草业学报 ›› 2023, Vol. 32 ›› Issue (7): 61-71.DOI: 10.11686/cyxb2022348
管瑾1(), 郭一荻1, 刘凌云1, 尹淑霞1(), 滕珂2()
收稿日期:
2022-08-30
修回日期:
2022-10-07
出版日期:
2023-07-20
发布日期:
2023-05-26
通讯作者:
尹淑霞,滕珂
作者简介:
E-mail: tengke.123@163.com基金资助:
Jin GUAN1(), Yi-di GUO1, Ling-yun LIU1, Shu-xia YIN1(), Ke TENG2()
Received:
2022-08-30
Revised:
2022-10-07
Online:
2023-07-20
Published:
2023-05-26
Contact:
Shu-xia YIN,Ke TENG
摘要:
为了结合转基因方法探索结缕草基因功能,建立高效、快速的结缕草原生质体制备及瞬时基因表达系统,利用正交试验对影响原生质体制备的主要因素进行优化,同时利用原生质体进行亚细胞定位和蛋白互作研究。结果表明,当酶配比为2% (w/v) cellulase R10和1.5% (w/v) macerozyme R10,酶解时间为7 h时,原生质体产量和活性均达到最高,分别为1.23×107个·mL-1和98%以上,满足后续转化所需。多次重复试验表明,加入5~10 μg质粒,原生质体转化率可达75%以上。利用原生质体转化体系进行了亚细胞定位检测,结果发现结缕草ZjNOL和ZjNYC1定位在叶绿体中,ZjZFN定位在细胞核中。双分子荧光互补分析证明ZjNOL和ZjNYC1在叶绿体中相互作用。原生质体制备及转化方法与遗传学和组学技术相结合,能够为结缕草基因功能研究和基因编辑提供支持。
管瑾, 郭一荻, 刘凌云, 尹淑霞, 滕珂. 结缕草叶肉细胞原生质体瞬时基因表达系统的构建[J]. 草业学报, 2023, 32(7): 61-71.
Jin GUAN, Yi-di GUO, Ling-yun LIU, Shu-xia YIN, Ke TENG. An efficient protocol for Zoysia japonica mesophyll protoplast isolation and transformation, and its application in subcellular localization and protein interaction analysis[J]. Acta Prataculturae Sinica, 2023, 32(7): 61-71.
图1 结缕草原生质体制备情况A:光学显微镜下原生质体的形态特征Morphological characteristics of protoplasts observed by light microscopy;B: 光学显微镜下FDA染色后的原生质体形态特征Morphological characteristics of protoplasts stained with FDA, observed by light microscopy.
Fig.1 Isolation of mesophyll protoplasts from Z. japonica
引物名称Primer name | 序列Sequence (5'-3') |
---|---|
ZjNYC1-F | CTCGTCCCGACCTTATCCGC |
ZjNYC1-R | GGGCACGGGGTCATCCAG |
ZjNOL-F | CTCCACACAAGACTCCATTCG |
ZjNOL-R | GAAAAAGGAATGGTTCAAAACAGAT |
ZjZFN-F | ATGTCGTCCGCCATGGAATT |
ZjZFN-R | TCACGCGGTCATGAGGAGGC |
3302Y3-ZjNYC1-F | cctactagtcctagggacgtcaATGGCCGCCGCCGTCGCGCA |
3302Y3-ZjNYC1-R | tgctcaccatacgcgttacagaTGTGCCTGGAAGAGGACCAC |
3302Y3-ZjNOL-F | cctactagtcctagggacgtcaATGGCTGCCAGCGTCAGCATCG |
3302Y3-ZjNOL-R | tgctcaccatacgcgttacagaATCTTCAACAACATACTTAT |
3302Y3-ZjZFN-F | cacgggggactcttgaccatggtaATGTCGTCCGCCATGGAATT |
3302Y3-ZjZFN-R | ggtacacgcgtactagtcagatcCGCGGTCATGAGGAGGCGGG |
35S-pSPY-ZjNYC1-F | caggcctggcgcgccactagtgATGGCCGCCGCCGTCGCGCA |
35S-pSPY-ZjNYC1-R | ggtcgacagtactatcgatggaTGTGCCTGGAAGAGGACCAC |
35S-pSPY-ZjNOL-F | caggcctggcgcgccactagtgATGGCTGCCAGCGTCAGCATCG |
35S-pSPY-ZjNOL-R | ggtcgacagtactatcgatggaATCTTCAACAACATACTTAT |
表1 用于基因克隆、表达分析和载体构建的引物
Table 1 Primers used for gene cloning, expression analysis and plasmids construction
引物名称Primer name | 序列Sequence (5'-3') |
---|---|
ZjNYC1-F | CTCGTCCCGACCTTATCCGC |
ZjNYC1-R | GGGCACGGGGTCATCCAG |
ZjNOL-F | CTCCACACAAGACTCCATTCG |
ZjNOL-R | GAAAAAGGAATGGTTCAAAACAGAT |
ZjZFN-F | ATGTCGTCCGCCATGGAATT |
ZjZFN-R | TCACGCGGTCATGAGGAGGC |
3302Y3-ZjNYC1-F | cctactagtcctagggacgtcaATGGCCGCCGCCGTCGCGCA |
3302Y3-ZjNYC1-R | tgctcaccatacgcgttacagaTGTGCCTGGAAGAGGACCAC |
3302Y3-ZjNOL-F | cctactagtcctagggacgtcaATGGCTGCCAGCGTCAGCATCG |
3302Y3-ZjNOL-R | tgctcaccatacgcgttacagaATCTTCAACAACATACTTAT |
3302Y3-ZjZFN-F | cacgggggactcttgaccatggtaATGTCGTCCGCCATGGAATT |
3302Y3-ZjZFN-R | ggtacacgcgtactagtcagatcCGCGGTCATGAGGAGGCGGG |
35S-pSPY-ZjNYC1-F | caggcctggcgcgccactagtgATGGCCGCCGCCGTCGCGCA |
35S-pSPY-ZjNYC1-R | ggtcgacagtactatcgatggaTGTGCCTGGAAGAGGACCAC |
35S-pSPY-ZjNOL-F | caggcctggcgcgccactagtgATGGCTGCCAGCGTCAGCATCG |
35S-pSPY-ZjNOL-R | ggtcgacagtactatcgatggaATCTTCAACAACATACTTAT |
因素 Factors | Cellulase R10 (%, w/v) | Macerozyme R10 (%, w/v) | 酶解时间 Enzymolysis time (h) |
---|---|---|---|
1 | 1 | 0.5 | 4 |
2 | 2 | 1.0 | 5 |
3 | 3 | 1.5 | 6 |
4 | 4 | 2.0 | 7 |
表2 原生质体制备酶配比和酶解时间
Table 2 The enzyme ratio and enzymolysis time in the protoplast isolation experiment
因素 Factors | Cellulase R10 (%, w/v) | Macerozyme R10 (%, w/v) | 酶解时间 Enzymolysis time (h) |
---|---|---|---|
1 | 1 | 0.5 | 4 |
2 | 2 | 1.0 | 5 |
3 | 3 | 1.5 | 6 |
4 | 4 | 2.0 | 7 |
溶液名称 Solution name | 溶液配方 Solution composition | 储存条件 Storage | 用途 Usage |
---|---|---|---|
酶解液Enzyme solution | cellulase R10,macerozyme R10,甘露醇Mannitol,10 mmol·L-1 MES,pH 5.7,10 mmol·L-1 CaCl2,0.1% (w/v) BSA | 现用现配,室温Freshly prepared, room temperature | 制备原生质体Preparation of protoplasts |
W5溶液 W5 solution | 154 mmol·L-1 NaCl,125 mmol·L-1 CaCl2,5 mmol·L-1 KCl,2 mmol·L-1 MES,pH 5.7 | 4 ℃ | 释放和清洗原生质体Release and wash protoplasts |
MMG溶液 MMG solution | 0.4 mol·L-1 甘露醇Mannitol, 15 mmol·L-1 MgCl2, 4 mmol·L-1 MES,pH 5.7 | 4 ℃ | 重悬原生质体Resuspend protoplasts |
PEG溶液 PEG solution | 40% (w/v) PEG 4000, 0.2 mol·L-1 甘露醇Mannitol,0.1 mol·L-1 CaCl2 | 现用现配,室温Freshly prepared, room temperature | 介导原生质体转化Transform plasmids into protoplasts |
WI溶液 WI solution | 0.5 mol·L-1 甘露醇Mannitol,20 mmol·L-1 KCl,4 mmol·L-1 MES,pH 5.7 | 4 ℃ | 重悬转化体系Resuspend transformation system |
表3 原生质体制备和转化的溶液配方
Table 3 Solution recipes for protoplast isolation and transformation
溶液名称 Solution name | 溶液配方 Solution composition | 储存条件 Storage | 用途 Usage |
---|---|---|---|
酶解液Enzyme solution | cellulase R10,macerozyme R10,甘露醇Mannitol,10 mmol·L-1 MES,pH 5.7,10 mmol·L-1 CaCl2,0.1% (w/v) BSA | 现用现配,室温Freshly prepared, room temperature | 制备原生质体Preparation of protoplasts |
W5溶液 W5 solution | 154 mmol·L-1 NaCl,125 mmol·L-1 CaCl2,5 mmol·L-1 KCl,2 mmol·L-1 MES,pH 5.7 | 4 ℃ | 释放和清洗原生质体Release and wash protoplasts |
MMG溶液 MMG solution | 0.4 mol·L-1 甘露醇Mannitol, 15 mmol·L-1 MgCl2, 4 mmol·L-1 MES,pH 5.7 | 4 ℃ | 重悬原生质体Resuspend protoplasts |
PEG溶液 PEG solution | 40% (w/v) PEG 4000, 0.2 mol·L-1 甘露醇Mannitol,0.1 mol·L-1 CaCl2 | 现用现配,室温Freshly prepared, room temperature | 介导原生质体转化Transform plasmids into protoplasts |
WI溶液 WI solution | 0.5 mol·L-1 甘露醇Mannitol,20 mmol·L-1 KCl,4 mmol·L-1 MES,pH 5.7 | 4 ℃ | 重悬转化体系Resuspend transformation system |
图3 正交试验A:原生质体产量Protoplast yield;B: 原生质体活性Protoplast activity;C:有效原生质体产量Active protoplast yield. 不同小写字母表示处理间差异显著(P<0.05)。Different lowercase lerrers indicate significant differernce among different treatments (P<0.05).
Fig.3 The results of the orthogonal experiment
处理编号 Treatment No. | Cellulase R10 | Macerozyme R10 | 酶解时间 Enzymolysis time (h) | 原生质体产量 Protoplast yield (×106 个protoplasts·mL-1) | 原生质体活性 Protoplast activity (%) | 有效原生质体产量 Active protoplast yield (×106 个protoplasts·mL-1) |
---|---|---|---|---|---|---|
1 | 1 | 1 | 1 | 2.25 | 78.03 | 1.76 |
2 | 1 | 2 | 2 | 2.51 | 81.64 | 2.05 |
3 | 1 | 3 | 3 | 4.05 | 86.13 | 3.49 |
4 | 1 | 4 | 4 | 4.54 | 85.25 | 3.87 |
5 | 2 | 1 | 2 | 6.54 | 85.25 | 5.58 |
6 | 2 | 2 | 1 | 3.91 | 84.14 | 3.28 |
7 | 2 | 3 | 4 | 12.25 | 98.87 | 12.11 |
8 | 2 | 4 | 3 | 7.81 | 88.07 | 6.87 |
9 | 3 | 1 | 3 | 6.03 | 81.84 | 4.94 |
10 | 3 | 2 | 4 | 7.08 | 84.56 | 5.99 |
11 | 3 | 3 | 1 | 4.47 | 88.31 | 3.94 |
12 | 3 | 4 | 2 | 6.63 | 84.71 | 5.62 |
13 | 4 | 1 | 4 | 6.43 | 81.29 | 5.23 |
14 | 4 | 2 | 3 | 6.08 | 85.16 | 5.18 |
15 | 4 | 3 | 2 | 6.63 | 90.11 | 5.97 |
16 | 4 | 4 | 1 | 5.25 | 85.94 | 4.52 |
原生质体产量 Protoplast yield (×106个protoplasts·mL-1) | K1 | 3.34 | 5.32 | 3.97 | ||
K2 | 7.63 | 4.90 | 5.58 | |||
K3 | 6.05 | 6.85 | 5.99 | |||
K4 | 6.10 | 6.06 | 7.58 | |||
范围Range | 4.29 | 1.53 | 3.61 | |||
排序Rank | cellulase R10>酶解时间Enzymolysis time>macerozyme R10 | |||||
原生质体活性 Protoplast activity (%) | K1 | 82.76 | 81.60 | 84.10 | ||
K2 | 89.08 | 83.87 | 85.43 | |||
K3 | 84.85 | 90.85 | 85.30 | |||
K4 | 85.62 | 85.99 | 87.49 | |||
范围Range | 6.32 | 9.25 | 2.07 | |||
排序Rank | macerozyme R10>cellulase R10>酶解时间Enzymolysis time | |||||
有效原生质体产量 Active protoplast yield (×106个protoplasts·mL-1) | K1 | 2.79 | 4.38 | 3.38 | ||
K2 | 6.96 | 4.13 | 4.80 | |||
K3 | 5.12 | 6.38 | 5.12 | |||
K4 | 5.23 | 5.22 | 6.80 | |||
范围Range | 4.17 | 2.00 | 3.42 | |||
排序Rank | cellulase R10>酶解时间Enzymolysis time>macerozyme R10 |
表4 影响原生质体制备效率的三因素正交试验
Table 4 Orthogonal experiment of three factors affecting the efficiency of protoplast isolation
处理编号 Treatment No. | Cellulase R10 | Macerozyme R10 | 酶解时间 Enzymolysis time (h) | 原生质体产量 Protoplast yield (×106 个protoplasts·mL-1) | 原生质体活性 Protoplast activity (%) | 有效原生质体产量 Active protoplast yield (×106 个protoplasts·mL-1) |
---|---|---|---|---|---|---|
1 | 1 | 1 | 1 | 2.25 | 78.03 | 1.76 |
2 | 1 | 2 | 2 | 2.51 | 81.64 | 2.05 |
3 | 1 | 3 | 3 | 4.05 | 86.13 | 3.49 |
4 | 1 | 4 | 4 | 4.54 | 85.25 | 3.87 |
5 | 2 | 1 | 2 | 6.54 | 85.25 | 5.58 |
6 | 2 | 2 | 1 | 3.91 | 84.14 | 3.28 |
7 | 2 | 3 | 4 | 12.25 | 98.87 | 12.11 |
8 | 2 | 4 | 3 | 7.81 | 88.07 | 6.87 |
9 | 3 | 1 | 3 | 6.03 | 81.84 | 4.94 |
10 | 3 | 2 | 4 | 7.08 | 84.56 | 5.99 |
11 | 3 | 3 | 1 | 4.47 | 88.31 | 3.94 |
12 | 3 | 4 | 2 | 6.63 | 84.71 | 5.62 |
13 | 4 | 1 | 4 | 6.43 | 81.29 | 5.23 |
14 | 4 | 2 | 3 | 6.08 | 85.16 | 5.18 |
15 | 4 | 3 | 2 | 6.63 | 90.11 | 5.97 |
16 | 4 | 4 | 1 | 5.25 | 85.94 | 4.52 |
原生质体产量 Protoplast yield (×106个protoplasts·mL-1) | K1 | 3.34 | 5.32 | 3.97 | ||
K2 | 7.63 | 4.90 | 5.58 | |||
K3 | 6.05 | 6.85 | 5.99 | |||
K4 | 6.10 | 6.06 | 7.58 | |||
范围Range | 4.29 | 1.53 | 3.61 | |||
排序Rank | cellulase R10>酶解时间Enzymolysis time>macerozyme R10 | |||||
原生质体活性 Protoplast activity (%) | K1 | 82.76 | 81.60 | 84.10 | ||
K2 | 89.08 | 83.87 | 85.43 | |||
K3 | 84.85 | 90.85 | 85.30 | |||
K4 | 85.62 | 85.99 | 87.49 | |||
范围Range | 6.32 | 9.25 | 2.07 | |||
排序Rank | macerozyme R10>cellulase R10>酶解时间Enzymolysis time | |||||
有效原生质体产量 Active protoplast yield (×106个protoplasts·mL-1) | K1 | 2.79 | 4.38 | 3.38 | ||
K2 | 6.96 | 4.13 | 4.80 | |||
K3 | 5.12 | 6.38 | 5.12 | |||
K4 | 5.23 | 5.22 | 6.80 | |||
范围Range | 4.17 | 2.00 | 3.42 | |||
排序Rank | cellulase R10>酶解时间Enzymolysis time>macerozyme R10 |
指标 Index | 原生质体产量 Protoplast yield | 原生质体活性 Protoplast activity |
---|---|---|
原生质体活性Protoplast activity | 0.774 | |
有效原生质体产量 Active protoplast yield | 0.993 | 0.830 |
表5 原生质体制备中各因素的相关性
Table 5 The correlation between the indexes of protoplast isolation
指标 Index | 原生质体产量 Protoplast yield | 原生质体活性 Protoplast activity |
---|---|---|
原生质体活性Protoplast activity | 0.774 | |
有效原生质体产量 Active protoplast yield | 0.993 | 0.830 |
图4 不同载体在结缕草原生质体中的亚细胞定位A:ZjNOL-YFP;B: ZjNYC1-YFP;C:ZjZFN-YFP; DAPI:二盐酸-4’,6-二脒基-2-苯基吲哚4’,6-diamidino-2-phenylindole dihydrochloride.
Fig.4 Subcellular localization for different vector in Z. japonica protoplasts
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