草业学报 ›› 2023, Vol. 32 ›› Issue (1): 165-177.DOI: 10.11686/cyxb2022189
• 研究论文 • 上一篇
姜凯(), 吴雪莉(), 刘奕君, 马越, 宋洋, 卢文杰, 王增裕
收稿日期:
2022-04-27
修回日期:
2022-06-16
出版日期:
2023-01-20
发布日期:
2022-11-07
通讯作者:
吴雪莉
作者简介:
E-mail: xueli0510qau@163.com基金资助:
Kai JIANG(), Xue-li WU(), Yi-jun LIU, Yue MA, Yang SONG, Wen-jie LU, Zeng-yu WANG
Received:
2022-04-27
Revised:
2022-06-16
Online:
2023-01-20
Published:
2022-11-07
Contact:
Xue-li WU
摘要:
海滨雀稗作为耐盐性较强的暖季型草坪草,具有极大的应用潜力。为建立高效稳定的海滨雀稗遗传转化体系,本研究以海滨雀稗成熟种子为外植体,确定了筛选剂潮霉素和草丁膦在海滨雀稗的愈伤组织继代培养和再生阶段的最佳筛选压。在进一步优化遗传转化条件的基础上,比较了以hpt与bar基因作为转基因筛选标记对海滨雀稗农杆菌介导的遗传转化效率的影响。结果表明:愈伤组织的再生率在继代36周后显著下降,继代48周的最高再生率为67.9%。潮霉素的最佳筛选压在继代培养阶段为120 mg·L-1,再生阶段为80 mg·L-1。草丁膦的最佳筛选压均为1.2 mg·L-1。选取继代24周的愈伤组织用于农杆菌介导的遗传转化,最佳的转化条件为菌液浓度OD600=0.6,添加100 μmol·L-1乙酰丁香酮和0.01%的Silwet L-77,并辅以超声波处理20 min或真空处理10 min,浸染30 min后共培养2 d。通过多次抗性愈伤组织和抗性再生苗的筛选,2种载体均获得了转基因再生植株。通过PCR检测证明hpt和bar基因分别成功在海滨雀稗基因组中表达。以潮霉素作为筛选剂,β-葡萄糖醛酸糖苷酶(GUS)基因稳定表达及PCR检测的转基因的平均效率为18.9%,最高转化效率为20.3%,均高于以草丁膦为筛选剂的转化效率。由此可见,hpt基因更适合作为海滨雀稗遗传转化中的筛选标记,能够获得更高的转基因效率。
姜凯, 吴雪莉, 刘奕君, 马越, 宋洋, 卢文杰, 王增裕. 海滨雀稗以hpt与bar基因为筛选标记的转化体系比较研究[J]. 草业学报, 2023, 32(1): 165-177.
Kai JIANG, Xue-li WU, Yi-jun LIU, Yue MA, Yang SONG, Wen-jie LU, Zeng-yu WANG. Comparative study on transformation systems of seashore paspalum using hpt and bar genes as selection markers[J]. Acta Prataculturae Sinica, 2023, 32(1): 165-177.
图1 植物表达载体图谱A、B分别为质粒pCAMBIA1305.2和pCAMBIA3301的结构示意图。A and B are structural representation of plasmid pCAMBIA1305.2 and pCAMBIA3301, respectively.
Fig.1 Map of plant expression vector
培养基类型Medium type | 培养基配方Formula of medium | 其他Others |
---|---|---|
诱导和继代培养基Induction and subculture media | MS基本培养基+2.5 mg·L-1 2,4-二氯苯氧乙酸+0.6 mg·L-1 CuSO4 MS basic medium+2.5 mg·L-1 2,4-dichlorophenoxy acetic acid (2,4-D)+0.6 mg·L-1 CuSO4 | 添加30 g·L-1蔗糖,调pH值为5.8,加入8 g·L-1琼脂,121 ℃高温灭菌20 min。Added 30 g·L-1 sucrose, adjusted the pH value to 5.8, add 8 g·L-1 agar, and sterilized at 121 ℃ for 20 min. |
选择继代培养基Selective subculture medium | MS基本培养基+2.5 mg·L-1 2,4-D+0.6 mg·L-1 CuSO4+200 mg·L-1头孢噻肟+适宜浓度筛选剂MS basic medium+2.5 mg·L-1 2,4-D+0.6 mg·L-1 CuSO4+200 mg·L-1 cefotaxime+suitable concentration screening agent | |
再生培养基Regeneration medium | MS基本培养基+0.05 mg·L-1激动素+8.0 mg·L-1 6-苄氨基嘌呤+10 mg·L-1 CuSO4 MS basic medium+0.05 mg·L-1 kinetin (KT)+8.0 mg·L-1 6-benzylaminopurine (6-BA)+10 mg·L-1 CuSO4 | |
选择再生培养基Select regeneration medium | MS基本培养基+0.05 mg·L-1激动素+8.0 mg·L-1 6-苄氨基嘌呤+10 mg·L-1 CuSO4+200 mg·L-1头孢噻肟+适宜浓度筛选剂MS basic medium+0.05 mg·L-1 KT+8.0 mg·L-1 6-BA+10 mg·L-1 CuSO4+200 mg·L-1 cefotaxime+suitable concentration screening agent | |
壮苗培养基Strong seedling medium | 1/2 MS基本培养基1/2 MS basic medium | |
选择壮苗培养基Select seedling growth medium | 1/2 MS基本培养基+适宜浓度筛选剂1/2 MS basic medium+suitable concentration screening agent |
表1 培养基的类型和配制方法
Table 1 Types and preparation methods of culture medium
培养基类型Medium type | 培养基配方Formula of medium | 其他Others |
---|---|---|
诱导和继代培养基Induction and subculture media | MS基本培养基+2.5 mg·L-1 2,4-二氯苯氧乙酸+0.6 mg·L-1 CuSO4 MS basic medium+2.5 mg·L-1 2,4-dichlorophenoxy acetic acid (2,4-D)+0.6 mg·L-1 CuSO4 | 添加30 g·L-1蔗糖,调pH值为5.8,加入8 g·L-1琼脂,121 ℃高温灭菌20 min。Added 30 g·L-1 sucrose, adjusted the pH value to 5.8, add 8 g·L-1 agar, and sterilized at 121 ℃ for 20 min. |
选择继代培养基Selective subculture medium | MS基本培养基+2.5 mg·L-1 2,4-D+0.6 mg·L-1 CuSO4+200 mg·L-1头孢噻肟+适宜浓度筛选剂MS basic medium+2.5 mg·L-1 2,4-D+0.6 mg·L-1 CuSO4+200 mg·L-1 cefotaxime+suitable concentration screening agent | |
再生培养基Regeneration medium | MS基本培养基+0.05 mg·L-1激动素+8.0 mg·L-1 6-苄氨基嘌呤+10 mg·L-1 CuSO4 MS basic medium+0.05 mg·L-1 kinetin (KT)+8.0 mg·L-1 6-benzylaminopurine (6-BA)+10 mg·L-1 CuSO4 | |
选择再生培养基Select regeneration medium | MS基本培养基+0.05 mg·L-1激动素+8.0 mg·L-1 6-苄氨基嘌呤+10 mg·L-1 CuSO4+200 mg·L-1头孢噻肟+适宜浓度筛选剂MS basic medium+0.05 mg·L-1 KT+8.0 mg·L-1 6-BA+10 mg·L-1 CuSO4+200 mg·L-1 cefotaxime+suitable concentration screening agent | |
壮苗培养基Strong seedling medium | 1/2 MS基本培养基1/2 MS basic medium | |
选择壮苗培养基Select seedling growth medium | 1/2 MS基本培养基+适宜浓度筛选剂1/2 MS basic medium+suitable concentration screening agent |
图2 愈伤组织年龄对再生的影响A:用于遗传转化的黄色颗粒状愈伤组织;B:生长12周的胚性愈伤组织;C:愈伤组织的再生;D:再生植株。不同小写字母表示差异显著(P<0.05)。下同。A: Yellow granules and firm structure callus that prepared for transformation; B: 12-week-old embryogenic callus; C: Callus regenerated; D: Regeneration plants. Different lowercase letters represented difference were significant (P<0.05). The same below.
Fig.2 Effect of callus age on regeneration
图3 潮霉素对胚性愈伤组织继代培养和再生的影响A:潮霉素浓度对愈伤组织继代培养的影响;B:潮霉素浓度对愈伤组织再生的影响。A: Effects of hygromycin concentration on callus subculture; B: Effects of hygromycin concentration on callus regeneration.
Fig.3 Effects of hygromycin on embryogenic callus subculture and regeneration
图4 草丁膦对胚性愈伤组织继代培养和再生的影响A:草丁膦浓度对愈伤组织继代培养的影响;B:草丁膦浓度对愈伤组织再生的影响。A: Effects of glufosinate concentration on callus subculture; B: Effects of glufosinate concentration on callus regeneration.
Fig.4 Effects of glufosinate on embryogenic callus subculture and regeneration
图5 不同因素对GUS瞬时表达效率的影响A~D:在添加AS、Silwet L-77、超声波处理、真空处理条件下,愈伤组织GUS瞬时表达的染色情况。A-D: Staining of GUS transient expression of callus that treated with AS, Silwet L-77, sonication or vacuum.
Fig.5 Factors affecting on percentage of transient GUS expression
图6 农杆菌介导的胚性愈伤组织的遗传转化及GUS染色A:潮霉素抗性的愈伤组织;B:GUS在潮霉素抗性愈伤组织中稳定表达;C:潮霉素抗性的再生苗;D:草丁膦抗性的愈伤组织;E:GUS在草丁膦抗性愈伤组织中稳定表达;F:草丁膦抗性的再生苗;G:抗性再生苗在温室中种植;H:GUS在抗性再生苗叶片中稳定表达。A: Hygromycin resistant calli; B: Stable expression of GUS gene in hygromycin resistant calli; C: Hygromycin resistant regenerated plants; D: Glufosinate resistant callus; E: Stable expression of GUS gene in glufosinate resistant calli; F: Glufosinate resistant regenerated plants; G: Resistant regenerated plants planted in greenhouse; H: Stable expression of GUS gene in leaves of resistant plants.
Fig.6 Agrobacterium-mediated transformation of embryogenic calli and result of GUS staining
筛选标记 Selectable marker | 接种愈伤组织数No. of infected calli | 抗性愈伤组织数No. of resistant calli | 愈伤组织GUS染色数No. of GUS (+) calli | 抗性再生植株的克隆数No. of resistant plant clones | 再生植株的GUS染色数No. of GUS (+) plant lines | 转基因效率 Transformation efficiency (%) | 平均转基因效率 Average transformation efficiency (%, mean±SD) |
---|---|---|---|---|---|---|---|
hpt | 63 | 10 | 10 | 6 | 6 | 9.52 | 14.46±4.69 |
59 | 17 | 15 | 15 | 12 | 20.34 | ||
49 | 10 | 7 | 7 | 6 | 12.24 | ||
77 | 15 | 11 | 9 | 9 | 11.69 | ||
81 | 22 | 22 | 18 | 15 | 18.52 | ||
bar | 89 | 7 | 5 | 5 | 3 | 3.37 | 5.65±2.61 |
60 | 9 | 9 | 7 | 6 | 10.00 | ||
79 | 8 | 7 | 7 | 4 | 5.06 | ||
76 | 4 | 3 | 3 | 3 | 3.95 | ||
51 | 6 | 6 | 5 | 3 | 5.88 |
表2 两种不同质粒的农杆菌介导的遗传转化效率
Table 2 Summary of transformation efficiencies of Agrobacterium transformation using embryogenic callus
筛选标记 Selectable marker | 接种愈伤组织数No. of infected calli | 抗性愈伤组织数No. of resistant calli | 愈伤组织GUS染色数No. of GUS (+) calli | 抗性再生植株的克隆数No. of resistant plant clones | 再生植株的GUS染色数No. of GUS (+) plant lines | 转基因效率 Transformation efficiency (%) | 平均转基因效率 Average transformation efficiency (%, mean±SD) |
---|---|---|---|---|---|---|---|
hpt | 63 | 10 | 10 | 6 | 6 | 9.52 | 14.46±4.69 |
59 | 17 | 15 | 15 | 12 | 20.34 | ||
49 | 10 | 7 | 7 | 6 | 12.24 | ||
77 | 15 | 11 | 9 | 9 | 11.69 | ||
81 | 22 | 22 | 18 | 15 | 18.52 | ||
bar | 89 | 7 | 5 | 5 | 3 | 3.37 | 5.65±2.61 |
60 | 9 | 9 | 7 | 6 | 10.00 | ||
79 | 8 | 7 | 7 | 4 | 5.06 | ||
76 | 4 | 3 | 3 | 3 | 3.95 | ||
51 | 6 | 6 | 5 | 3 | 5.88 |
图7 抗性再生植株的PCR检测A:PCR检测抗性再生植株的hpt基因;B:PCR检测抗性再生植株的bar基因;M:标尺;P:质粒阳性对照;WT:野生型阴性对照;1~11:抗性再生植株。A: PCR detection of the hpt gene in resistant plants; B: PCR detection of the bar gene in resistant plants; M: Marker; P: Plasmid as positive control; WT: Wild type as negative control; 1-11: Resistant transgenic lines.
Fig.7 PCR test results of resistant regenerated plants
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