
Gene		 区域
Region
Target TGGGCGAGAAGCGTTTATATAGG 0.076 - Manes.06G123400 EBE
Off-target-1 TTGGCAAGAAGCATTTATATAGG 0.587 3MMs Manes.14G047700 Intergenic
Off-target-2 TGGGAGAGAAGAGATTATAAAGG 0.142 4MMs Manes.15G162100 utr
Off-target-3 TGGGCGTGAGGCCTTTATATGGG 0.102 3MMs Manes.10G134400 intron
Off-target-4 TAGGAGAGAAGTGTTTATGTGGG 0.098 4MMs Manes.10G045900 intron

Note: Shaded letters indicate PAM areas, Underlined letters indicate bases mismatched with the target.

注：带阴影的字母表示PAM区,带下划线字母表示与靶点错配的碱基。

图2 sgRNA二级结构预测

Fig. 2 Prediction secondary structure of sgRNA

2.2 基因编辑载体的构建

本研究所使用的基因编辑载体为pCAMBIA 1301-Cas9-sgRNA,采用线性化的质粒与退火后的EBE区靶点引物连接,构建AtU6-26-EBE- sgRNA表达盒。通过菌液PCR筛选阳性大肠杆菌克隆,获得约350 bp目的条带（图3）。测序分析进一步证明基因编辑载体pCAMBIA1301- Cas9-EBE-sgRNA已构建成功。提取质粒后,转LBA4404农杆菌备用。
图3 重组质粒菌液PCR检测

M：DL2000 DNA marker;1~5：单克隆;6：阳性对照;7：阴性对照。

Fig. 2 PCR detection of recombinant plasmid

M: DL2000 DNA marker; 1-5: Monoclones; 6: Positive control; 7: Negative control.

2.3 编辑载体转化木薯脆性胚性愈伤组织及编辑效果验证

以‘SC8’木薯无菌苗为外植体,诱导腋芽膨大、然后诱导体细胞胚形成,最终诱导获得脆性胚性愈伤组织（图4）。将携带pCAMBIA1301- Cas9-EBE-sgRNA质粒的LBA4404转化木薯脆性胚性愈伤组织21 d,PCR扩增MeSWEET10a基因启动子EBE区域的片段,测序分析该区域的序列变化情况。结果发现（图5）,转化后的样品中,PAM区附近开始出现多峰,然而未转化样品中并无杂峰出现。说明本研究选择的基因编辑靶点序列形成的sgRNA可引导Cas9蛋白对MeSWEET10a基因启动子EBE区域进行编辑,造成了核苷酸序列的变异。
图4 木薯脆性胚性愈伤组织诱导及侵染过程

A：无菌苗;B：茎段腋芽膨大;C：腋芽诱导出的体细胞胚;D：纯化后的体细胞胚;E：脆性胚性愈伤组织;F：侵染后的脆性胚性愈伤组织。

Fig. 4 Induction and infection process of cassava friable embryogenic calli

A: Sterile seedlings; B: Swelling oflateral buds on the stem segment; C: Somatic embryos induced by lateral buds; D: Purified somatic embryos; E: Friable embryogenic calli; F: Transformed friable embryogenic calli.

图5 基因编辑效果分析

红色线条：PAM区;绿色线条：靶点序列。

Fig. 5 Analysis of gene editing effect

Red line: PAM area; Green line: Target sequence.

2.4 脱靶分析

以转化和未转化基因编辑载体的木薯脆性胚性愈伤组织DNA为模板,PCR扩增MeSWEET10a基因启动子EBE区编辑靶点的4个潜在脱靶位点区域片段,Sanger测序分析该靶点是否有脱靶的现象。结果表明（图6）,潜在脱靶位点的序列在已转化和未转化的样品中没有发生改变。说明本研究构建的基因编辑载体pCAMBIA1301- Cas9-EBE-sgRNA可对MeSWEET10a基因启动子EBE区进行特异编辑,并且无脱靶现象。
图6 基因编辑位点的潜在脱靶效果分析

A：Off-target-1测序峰值图;B：Off-target-2测序峰值图;C：Off-target-3测序峰值图;D：Off-target-4测序峰值图;红色线条：PAM区;绿色线条：靶点序列。

Fig. 6 Analysis of potential off-target effects of gene editing sites

A: Peak map of Off-target-1 sequencing; B: Peak map of Off-target-2 sequencing; C: Peak map of Off-target-3sequencing; D: Peak map of Off-target-4 sequencing; Red line: PAM area; Green line: Target sequence.

3 讨论

植物病原微生物引起的病害对作物的产量构成严重威胁。病原微生物利用宿主的易感（S）基因来促进增殖,敲除或降低S基因可能会干扰宿主和病原体之间的亲和性,从而为宿主提供广谱和持久的抗病能力。利用基因组编辑技术编辑作物S基因的编码区或其启动子,提高抗病性的报道主要集中于水稻、小麦、西红柿、柑橘等作物。霉菌抗性位点O（mildew resistance locus O, MLO）的突变可赋予植物对白粉病的抗病性,如编辑番茄SlMlo1基因[9],小麦TaMLO-A1、TaMLO-B1、TaMLO-D1可提高小麦和番茄的抗白粉病能力[10]。小麦TaEDR1的3个同源基因被编辑突变,可增强小麦对白粉病的抗病能力[11]。转录因子CsLOB1在促进柑橘溃疡病病菌生长和脓包形成过程中起关键作用,编辑CsLOB1基因增强柑橘抗柑橘溃疡病的能力[12]。在敲除S基因启动子关键元件,干扰病原微生物对作物S基因表达的调控,可创制抗病种质。水稻白叶枯病S基因OsSWEET11、OsSWEET14和OsSWEET13启动子的EBE区敲除,阻止TAL效应蛋白激活这些S基因表达,赋予水稻对白叶枯病原菌的广谱抗病能力[13]。
效应蛋白在病原菌与植物的互作过程中扮演着重要的作用,病原菌分泌的效应蛋白可调控宿主感病相关基因的表达,干扰植物细胞的正常活动,创造病原菌侵染和繁殖的有利条件,同时植物可协同进化,获得识别和监控效应蛋白的能力,从而激活植物的免疫反应,增强宿主的抗病性。病原菌通过效应蛋白结合植物的SWEET基因启动子,劫持SWEET基因为病原菌自身的生长提供养分。白粉菌浸染拟南芥后诱导AtSWEET12基因表达升高,灰霉菌侵染后则诱导AtSWEET4、AtSWEET15、AtSWEET17;灰霉菌侵染葡萄诱导VvSWEET4基因表达量升高[14,15,16]。木薯Xam病原菌分泌的TAL20效应蛋白,结合MeSWEET10a基因启动子的EBE区,调控该基因表达。为了创制抗CBB的木薯新种质,本研究成功构建了EBE区的CRISPR/Cas9编辑载体。李崭等[8]证明了利用基因编辑载体,遗传转化‘SC8’木薯脆性胚性愈伤组织,可快速检测淀粉合成酶基因SSIII-1和SSIII-2编码区的编辑效果。本研究证明脆性胚性愈伤组织转化检测方式,同样适用于检测GC含量较低的MeSWEET10a基因启动子EBE区的编辑效果。脱靶是CRISPR/Cas9基因编辑技术面临的一个重要问题。本研究设计的EBE区编辑靶点在木薯基因组中存在4个潜在脱靶位点,其中Off-target-1和4与设计的靶点仅有3个碱基差异。脱靶检测分析发现,EBE区被编辑的同时,潜在脱靶位点的序列没有发生突变,表明本研究设计的EBE区编辑靶点具有良好的特异性。本研究有助于进一步获得木薯MeSWEET10a基因启动子EBE区突变体,以增强木薯的抗细菌性枯萎病能力,为木薯抗病育种奠定材料和研究基础。
责任编辑：黄东杰

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