园艺学报 ›› 2023, Vol. 50 ›› Issue (5): 985-999.doi: 10.16420/j.issn.0513-353x.2022-0273
李国斌, 蔡梁玉, 肖立成, 王家发, 张得迪, 张俊红()
收稿日期:
2022-11-27
修回日期:
2023-01-16
出版日期:
2023-05-25
发布日期:
2023-05-31
通讯作者:
*(E-mail:zhangjunhng@hzau.edu.cn)基金资助:
LI Guobin, CAI Liangyu, XIAO Licheng, WANG Jiafa, ZHANG Dedi, ZHANG Junhong()
Received:
2022-11-27
Revised:
2023-01-16
Published:
2023-05-25
Online:
2023-05-31
摘要:
以栽培番茄品种(系)Ailsa Craig(AC)、B5和B9为试材,利用CRISPR/Cas9双元表达载体系统,对番茄品质调控关键基因SlGLK2和SlMYB12进行定点敲除。获得AC背景下SlGLK2和SlMYB12的基因编辑植株各9株,B5背景下SlGLK2的基因编辑植株3株,B9背景下SlMYB12的基因编辑植株4株。通过对靶点测序发现,在靶位点的PAM上游3 ~ 4 bp处发生了不同形式的碱基缺失、插入或替换,主要以1 ~ 10 bp的缺失和单碱基G和T的插入为主;也产生了两靶点间的大片段缺失,最大片段为334 bp。AC背景下敲除SlMYB12产生粉色果实,敲除SlGLK2基因,果实肩部的绿色明显变浅,B5背景下的3株基因编辑番茄均无“绿肩”。本研究中利用CRISPR/Cas9技术在不同番茄种质中成功创制了敲除SlGLK2或SlMYB12的基因编辑材料,以期为番茄品质育种提供优良种质。
中图分类号:
李国斌, 蔡梁玉, 肖立成, 王家发, 张得迪, 张俊红. 利用CRISPR/Cas9技术创制番茄粉果和无绿肩材料[J]. 园艺学报, 2023, 50(5): 985-999.
LI Guobin, CAI Liangyu, XIAO Licheng, WANG Jiafa, ZHANG Dedi, ZHANG Junhong. Creating Pink Fruit and Fruit Without Green Shoulder Using CRISPR/ Cas9 Technology in Tomato[J]. Acta Horticulturae Sinica, 2023, 50(5): 985-999.
基因 Gene | sgRNA 靶标序列 Target sequence of sgRNA |
---|---|
SlGLK2-sgRNA1 | TGGTGTCGATTAAATTCCCA |
SlGLK2-sgRNA2 | TGAATAGCAGGTTCTTCACA |
SlMYB12-sgRNA1 | AACACCTTGTTGTGAAAAA |
SlMYB12-sgRNA2 | CCAAGAGCCTTCTCCATTAG |
表1 SlGLK2和SlMYB12的靶标序列
Table 1 Target sites of SlGLK2 and SlMYB12 gene
基因 Gene | sgRNA 靶标序列 Target sequence of sgRNA |
---|---|
SlGLK2-sgRNA1 | TGGTGTCGATTAAATTCCCA |
SlGLK2-sgRNA2 | TGAATAGCAGGTTCTTCACA |
SlMYB12-sgRNA1 | AACACCTTGTTGTGAAAAA |
SlMYB12-sgRNA2 | CCAAGAGCCTTCTCCATTAG |
引物 Primer | 用途 Purpose | 序列(5′-3′) Sequence |
---|---|---|
SlGLK2-CRISPR | 构建载体Construction vector | F:GAATCTAACAGTGTAGTTTGTGGTGTCGATTAAATTCCCAGTTT TAGAGC TAGAAATAG |
R:GCTATTTCTAGCTCTAAAACTGAATAGCAGGTTCTTCACACAA ACTACACTG TTAGATT | ||
SlMYB12-CRISPR | 构建载体Construction vector | F:GAATCTAACAGTGTAGTTTGAACACCTTGTTGTGAAAAAGGTT TTAGAGCT AGAAATAG |
R:GCTATTTCTAGCTCTAAAACCCAAGAGCCTTCTCCATTAGCAA ACTACACTG TTAGATT | ||
SlGLK2 | 检测靶点Detection target | F:GATGCTGGCATTTGTGTGATTC |
R:TTAATTACGACTAGAACCGTCAACG | ||
SlMYB12 | 检测靶点Detection target | F:TAACTAGACTAAAGTGAAGAGCGGG |
R:CTAATTACCTGTTACCCAAAGTTGC |
表2 构建CRISPR所用的引物序列
Table 2 Primer sequence used for construct CRISPR vector
引物 Primer | 用途 Purpose | 序列(5′-3′) Sequence |
---|---|---|
SlGLK2-CRISPR | 构建载体Construction vector | F:GAATCTAACAGTGTAGTTTGTGGTGTCGATTAAATTCCCAGTTT TAGAGC TAGAAATAG |
R:GCTATTTCTAGCTCTAAAACTGAATAGCAGGTTCTTCACACAA ACTACACTG TTAGATT | ||
SlMYB12-CRISPR | 构建载体Construction vector | F:GAATCTAACAGTGTAGTTTGAACACCTTGTTGTGAAAAAGGTT TTAGAGCT AGAAATAG |
R:GCTATTTCTAGCTCTAAAACCCAAGAGCCTTCTCCATTAGCAA ACTACACTG TTAGATT | ||
SlGLK2 | 检测靶点Detection target | F:GATGCTGGCATTTGTGTGATTC |
R:TTAATTACGACTAGAACCGTCAACG | ||
SlMYB12 | 检测靶点Detection target | F:TAACTAGACTAAAGTGAAGAGCGGG |
R:CTAATTACCTGTTACCCAAAGTTGC |
图3 番茄AC和B5品种基因编辑植株中SlGLK2编辑情况检测 红色标记为靶点;加粗字体为PAM;蓝色加号和横线分别为插入和缺失;绿色标记为替换。下同。
Fig. 3 Targeted mutagenesis of SlGLK2 gene in gene-edited tomato The red marker is the target. The bold font is PAM. The blue plus and the horizontal lines are the base insertions and deletions respectively. The green markers are the base substitutions. The same below.
图5 栽培番茄品种AC、B5和B9的基因编辑植株SlGLK2和SlMYB12氨基酸序列分析 红色标记为靶点;蓝色为突变的氨基酸;短横线表示终止密码子。
Fig. 5 Amino acid sequence analysis of the SlGLK2 and SlMYB12 in gene editing transgenic plants of AC,B5 and B9 tomatos The red marker is the target. Blue represents the mutated amino acid. The dash indicates the stop codon.
图8 SlGLK2-AC基因编辑材料果实中叶绿素含量 * 指与对照AC的白色柱形所示数值有显著性差异(P < 0.05)。
Fig. 8 The chlorophyll content of fruits in SlGLK2-AC gene-edited plants * indicates that there is a significant difference in the values shown of white histogram as the control AC(P < 0.05).
材料 Material | 单果质量/g Fruit weight | 果实纵径/mm Fruit length | 果实横径/mm Fruit diameter | 果形指数 Index of fruit shape | 果实硬度/(× 105 Pa) Fruit hardness |
---|---|---|---|---|---|
AC | 28.95 ± 0.83 b | 34.51 ± 0.37 c | 38.02 ± 0.77 c | 0.90 ± 0.03 a | 8.14 ± 0.37 b |
SlMYB12-AC-3 | 28.82 ± 0.75 b | 34.45 ± 0.76 c | 37.79 ± 0.64 c | 0.91 ± 0.02 a | 7.86 ± 0.52 b |
SlMYB12-AC-4 | 29.46 ± 1.02 b | 34.04 ± 0.25 c | 38.03 ± 0.92 c | 0.89 ± 0.02 a | 8.32 ± 0.41 b |
SlMYB12-AC-7 | 28.03 ± 0.62 b | 34.26 ± 0.43 c | 37.65 ± 0.43 c | 0.91 ± 0.01 a | 8.22 ± 0.26 b |
SlGLK2-AC-3 | 27.97 ± 0.69 b | 33.52 ± 0.21 c | 37.63 ± 0.26 c | 0.89 ± 0.03 a | 8.47 ± 0.67 b |
SlGLK2-AC-4 | 28.84 ± 1.25 b | 33.96 ± 0.64 c | 38.12 ± 0.71 c | 0.89 ± 0.02 a | 7.92 ± 0.85 b |
SlGLK2-AC-5 | 28.88 ± 0.48 b | 34.62 ± 0.41 c | 38.46 ± 0.65 c | 0.90 ± 0.01 a | 8.08 ± 0.56 b |
B5 | 80.82 ± 0.67 a | 41.42 ± 1.21 ab | 53.96 ± 1.84 ab | 0.76 ± 0.04 b | 11.33 ± 1.08 a |
SlGLK2-B5-1 | 79.94 ± 1.23 a | 39.61 ± 1.73 b | 52.75 ± 2.17 b | 0.75 ± 0.06 b | 10.76 ± 0.84 a |
SlGLK2-B5-2 | 81.89 ± 1.06 a | 43.30 ± 1.55 a | 55.53 ± 1.49 a | 0.77 ± 0.03 b | 11.59 ± 0.86 a |
SlGLK2-B5-3 | 81.03 ± 1.35 a | 41.42 ± 1.83 ab | 55.02 ± 2.27 a | 0.75 ± 0.05 b | 10.94 ± 1.23 a |
表3 基因编辑材料的果实性状
Table 3 Fruit traits of gene-edited plants
材料 Material | 单果质量/g Fruit weight | 果实纵径/mm Fruit length | 果实横径/mm Fruit diameter | 果形指数 Index of fruit shape | 果实硬度/(× 105 Pa) Fruit hardness |
---|---|---|---|---|---|
AC | 28.95 ± 0.83 b | 34.51 ± 0.37 c | 38.02 ± 0.77 c | 0.90 ± 0.03 a | 8.14 ± 0.37 b |
SlMYB12-AC-3 | 28.82 ± 0.75 b | 34.45 ± 0.76 c | 37.79 ± 0.64 c | 0.91 ± 0.02 a | 7.86 ± 0.52 b |
SlMYB12-AC-4 | 29.46 ± 1.02 b | 34.04 ± 0.25 c | 38.03 ± 0.92 c | 0.89 ± 0.02 a | 8.32 ± 0.41 b |
SlMYB12-AC-7 | 28.03 ± 0.62 b | 34.26 ± 0.43 c | 37.65 ± 0.43 c | 0.91 ± 0.01 a | 8.22 ± 0.26 b |
SlGLK2-AC-3 | 27.97 ± 0.69 b | 33.52 ± 0.21 c | 37.63 ± 0.26 c | 0.89 ± 0.03 a | 8.47 ± 0.67 b |
SlGLK2-AC-4 | 28.84 ± 1.25 b | 33.96 ± 0.64 c | 38.12 ± 0.71 c | 0.89 ± 0.02 a | 7.92 ± 0.85 b |
SlGLK2-AC-5 | 28.88 ± 0.48 b | 34.62 ± 0.41 c | 38.46 ± 0.65 c | 0.90 ± 0.01 a | 8.08 ± 0.56 b |
B5 | 80.82 ± 0.67 a | 41.42 ± 1.21 ab | 53.96 ± 1.84 ab | 0.76 ± 0.04 b | 11.33 ± 1.08 a |
SlGLK2-B5-1 | 79.94 ± 1.23 a | 39.61 ± 1.73 b | 52.75 ± 2.17 b | 0.75 ± 0.06 b | 10.76 ± 0.84 a |
SlGLK2-B5-2 | 81.89 ± 1.06 a | 43.30 ± 1.55 a | 55.53 ± 1.49 a | 0.77 ± 0.03 b | 11.59 ± 0.86 a |
SlGLK2-B5-3 | 81.03 ± 1.35 a | 41.42 ± 1.83 ab | 55.02 ± 2.27 a | 0.75 ± 0.05 b | 10.94 ± 1.23 a |
材料 Material | 可溶性固形物/% Soluble solid | 可滴定酸/% Titrable acid | 固酸比 S/T | 总维生素C/(mg · g-1) Total vitamin C | 还原维生素C/(mg · g-1) Reduced vitamin C |
---|---|---|---|---|---|
AC | 5.7 ± 0.34 a | 0.62 ± 0.21 b | 9.19 ± 0.87 a | 0.09 ± 0.01 b | 0.09 ± 0.01 c |
SlMYB12-AC-3 | 5.3 ± 0.27 b | 0.69 ± 0.17 b | 7.68 ± 1.21 b | 0.08 ± 0.02 b | 0.08 ± 0.01 c |
SlMYB12-AC-4 | 5.5 ± 0.61 a | 0.64 ± 0.13 b | 8.59 ± 0.96 ab | 0.12 ± 0.02 b | 0.09 ± 0.01 c |
SlMYB12-AC-7 | 5.8 ± 0.29 a | 0.63 ± 0.24 b | 9.21 ± 1.35 a | 0.10 ± 0.01 b | 0.09 ± 0.01 c |
SlGLK2-AC-3 | 5.9 ± 0.53 a | 0.68 ± 0.30 b | 8.67 ± 1.07 ab | 0.11 ± 0.01 b | 0.10 ± 0.01 c |
SlGLK2-AC-4 | 5.6 ± 0.28 a | 0.66 ± 0.27 b | 8.48 ± 0.67 ab | 0.11 ± 0.02 b | 0.09 ± 0.01 c |
SlGLK2-AC-5 | 5.5 ± 0.36 a | 0.63 ± 0.18 b | 8.73 ± 1.26 a | 0.11 ± 0.02 b | 0.09 ± 0.01 c |
B5 | 6.0 ± 0.27 a | 1.10 ± 0.22 a | 5.45 ± 0.93 c | 0.20 ± 0.03 a | 0.19 ± 0.02 a |
SlGLK2-B5-1 | 6.0 ± 0.34 a | 1.04 ± 0.17 a | 5.77 ± 0.72 c | 0.16 ± 0.04 a | 0.15 ± 0.03 b |
SlGLK2-B5-2 | 5.6 ± 0.51 a | 1.08 ± 0.31 a | 5.18 ± 0.64 c | 0.20 ± 0.03 a | 0.17 ± 0.02 a |
SlGLK2-B5-3 | 5.8 ± 0.43 a | 1.21 ± 0.37 a | 4.79 ± 1.37 d | 0.18 ± 0.03 a | 0.16 ± 0.03 ab |
表4 基因编辑材料的营养品质
Table 4 Fruits quality traits of gene-edited plants
材料 Material | 可溶性固形物/% Soluble solid | 可滴定酸/% Titrable acid | 固酸比 S/T | 总维生素C/(mg · g-1) Total vitamin C | 还原维生素C/(mg · g-1) Reduced vitamin C |
---|---|---|---|---|---|
AC | 5.7 ± 0.34 a | 0.62 ± 0.21 b | 9.19 ± 0.87 a | 0.09 ± 0.01 b | 0.09 ± 0.01 c |
SlMYB12-AC-3 | 5.3 ± 0.27 b | 0.69 ± 0.17 b | 7.68 ± 1.21 b | 0.08 ± 0.02 b | 0.08 ± 0.01 c |
SlMYB12-AC-4 | 5.5 ± 0.61 a | 0.64 ± 0.13 b | 8.59 ± 0.96 ab | 0.12 ± 0.02 b | 0.09 ± 0.01 c |
SlMYB12-AC-7 | 5.8 ± 0.29 a | 0.63 ± 0.24 b | 9.21 ± 1.35 a | 0.10 ± 0.01 b | 0.09 ± 0.01 c |
SlGLK2-AC-3 | 5.9 ± 0.53 a | 0.68 ± 0.30 b | 8.67 ± 1.07 ab | 0.11 ± 0.01 b | 0.10 ± 0.01 c |
SlGLK2-AC-4 | 5.6 ± 0.28 a | 0.66 ± 0.27 b | 8.48 ± 0.67 ab | 0.11 ± 0.02 b | 0.09 ± 0.01 c |
SlGLK2-AC-5 | 5.5 ± 0.36 a | 0.63 ± 0.18 b | 8.73 ± 1.26 a | 0.11 ± 0.02 b | 0.09 ± 0.01 c |
B5 | 6.0 ± 0.27 a | 1.10 ± 0.22 a | 5.45 ± 0.93 c | 0.20 ± 0.03 a | 0.19 ± 0.02 a |
SlGLK2-B5-1 | 6.0 ± 0.34 a | 1.04 ± 0.17 a | 5.77 ± 0.72 c | 0.16 ± 0.04 a | 0.15 ± 0.03 b |
SlGLK2-B5-2 | 5.6 ± 0.51 a | 1.08 ± 0.31 a | 5.18 ± 0.64 c | 0.20 ± 0.03 a | 0.17 ± 0.02 a |
SlGLK2-B5-3 | 5.8 ± 0.43 a | 1.21 ± 0.37 a | 4.79 ± 1.37 d | 0.18 ± 0.03 a | 0.16 ± 0.03 ab |
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