马铃薯川芋50遗传转化及基因编辑体系的建立

doi:10.13560/j.cnki.biotech.bull.1985.2024-1178

生物技术通报 ›› 2025, Vol. 41 ›› Issue (4): 88-97.doi: 10.13560/j.cnki.biotech.bull.1985.2024-1178

马铃薯川芋50遗传转化及基因编辑体系的建立

文博霖¹(), 万敏¹^,², 胡建军³^,⁴, 王克秀³^,⁴, 景晟林¹^,², 王心悦¹, 朱博¹^,², 唐铭霞³^,⁴, 李兵³^,⁴, 何卫³^,⁴, 曾子贤¹^,²()

^1.四川师范大学生命科学学院，成都 610101
^2.四川师范大学植物功能基因组及生物信息学研究中心，成都 610101
^3.四川省农业科学院作物研究所（四川省种质资源中心），成都 610066
^4.粮油作物绿色种质创新与遗传改良四川省重点实验室，成都 610066

收稿日期:2024-12-05 出版日期:2025-04-26 发布日期:2025-04-25
通讯作者: 曾子贤，男，教授，研究方向：马铃薯功能基因组学、表观基因组学及生物信息学；E-mail: zengzixian@sicnu.edu.cn
作者简介:文博霖，男，硕士研究生，研究方向：马铃薯功能基因组学；E-mail: w1666425267@163.com
基金资助:
国家自然科学基金面上项目(32170573);“十四五”四川省科技厅薯类育种攻关项目(2021YFYZ0019)

Establishment of Genetic Transformation and Gene Editing System for a Potato Cultivar Chuanyu 50

WEN Bo-lin¹(), WAN Min¹^,², HU Jian-jun³^,⁴, WANG Ke-xiu³^,⁴, JING Sheng-lin¹^,², WANG Xin-yue¹, ZHU Bo¹^,², TANG Ming-xia³^,⁴, LI Bing³^,⁴, HE Wei³^,⁴, ZENG Zi-xian¹^,²()

^1.Department of Biological Science, College of Life Sciences, Sichuan Normal University, Chengdu 610101
^2.Plant Functional Genomics and Bioinformatics Research Center, Sichuan Normal University, Chengdu 610101
^3.Crop Research Institute of Sichuan Academy of Agriculture Sciences (Sichuan Germplasm Resource Center), Chengdu 610066
^4.Environment-friendly Crop Germplasm Innovation and Genetic Improvement Key Laboratory of Sichuan Province, Chengdu 610066

Received:2024-12-05 Published:2025-04-26 Online:2025-04-25

摘要/Abstract

摘要：

目的构建马铃薯川芋50的遗传转化体系和基因编辑体系。方法以四川省主导品种川芋50无菌脱毒组培苗茎段和叶片为外植体，用农杆菌介导法将含有植物敲除载体质粒pJCV55-StU6-200-StUBI10-T#01转化马铃薯外植体，分析4种不同植物激素配比的培养基体系对马铃薯愈伤组织诱导和分化的影响，探究川芋50的基因编辑、阳性植株鉴定方法。结果 1）最适马铃薯川芋50遗传转化的培养基体系为B体系。茎段和叶片预培养及愈伤组织诱导的培养基配方分别为：MS 基础盐+20 g/L蔗糖+1 mL/L 1 000× N&N维生素（1 000× Nitsch & Nitsch Vitamin Solution, 1 000× N&N维生素）+1.0 mg/L TZR（trans-zeatin-riboside, TZR）+0.027 8 mg/L GA₃（gibberellin A3, GA₃）+0.02 mg/L NAA（1-naphthaleneacetic acid, NAA）+2.0 g/L Phytagel和MS基础盐+20 g/L蔗糖+1 mL/L 1 000× N&N维生素+0.5 mg/L TZR+2.5 mg/L IAA（indole acetic acid, IAA）+2.0 g/L Phytagel，愈伤诱导率分别为93%和88%。愈伤组织分化的培养基为：MS基础盐+20 g/L蔗糖+1 mL/L 1 000× N&N维生素+2.0 mg/L TZR+10 mg/L GA₃+2.0 g/L Phytagel；2）证明了二次生根筛选法可准确鉴定马铃薯转基因阳性植株，准确率100%；3）建立了针对川芋50的农杆菌介导CRISPR/Cas9基因编辑体系，编辑率63%。结论通过对愈伤再生过程中各个影响因素进行试验以及利用农杆菌介导的CRISPR/Cas9技术进行编辑，建立了马铃薯川芋50的遗传转化体系和基因编辑体系。

关键词: 马铃薯, 川芋50, 根癌农杆菌, 遗传转化, 基因编辑, CRISPR/Cas9

Abstract:

Objective To construct a genetic transformation system and gene editing system of potato Chuanyu 50. Method Aseptic histocultured seedling stems and leaves of Chuanyu 50, the main potato (Solanum tuberosum L.) variety promoted in Sichuan province, were used as explants. The potato explants were transformed with Agrobacterium-mediated transformation containing the plant CRISPR/Cas9 vector pJCV55-StU6-200-StUBI10-T#01. Four different media with various phytohormone ratios were analyzed and screened for callus induction and differentiation. CRISRR-Cas9-based editing method was applied for creating mutants. Result 1) The optimal medium among the tested ones for genetic transformation Chuanyu 50 was B system. The medium formulation for stem and leaf pre-cultivation and callus induction, respectively, were as follows: MS basic salt+20 g/L sucrose +1 mL/L 1 000× N&N vitamins+1.0 mg/L TZR(trans-zeatin-riboside,TZR)+0.027 8 mg/L GA3(gibberellin A3,GA3)+0.02 mg/L NAA(1-naphthaleneacetic acid,NAA)+2.0 g/L phytagel, and MS basic salt+20 g/L sucrose +1 mL/L 1 000× N&N vitamins+0.5 mg/L TZR+2.5 mg/L IAA(indole acetic acid,IAA)+2.0 g/L phytagel, the callus induction rates using stem and leaf were 93% and 88%, respectively. The medium for differentiation was MS basic salt+20 g/L sucrose+1 mL/L 1 000× N&N vitamins+2.0 mg/L TZR+10 mg/L GA3+2.0 g/L phytagel. 2) It is demonstrated that the two-round rooting screening method accurately identified transgenic-positive plants with a 100% accuracy. 3) An Agrobacterium-mediated CRISPR/Cas9 gene editing system for Chuanyu 50 with 63% editing rate was established. Conclusion The genetic transformation and gene editing system of potato Chuanyu 50 are initially established and multiple factors affecting the process of callus induction and regeneration are evaluated.

Key words: potato, Chuanyu 50, Agrobacterium tumefaciens, genetic transformation, gene editing, CRISPR/Cas9

文博霖, 万敏, 胡建军, 王克秀, 景晟林, 王心悦, 朱博, 唐铭霞, 李兵, 何卫, 曾子贤. 马铃薯川芋50遗传转化及基因编辑体系的建立[J]. 生物技术通报, 2025, 41(4): 88-97.

WEN Bo-lin, WAN Min, HU Jian-jun, WANG Ke-xiu, JING Sheng-lin, WANG Xin-yue, ZHU Bo, TANG Ming-xia, LI Bing, HE Wei, ZENG Zi-xian. Establishment of Genetic Transformation and Gene Editing System for a Potato Cultivar Chuanyu 50[J]. Biotechnology Bulletin, 2025, 41(4): 88-97.

图/表 9

表1 试验用培养基

Table 1 Media used in the study

培养基体系 Medium system	培养基 Medium	培养基成分 Medium composition	pH
A	A1	MS基础盐+20 g/L 蔗糖+1 mL/L 1 000× N&N维生素+2.49 mg/L TZR+0.027 8 mg/L GA3+0.2 mg/L NAA+2 g/L Phytagel	5.7
A	A2	MS基础盐+20 g/L 蔗糖+1 mL/L 1 000× N&N维生素+2.49 mg/L TZR+1.0 mg/L NAA+2.0 g/L Phytagel	5.7
B	BS（茎段）	MS基础盐+20 g/L 蔗糖+1 mL/L 1 000× N&N维生素+1.0 mg/L TZR+0.027 8 mg/L GA3+0.02 mg/L NAA+2.0 g/L Phytagel	5.7
	BL（叶片）	MS基础盐+20 g/L 蔗糖+1 mL/L 1 000× N&N 维生素+0.5 mg/L TZR+2.5 mg/L IAA+2.0 g/L Phytagel	5.7
	B2	MS基础盐+20 g/L 蔗糖+1 mL/L 1 000× N&N维生素+2.0 mg/L TZR+10 mg/L GA3 +2.0 g/L Phytagel	5.7
C	C1	MS基础盐+20 g/L 蔗糖+1 mL/L 1 000× N&N维生素+1.0 mg/L TZR+2.0 mg/L NAA+2.0 g/L Phytagel	5.7
C	C2	MS基础盐+20 g/L 蔗糖+1 mL/L 1 000× N&N维生素+0.5 mg/L GA3+2.0 mg/L 6-BA +0.02 mg/L NAA+2.0 g/L Phytagel	5.7
D	D	MS基础盐+20 g/L 蔗糖+1 mL/L 1 000× N&N维生素+4.78 mg/L TZR+0.2 mg/L GA3 +0.01 mg/L IAA+2.0 g/L Phytagel	5.7
	MS 液体 Liquid MS	4.33 g/L MS基础盐（Phyto NO.524）+ 30 g/L蔗糖	5.8
	生根培养基 Rooting medium	4.74 g/L MS培养基（不含琼脂和蔗糖）+30 g/L蔗糖+2.0 g/L植物凝胶（Phytagel）	5.6

图1 川芋50茎段和叶片的预培养

Fig. 1 Preculture of Chuanyu 50 stem segments and leaves

图2 川芋50茎段和叶片的愈伤诱导情况

Fig. 2 Callus induction of Chuanyu 50 stem segments and leaves

表2 不同培养基对茎段和叶片愈伤组织形成和分化的影响

Table 2 Effects of different media on the callus formation and differentiation of stem segments and leaves

外植体类型 Explant type	培养基体系 Media system	外植体数 Number of explants	愈伤数 Number of calluses	诱导率 Induction rate/%	分化率 Differentiation rate/%
茎段 Stem	A	100	90	90	9
	B	100	93	93	25
	C	100	65	65	0
	D	100	43	43	0
叶片 Leaf	A	50	40	80	0
	B	50	44	88	22
	C	50	29	58	0
	D	50	31	62	0

图3 川芋50茎段和叶片培养50 d后的愈伤分化情况

Fig. 3 Callus differentiation of Chuanyu 50 stem segments and leaves upon 50-day treatment

图4 gRNA位置与基因编辑载体结构A：gRNA在T#01上的位置及测序验证；B：pJCV55-StU6-200-StUBI10-T#01载体示意图。KanR ：卡那霉素抗性基因；StUBI10：马铃薯泛素10启动子；StU6：马铃薯 U6 snoRNA启动子；NosT：胭脂碱合酶终止子；LB：T-DNA左边界；RB：T-DNA右边界

Fig. 4 gRNA location and structure of gene editing vectorA: Position of the gRNAs on T#01 and sequencing validation. B: Schematic diagram of pJCV55-StU6-200-StUBI10-T#01. KanR : Kanamycin resistance gene; StUBI10: potato ubiquitin 10 promoter; StU6: potato U6snoRNA promoter; NosT: nopaline synthase terminator; LB: T-DNA left border; RB: T-DNA right border

图5 转基因马铃薯株系的PCR检测M：100 bp plus DNA marker；#1-#40：转基因马铃薯株系；WT：阴性对照（野生型）；CK：空白对照（ddH2O）；P：阳性对照（质粒pJCV55-StU6-200-StUBI10-T#01）

Fig. 5 PCR detection of transgenic potato linesM: 100 bp plus DNA marker; #1-#40: transgenic potato lines; WT: negative control (wild type); C: blank control (ddH2O);P: positive control (plasmid pJCV55-StU6-200-StUBI10-T#01)

图6 T#01突变体PCR产物的序列分析

Fig. 6 Sequence analysis of PCR products from the T#01 mutants

图7 转基因马铃薯株系突变位点和类型#1、#12、#19的突变序列与野生型序列进行了比对。gRNA1区域突变类型/gRNA2区域突变类型，插入（+）或缺失（-）用红色表示。PAM位点用蓝色表示。gRNA 位置用紫色表示

Fig. 7 Mutated sites and types in transgenic potato linesMutant sequences of #1, #12, and #19 were aligned with wild-type sequences. gRNA1 region mutation type/gRNA2 region mutation type with insertion (+) or deletion (-) in red. PAM sites are indicated in blue. The gRNA positions are indicated in purple

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马铃薯川芋50遗传转化及基因编辑体系的建立

Establishment of Genetic Transformation and Gene Editing System for a Potato Cultivar Chuanyu 50

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