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2022 , Vol. 43 >Issue 3: 463 - 472

DOI: https://doi.org/10.3969/j.issn.1000-2561.2022.03.004

Omics ＆ Biotechnology

Construction of MeSTP7 and MeSTP15 Double Mutants in Cassava Based on CRISPR/Cas9 Technology

  • GENG Sha 1, 2 ,
  • ZHANG Jianyu 1, 2 ,
  • WANG Xiaotong 1, 2 ,
  • REN Siyang 1, 2 ,
  • WU Zhihao 1, 2 ,
  • YAO Yuan 2 ,
  • LI Ruimei 2 ,
  • GUO Jianchun 2 ,
  • LIU Jiao , 2, * ,
  • LUO Lijuan , 1, *
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  • 1. College of Tropical Crops, 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
* LIU Jiao,E-mail： ;
LUO Lijuan,E-mail：

Received date: 2021-09-09

  Request revised date: 2021-11-29

  Online published: 2022-03-23

Copyright

Copyright reserved © 2022. Office of Acta Agronomica Sinica All articles published represent the opinions of the authors, and do not reflect the official policy of the Chinese Medical Association or the Editorial Board, unless this is clearly specified.
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Abstract

Cassava (Manihot esculenta Crantz) is an important food and energy crop in the tropics and subtropics, and improving cassava production is essential for the development of the cassava industry. The development of cassava storage root directly affects its yield, and different adversity stresses can affect the development of cassava storage root. Therefore, the analysis of cassava storage root development mechanism can help to achieve high cassava yield through molecular breeding, as well as to obtain excellent germplasm with certain resistance quality and enhance the adaptability of cassava, so as to expand the extension of cassava cultivation. Hexose transport proteins (STPs) are a subfamily of MSTs that regulate plant growth and development as well as abiotic stresses by transporting sugars. The MeSTP7 and MeSTP15 genes have been identified to play an important role in sugar accumulation during tuber development and in response to abiotic stresses. To obtain MeSTP7 MeSTP15 cassava double mutants, CRISPR/Cas9 double gene editing vectors for MeSTP7 and MeSTP15 were successfully constructed by designing the sgRNAs of both target genes, MeSTP7 and MeSTP15, using the online software CRISPR-P v2.0. After transformation of the editing vector into Agrobacterium tumefaciens LBA4404, ‘SC8’ cassava brittle embryonic healing tissues were infiltrated, and MeSTP7 and MeSTP15 were successfully edited by Sanger sequencing analysis, while the potential off-target sites were not edited, indicating that the dual editing vector could edit both MeSTP7 and MeSTP15 genes without causing off-target phenomenon. The rooted seedlings were then screened with 15 mmol/L Hygromycin B to produce rooted seedlings, which were then characterized detection to obtain positive transgenic cassava plants with the expression frame of the double-edited vector. The genomes of positive plants were used as the templates for PCR amplification of 100 bp nucleotide sequences before and after each target site of the two genes, and after Hi-TOM sequencing, the results showed that there were 26 strains with mutations, including 23 MeSTP7/15 double mutants, 2 MeSTP7 single mutants and 1 MeSTP15 single mutant, and the editing types were mostly single. The types of editing were mostly single base deletions or insertions, with a small percentage of deletions of large segments of bases. Our preliminary observations of the mutant variants in histoponic flasks revealed that root growth was inhibited and plants were dwarfed in both the single and double mutants, and that the MeSTP7 and MeSTP15 double mutants were more severely damaged. These results not only lay the foundation for further analysis of the mechanism of cassava tuber development, but also provide material for obtaining new germplasm for cassava disease and stress resistance.

Key words： cassava; hexose transport proteins; CRISPR/Cas9; double gene editing

Cite this article

GENG Sha , ZHANG Jianyu , WANG Xiaotong , REN Siyang , WU Zhihao , YAO Yuan , LI Ruimei , GUO Jianchun , LIU Jiao , LUO Lijuan . Construction of MeSTP7 and MeSTP15 Double Mutants in Cassava Based on CRISPR/Cas9 Technology[J]. Chinese Journal of Tropical Crops, 2022 , 43(3) : 463 -472 . DOI: 10.3969/j.issn.1000-2561.2022.03.004

木薯（Manihot esculenta Crantz）是一种热带、亚热带重要的粮食与能源作物[1]。木薯产量与其块根发育和逆境适应密切相关[2]。在植物中,单糖可作为植物生命活动的能量供给,还可作为信号分子与激素协同作用、渗透保护剂、抗氧化剂等影响植物的生长发育[3,4]。而糖类物质的运输由糖外排转运蛋白（SWEETs）、蔗糖转运蛋白（SUTs）、单糖转运蛋白（MSTs）三类蛋白负责运输[5]。己糖转运蛋白（STPs）属于MSTs亚家族,具有典型的12次跨膜结构域,是质膜上H+/糖共转体,不同的己糖转运蛋白STPs具有的氨基端和羧基端跨膜蛋白序列有很高的同源性,但在一些结构域有保守的氨基酸序列,其功能并不一致[6,7]。研究表明STPs主要参与植物库器官的发育和库强的构建,还参与植物的开花结果、信号传导、生物和非生物胁迫等多种生理过程[8,9]。为研究MeSTPs在木薯块根发育和非生物胁迫中的作用,本实验室前期在木薯中成功鉴定出20个己糖转运蛋白基因MeSTPs。对其组织特异性和木薯块根发育过程中的表达模式分析,发现其中MeSTP7（Manes.03G180400）和MeSTP15（Manes. 15G027300）在早期贮藏根的根尖中高表达[10],且受干旱胁迫和低温胁迫诱导表达上调最高[11,12],推测MeSTP7和MeSTP15基因参与块根发育时期的糖分累积和应对非生物胁迫过程中起到重要作用。此外,MeSTP7和MeSTP15有相似的糖转运功能,对葡萄糖、戊糖、木糖、核糖、半乳糖、果糖和甘露糖这7种己糖都有转运特性[10]。
MeSTP7和MeSTP15的蛋白序列相近,功能可能存在冗余,为更好地解析这2个基因功能,对其同时进行基因编辑,创制MeSTP7和MeSTP15双突变体。CRISPR/Cas9基因编辑技术不仅编辑效率高、稳定和特异性强,还能同时编辑多个基因[13],被广泛地应用到医学研究和生命科学领域,目前用于提高作物的抗性及营养价值,已在玉米、水稻、木薯等植物中应用[14,15]。在木薯中,利用CRISPR/Cas9双基因编辑技术同时介导编辑木薯eIF4E异构体nCBP-1和nCBP-2,可降低木薯褐条病症状严重程度和发病率[16],为本研究提供了设计思路。本研究利用CRISPR/Cas9技术构建木薯MeSTP7和MeSTP15双基因编辑载体,转化‘华南8号’（SC8）木薯脆性胚性愈伤,验证载体的编辑效果,获得了MeSTP7和MeSTP15双基因突变体,有助于后续深入解析MeSTPs在木薯块根发育过程中的作用。

1 材料与方法

1.1 材料

本研究选用具有适应性强、抗风性强、出苗率高的木薯品种‘华南8号’（SC8）,种植于中国热带农业科学院木薯种质资源圃。菌株LBA4404根癌农杆菌菌株、DH5α大肠杆菌菌株购于上海唯地生物技术有限公司,载体质粒pCAMBIA1301-Cas9-sgRNA为本实验室所保存。

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MeSTP7（Manes.03G180400）和MeSTP15（Manes.15G027300）基因的CDS序列,利用CRISPR-P v2.0在线软件（ http://crispr.hzau.edu.cn/cgi-bin/CRISPR2/CRISPR）分析CDS区靶点及潜在脱靶位点,选择满足以下条件的靶点：靶点得分高于0.6,相对应脱靶位点得分低;靶点GC含量为50%~70%;sgRNA中至少含有完整的茎环RAR、茎环2、茎环3。合成靶点退火引物（表1）,参照李崭等[17]的方法构建MeSTP7和MeSTP15双基因编辑载体。根据载体序列,在AtU6-26启动子前设计阳性克隆F,在gRNA2后设计阳性克隆R,通过菌液PCR扩增目的片段（538 bp）,将测序正确的质粒命名为pCAMBIAP1301-Cas9- MeSTP7/15-sgRNA,并将该质粒转化LBA4404农杆菌,菌液PCR验证正确后备用。
表1 本研究所用引物

Tab. 1 Primers for this study

引物
Primer		 上游序列（5°-3°）
Upstream sequence (5°-3°)		 下游序列（5°-3°）
Downstream sequence (5°-3°)		 产物大小
Product size/bp		 用途
Use
靶点1退火 CACCGGCACCCACAAGAATCCGTGGAGG CGTGGGTGTTCTTAGGCACCTCCCAAA 25 载体构建
靶点2退火 CACCGACACCACTAGAAAGTTAGGCAGG TGTGGTGATCTTTCAATCCGTCCCAAA 25 载体构建
阳性克隆筛选 TAATCATCGCAAGACCGGCA CCTGTACGAGACACGGATCG 538 PCR检测
target-1 TCATCTCACAGGCACGTGGT CTGGGCTTACTTTGCAGTCCC 217 编辑效果分析
target-2 CAAGGGTTGCAATTGTTCACATCT CTGATTAGCAAAACCGACACCAC 219 编辑效果分析
Off-target-1 TGATTTTCTTGCTCATCACACA AGCTTGGTGAGCTAAGATTG 384 脱靶分析
Off-target-2 TATTTCTCGAGAGGTGCTTGC TGAGCGTTGGATTGACTGCC 368 脱靶分析
Off-target-3 TGCTCCAACATATTCCTTACGA CCTAGGTGAATCACGAGGCA 393 脱靶分析
Off-target-4 CTGCGACTCCAAAGAGACAGA ACTGGAAGCTGCCAATATGAG 363 脱靶分析
1.2.2 木薯脆性胚性愈伤组织的诱导及侵染转化 将鉴定正确的pCAMBIAP1301-Cas9-MeSTP7/15- sgRNA质粒的LBA4404农杆菌侵染木薯的脆性愈伤组织,将农杆菌摇菌至对数,调整侵染浓度为OD600为0.65,乙酰丁香酮浓度为250 mmol/L,侵染时间为40 min。侵染后的脆性胚性愈伤组织转移至GD固体培养基上,在22℃黑暗条件培养3 d后洗菌。然后移至GD固体培养基上,第1周不加潮霉素筛选,从第2周开始每周更换一次潮霉素筛选压（5、8、15 mg/L潮霉素）,培养4周后,移至培养基（MS+1 mg/L NAA）诱导子叶,子叶在生根（MS+15 mg/L潮霉素）筛选后再经过DNA鉴定,确定阳性植株。
1.2.3 编辑效果检测及脱靶分析 根据张彤等[18]的方法,设计MeSTP7和MeSTP15 CDS区编辑靶点和4个潜在脱靶位点区域的PCR扩增引物（表1）。提取未侵染的木薯脆性胚性愈伤组织DNA和含有pCAMBIAP1301-Cas9-MeSTP7/15-sgRNA质粒农杆菌侵染后的木薯脆性胚性愈伤组织DNA,用MeSTP7靶点引物（target-1）和MeSTP15靶点引物（target-2）PCR扩增获得目的片段并测序。基于Sanger测序峰值图,分析编辑效果及验证是否存在脱靶情况。
1.2.4 阳性苗的鉴定及突变体的鉴定 提取经过潮霉素生根筛选的再生植株叶片的DNA,用阳性克隆筛选引物,PCR扩增包含AtU6-26、靶点1、gRNA1、靶点2、gRNA2载体片段,凝胶电泳片段大小为538 bp的则为转基因苗。提取阳性苗的DNA,用MeSTP7靶点引物（target-1）和MeSTP15 靶点引物（target-2）PCR扩增基因靶点前后100 bp,送高通量测序（Hi-TOM测序）,检测靶点是否发生插入或缺失,分析是否导致基因突变。

2 结果与分析

2.1 双基因编辑靶点的设计及潜在脱靶位点分析

利用木薯基因MeSTP7和MeSTP15的DNA序列,通过在线设计软件CRISPR-P2.0,获得MeSTP7基因编辑靶点位于该基因的第3个外显子,命名靶点1（图1）,GC含量为60%,有1个潜在脱靶位点,位于10号染色体Manes. 10G030300基因的CDS区,脱靶分值为0.106,命名为Off-target-1;MeSTP15基因编辑靶点位于该基因的第2个外显子,命名靶点2（图1）,GC含量为48%,有3个潜在脱靶位点,Off-target-2位于10号染色体Manes.10G105000基因的内含子,脱靶分值为0.312,Off-target-3位于号染色体Manes.08G047800基因的内含子,脱靶分值为0.115,Off-target-4位于1号染色体Manes. 01G204800基因的内含子,脱靶分值为0.092。潜在脱靶位点只有Off-target-1位于CDS区且得分较低,其余脱靶位点位于内含子,表明即使脱靶也不会造成基因功能的缺失（表2）。对靶点1和靶点2的sgRNA的二级结构预测分析结果显示,靶点序列二级结构较为松散,含有完整的茎环RAR、茎环2、茎环3,利于结合靶位点（图2）。
图1 基因编辑靶点位置

灰色框：靶点序列;红色字母：PAM区。

Fig. 1 Location of gene editing target

Gray boxes: Target sequence; Red letters: PAM area.

表2 基因编辑靶点和潜在脱靶位点

Tab. 2 Editing target and potential off-target sites

位点
Site		 序列（5°-3°）
Sequence (5°-3°)		 编辑靶点或潜在脱靶位点得分
Score of editing or off-target potential		 错配数量
Number of
mismatch		 基因
Gene		 区域
Region
Target-1 GCACCTACAAGAATCCGTGGAGG 0.642 MeSTP7 CDS
Target-2 ACACCACTAGAAAGTTAGGCAGG 0.697 MeSTP15 CDS
Off-target-1 CAAACCACAAGAATCCATGGAAG 0.106 4 Manes.10G030300 CDS
Off-target-2 ATATAAATAGAAAGTTAGGCTAG 0.312 5 Manes.10G105000 Intron
Off-target-3 AATACACTAGAAATTTAGGCAGG 0.115 3 Manes.08G047800 Intron
Off-target-4 ACACCACTAGAAAGAGAATCAGG 0.092 4 Manes.01G204800 Intron
图2 sgRNA二级结构预测

Fig. 2 Prediction secondary structure of sgRNA

2.2 基因编辑载体构建

本研究所采用的基因编辑载体为pCAMBIA1301-Cas9-sgRNA。利用限制性内切酶BsaⅠ酶切将载体线性化,再将线性化的质粒与退火后的靶点引物连接,得到AtU6-26-MeSTP7/15-sgRNA表达盒（图3）。通过菌液PCR扩增目的条带,经测序分析,长约538 bp,进一步证明载体pCAMBIA1301-Cas9-MeSTP7/15-sgRNA已构建成功。最后提取阳性质粒转LBA4404农杆菌,备用。
图3 pCAMBIA1301-Cas9-MeSTP7/15-sgRNA载体构建

A：pCAMBIA1301-Cas9-STPs1-sgRNA载体T-DNA图;B：重组质粒菌液PCR检测,M：DL2000 DNA marker;1~7：单克隆;8：阴性对照;9：阳性对照。

Fig. 3 T-DNA map of pCAMBIA1301-Cas9-MeSTP7/15-sgRNA vector

A: T-DNA map of pCAMBIA1301-Cas9-STPs1-sgRNA vector; B: PCR assay of recombinant plasmid bacterial solution, M: DL2000 DNA marker; 1-7: Monoclones; 8: Negative control; 9: Positive control.

2.3 木薯脆性胚性愈伤组织的诱导及转化

将培养2个月的‘SC8’无菌苗切茎段,诱导侧芽膨大,挑取侧芽诱导体细胞胚,体细胞胚诱导得到脆性胚性愈伤组织（图4A、图4B、图4C）。将携带编辑质粒pCAMBIA1301-Cas9- MeSTP7/15-sgRNA的LBA4404农杆菌转化木薯脆性胚性愈伤组织,转化后培养脆性胚性愈伤组织4周,再诱导出子叶,诱导子叶分化形成植株,在MS+15 mg/L潮霉素的培养基中筛选再生根苗（图4D、图4E、图4F）。
图4 木薯脆性胚性愈伤组织诱导及遗传转化过程

A：茎段腋芽膨大;B：腋芽诱导出的体细胞胚;C：脆性胚性愈伤组织;D：侵染后的脆性胚性愈伤组织;E：侵染后脆性胚性愈伤组织诱导子叶;F：再生苗。

Fig. 4 Induction and genetic transformation process of cassava friable embryogenic calli

A: Swelling oflateral buds on the stem segment; B: Somatic embryos induced by lateral buds; C: Friable embryogenic calli; D: Transformed friable embryogenic calli; E: Cotyledon induction from transformed friable embryogenic calli; F: Regrowth.

2.4 编辑效果检测及脱靶分析

以未转化和转化4周后的脆性胚性愈伤组织DNA为模板,通过PCR扩增MeSTP7和MeSTP15基因的编辑靶点上下100 bp左右的基因组片段,并测序分析该区域的序列变化情况。结果显示,在转化后的样品中,MeSTP7靶点（Target-1）的PAM区附近开始出现多峰,MeSTP15靶点（Target-2）的PAM区及后续片段都发生了移码,峰值错乱,然而未转化样品中并无杂锋出现（图5）,表明携带编辑质粒的农杆菌成功转化进入脆性胚性愈伤组织,在2个靶点处发生编辑。再以未转化和转化4周后的木薯脆性胚性愈伤组织DNA为模板,通过PCR扩增MeSTP7和MeSTP15基因编辑靶点的4个潜在脱靶位点区域片段,利用Sanger测序分析该靶点是否存在脱靶现象。结果表明,潜在脱靶位点的序列在已转化和未转化的样品中未发生改变（图6）。说明本研究构建的双基因编辑载体pCAMBIA1301-Cas9-MeSTP7/ 15- sgRNA可对MeSTP7和MeSTP15基因同时进行编辑,均无脱靶现象。
图5 基因编辑效果分析

红色框：PAM区;黑色线条：靶点序列;红色箭头：编辑位点。

Fig. 5 Analysis of gene editing effect

Red box: PAM area; Black line: Target sequence; Red arrows: Edit loci.

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;红色框：PAM区;黑色下划线：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-3 sequencing; D: Peak map of Off-target-4 sequencing; Red box: PAM area; Black line: Target sequence.

2.5 阳性苗筛选及突变体编辑分析

将鉴定发生编辑的木薯脆性胚性愈伤组织诱导产生子叶,子叶诱导成苗,经过15 mg/L的潮霉素生根筛选,提取生根苗的DNA进行PCR检测,成功得到带有编辑表达框的阳性植株（图7）。阳性苗Hi-TOM测序检测是否发生编辑,于‘SC8’比对分析,2个靶点的编辑类型主要是单个碱基的缺失或插入,大片段的编辑类型较少;突变类型有纯和突变、杂合突变及双等位突变的多种突变形式,编辑率比对发现,MeSTP7靶点Target-1编辑效率明显高于MeSTP15靶点Target-2,可能和靶点在载体中的前后位置有关。本研究一共筛选到26个突变体,获得MeSTP7单突变体mestp7-1和mestp7-2,MeSTP15的单基因突变体mestp15-1,其余的株系为不同类型的双基因突变体（表3）。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MeSTP7和MeSTP15基因CDS区进行编辑,造成基因序列的突变,得到不同编辑类型的突变体,为后续实验提供了丰富的材料。
图7 mestp7/15突变体PCR检测

M：DL2000 DNA marker;1~19：单克隆; 20：阴性对照;21：阳性对照。

Fig. 7 PCR detection of mestp7/15 mutant

M: DL2000 DNA marker; 1-19: Monoclonal; 20: Negative control; 21: Positive control.

表3 mestp7/15 突变体与SC8野生型DNA靶点序列比对

Tab. 3 mestp7/15 mutant comparison with SC8 wild-DNA Target sequences

注:阳性植株编辑位点的PCR产物测序序列与野生型(WT)靶点序列的比对结果,小写字母为剪切位置,D表示缺失,I表示插人,WT为野生型。

Noto: Comparison of sequenced sequences of PCR products from the editing site of positive plants with wild-type (WT) target sequences, lowercase letters are cut positions, D indicates deletion, I indicates insertion, WT is wild-type.

2.6 突变体表型分析

MeSTP7/15突变体茎段培养一个月后,与野生型（SC8）相比,所有株系均表现出生长缓慢,植株矮小,根系生长受抑制。MeSTP7单突变体mestp7-1与MeSTP15的单基因突变体mestp15-1相比,株高稍高但根系发育相对缓慢;推测MeSTP7主要在根中发挥作用;而双突变体mestp7/15-4、mestp7/15-24株系相比于野生型和单突变体,根系发育严重受损,植株低矮,叶片窄小。同时观察到mestp7/15-24的根系受损比mestp7/15-4严重,推测由于mestp7/15-24突变比率高于mestp7/15-4（图8）。初步证明MeSTP7和MeSTP15功能存在冗余,影响木薯根的发育。
图8 部分突变体组培苗

A: SC8; B: mestp7-1; C: mestp15-1; D: mestp7/15-4; E: mestp7/15-24.

Fig. 8 Phenotypes of some mutant histoculture seedlings

3 讨论

植物进行光合作用,在叶片中合成糖类物质,这些糖类物质从源叶组织经过糖转运体运输、转运到下沉库组织利用和储存。这一过程对植物应对非生物胁迫和生物胁迫至关重要[19]。因此,了解糖分配及其遗传调控对于实现突破以提高作物产量和非生物胁迫耐受性至关重要。KOMAITIS等[20]在蒺藜苜蓿中研究发现MSTs家族通过调控糖分的运输,进而影响苜蓿根瘤菌对氮的固定。董元花[21]在苹果中发现过表达MdSTP1后,促进植株根的生长发育,同时抗逆性降低。拟南芥中,利用RNAi干扰沉默AtSTP1和AtSTP4后,切断对保卫细胞的能量供应,抑制光合作用[22]。Pro:AtSTP12定位于拟南芥侧根结点处,和侧根生长相关[23]。过表达AtSTP13后增加拟南芥叶片光合作用,从而提高氮的利用率,并且能抑制灰霉病[24,25]。在水稻中,OsSTP4、OsSTP11、OsSTP14、OsSTP19和OsSTP28在盐、渗透性和干旱胁迫下高表达[26]。在小麦中,小麦条锈病可刺激宿主细胞中的ABA生物合成,从而上调TaSTP3、TaSTP6和TaSTP13的表达,从而增加真菌己糖的供应,促进感染[27],在大麦中发现Lr67res突变体（自然突变的HvSTP13）,削弱了己糖转运活性,对锈病和白粉病有一定的抗性,反义沉默TaSTP6也能抑制条锈病的感染[28]。
CRISPR/Cas9是一项特异识别编辑技术,仅需gRNA引导序列和Cas9核酸酶即可对靶基因DN序列进行剪切编辑,CRISPR/Cas9双基因编辑已经使用于多个物种[29]。为研究MeSTP7和MeSTP15的生物学功能,利用CRISPR/Cas9技术分别设计MeSTP7和MeSTP15的靶位点,将2个靶位点串联,构建双基因编辑载体,转化木薯胚性脆性愈伤组织,一个月后检测靶点的编辑效率,证明本研究构建的双基因编辑载体pCAMBIA1301-Cas9-MeSTP7/15-sgRNA可对MeSTP7和MeSTP15基因同时编辑,无脱靶现象,说明本研究选择的2个靶位点的编辑效率高。将编辑后的脆性胚性愈伤组织诱导成苗,经过15 mg/L潮霉素生根筛选和DNA分子检测得到阳性苗,再将单株阳性苗Hi-TOM测序序列比对分析,成功得到26个突变体,其中有23株双突变体,2株MeSTP7突变体,1株MeSTP15突变体,突变体的编辑类型有纯和突变、杂合突变、双等位突变等多种编辑类型,2个靶位点的突变类型多为单碱基插入或缺失突变,小片段突变率较少,这与前人报道的编辑现象基本相似[30,31]。对突变体的表型初步观察,发现单突变体和双突变体的根系生长均受抑制,植株矮小,这与前人报道的表型相似[21]。初步确定MeSTP7和MeSTP15的功能存在冗余,MeSTP7和MeSTP15均影响木薯的根系发育,但mestp7-1突变体根系发育更迟缓,MeSTP7主要在根顶端分生组织中表达,MeSTP15在须根中特异表达,推测MeSTP7主要在根系形成发挥作用,而MeSTP15在根系发育中起作用[10]。
本研究通过构建CRISPR/Cas9介导的双基因编辑载体,对MeSTP7和MeSTP15基因同时进行编辑。初步确定MeSTP7和MeSTP15对木薯根系发育的影响。为后续研究块根发育机制提供了丰富的材料,而STPs家族在植物应对非生物胁迫和抗击病原菌中也起到关键作用,推测突变体在应对非生物胁迫和抵抗病原菌起作用。本研究对其功能分析尚浅,后续将在大田里筛选可稳定遗传的株系作为研究材料,分析不同阶段的木薯根系形态及地上部分的生长情况,检测块根淀粉和糖类物质含量,分析MeSTP7和MeSTP15基因对植物生长发育的影响;对其突变体进行干旱、盐和低温胁迫并分析其抗逆性,解析MeSTP7和MeSTP15基因在非生物胁迫中的作用;接种木薯的主要病原菌：细菌性枯萎病、花叶病、褐斑病、锈病和白粉病,分析突变体对这些病害是否有抗病性。

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