生物技术通报 ›› 2021, Vol. 37 ›› Issue (12): 13-21.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0497
收稿日期:
2021-04-15
出版日期:
2021-12-26
发布日期:
2022-01-19
作者简介:
石欣玥,女,硕士研究生,研究方向:草地植物遗传育种与生物技术;E-mail: 基金资助:
SHI Xin-yue(), SHANG Xiao-yao, ZHOU Ling-fang, ZHANG Tie-jun, CHAO Yue-hui()
Received:
2021-04-15
Published:
2021-12-26
Online:
2022-01-19
摘要:
紫花苜蓿(Medicago sativa)为重要的豆科牧草植物,研究紫花苜蓿转录因子基因MsAP2的功能,为探究MsAP2的信号转导网络提供理论指导和材料基础,也为紫花苜蓿生物技术育种提供一定的参考和借鉴。运用RT-PCR技术克隆MsAP2,利用DNA重组技术构建3302-3flag-AP2植物表达载体,并利用农杆菌介导法对紫花苜蓿进行遗传转化,对再生植株进行转基因鉴定、基因表达量分析、内源激素的测定(脱落酸、细胞分裂素、生长素和赤霉素)和表型鉴定。结果表明,成功获得转MsAP2紫花苜蓿植株,与野生型相比,转基因植株的MsAP2表达量和激素水平均发生明显改变,转基因植株呈现早衰性状,叶片形态和大小发生改变,根系生长受到抑制,分枝数发生改变。
石欣玥, 尚骁尧, 周玲芳, 张铁军, 晁跃辉. 紫花苜蓿转录因子基因MsAP2的克隆及转化[J]. 生物技术通报, 2021, 37(12): 13-21.
SHI Xin-yue, SHANG Xiao-yao, ZHOU Ling-fang, ZHANG Tie-jun, CHAO Yue-hui. Cloning and Transformation of MsAP2 Gene in Medicago sativa[J]. Biotechnology Bulletin, 2021, 37(12): 13-21.
引物名称Primer name | 序列Sequence(5'-3') | 用途Usage |
---|---|---|
MsAP2-f | CTTCCACAAACGGTAGACTTTAGAT | 获得MsAP2基因序列 Obtain MsAP2 gene sequence |
MsAP2-r | GCAACTACACTATGAGCTCTAGATT | |
M13f | CGCCAGGGTTTTCCCAGTCACGAC | 检测克隆载体 Detection of clone vector |
M13r | AGCGGATAACAATTTCACACAGGA | |
3302-AP2-f | GAACACGGGGGACTCTTGACATGGCTTCAACCACCAAAGA | 构建表达载体 Construction of expression vector |
3302-AP2-r | TGTAATCCAGATCTACCATGCTAGATTAGAGTGCAATCAT | |
3302-f | TGACGCACAATCCCACTATCCTT | 检测表达载体 Detection of expression vector |
Nos181-r | CGTATTAAATGTAATTGCGGGAC | |
AP2-RT-f | GGCTTCAACCACCAAAGACTCAG | 基因表达分析 Gene expression analysis |
AP2-RT-r | CACTCCCTTGTACCGCTTCCATA | |
actin-f | TGATCTGGCTGGTCGTGACCTTA | 扩增内参 Amplified internal reference |
actin-r | ATGCCTGCTGCTTCCATTCCTAT |
表1 所用引物的序列
Table 1 Primer sequence
引物名称Primer name | 序列Sequence(5'-3') | 用途Usage |
---|---|---|
MsAP2-f | CTTCCACAAACGGTAGACTTTAGAT | 获得MsAP2基因序列 Obtain MsAP2 gene sequence |
MsAP2-r | GCAACTACACTATGAGCTCTAGATT | |
M13f | CGCCAGGGTTTTCCCAGTCACGAC | 检测克隆载体 Detection of clone vector |
M13r | AGCGGATAACAATTTCACACAGGA | |
3302-AP2-f | GAACACGGGGGACTCTTGACATGGCTTCAACCACCAAAGA | 构建表达载体 Construction of expression vector |
3302-AP2-r | TGTAATCCAGATCTACCATGCTAGATTAGAGTGCAATCAT | |
3302-f | TGACGCACAATCCCACTATCCTT | 检测表达载体 Detection of expression vector |
Nos181-r | CGTATTAAATGTAATTGCGGGAC | |
AP2-RT-f | GGCTTCAACCACCAAAGACTCAG | 基因表达分析 Gene expression analysis |
AP2-RT-r | CACTCCCTTGTACCGCTTCCATA | |
actin-f | TGATCTGGCTGGTCGTGACCTTA | 扩增内参 Amplified internal reference |
actin-r | ATGCCTGCTGCTTCCATTCCTAT |
培养基名称 Name of culture medium | 培养基配方 Ingredient culture |
---|---|
Ms-COLQ | 4.43 g MS+30 g蔗糖+4 mg/L 2,4-D+0.5 mg/L 6-BA+100 µmol/L乙酰丁香酮 |
Ms-CO | 4.43 g MS+30 g蔗糖+4 mg/L 2,4-D+0.5 mg/L 6-BA+1 mg/L KT+200 mg/L头孢霉素+2 mg/L草铵膦+4 g/L植物凝胶,pH5.8 |
Ms-YD | 4.43 g MS+30 g蔗糖+4 mg/L 2,4-D+1 mg/L 6-BA+200 mg/L头孢霉素+2 mg/L草铵膦+4 g/L植物凝胶,pH5.8 |
Ms-SY | 4.43 g MS+30 g蔗糖+200 mg/L头孢霉素+1 mg/L草铵膦+4 g/L植物凝胶,pH5.8 |
Ms-SG | 2.22 g MS+15 g蔗糖+1 mg/L IAA+200 mg/L头孢霉素+7 g琼脂,pH5.8 |
表2 紫花苜蓿遗传转化培养基成分
Table 2 A variety of culture medium in the genetic trans-formation experiment of M. sativa
培养基名称 Name of culture medium | 培养基配方 Ingredient culture |
---|---|
Ms-COLQ | 4.43 g MS+30 g蔗糖+4 mg/L 2,4-D+0.5 mg/L 6-BA+100 µmol/L乙酰丁香酮 |
Ms-CO | 4.43 g MS+30 g蔗糖+4 mg/L 2,4-D+0.5 mg/L 6-BA+1 mg/L KT+200 mg/L头孢霉素+2 mg/L草铵膦+4 g/L植物凝胶,pH5.8 |
Ms-YD | 4.43 g MS+30 g蔗糖+4 mg/L 2,4-D+1 mg/L 6-BA+200 mg/L头孢霉素+2 mg/L草铵膦+4 g/L植物凝胶,pH5.8 |
Ms-SY | 4.43 g MS+30 g蔗糖+200 mg/L头孢霉素+1 mg/L草铵膦+4 g/L植物凝胶,pH5.8 |
Ms-SG | 2.22 g MS+15 g蔗糖+1 mg/L IAA+200 mg/L头孢霉素+7 g琼脂,pH5.8 |
图4 MsAP2对紫花苜蓿的遗传转化过程 A:在Ms-CO培养基上培养3 d的外植体;B:在Ms-YD培养基上培养5周的愈伤组织;C:在Ms-SY培养基上培养3周愈伤组织长出嫩芽;D:在Ms-SY培养基上培养6周的嫩芽;E:在Ms-SG培养基上培养4周后的幼苗;F:在无菌土壤中生长6周的再生植株
Fig.4 MsAP2 genetic transformation to M. sativa A: Explants were cultured on Ms-CO medium for 3 days. B: Calli were cultured on Ms-YD medium for 5 weeks. C: The callus grew buds after cultured on Ms-SY medium for 3 weeks. D: Buds were cultured on Ms-SY medium for 6 weeks. E: Seedlings cultured on Ms-SG medium for 4 weeks. F: Regenerated plants grown in sterile soil for 6 weeks
图5 转基因紫花苜蓿DNA检测 M:DNA marker DL2000 Plus II;1-7:转基因紫花苜蓿;W:野生型;E:空载
Fig. 5 DNA detection of transgenic M. sativa M:DNA marker DL2000 Plus II. 1-7: Transgenic M. sativa. W: Wild type. E: No load
图6 转基因紫花苜蓿的RT-PCR检测 M:DNA marker DL2000 Plus II;1-4:转基因紫花苜蓿;W:野生型
Fig.6 RT-PCR detection of transgenic M. sativa M:DNA marker DL2000 Plus II. 1-4: Transgenic alfalfa. W: Wild type
图7 转基因紫花苜蓿的蛋白质检测 M:蛋白分子量标准;1-4:转基因紫花苜蓿;W:野生型
Fig.7 Protein detection of transgenic M. sativa M: Protein molecular weight standard. 1-4: Transgenic alfalfa. W: Wild type
图8 转基因紫花苜蓿的MsAP2相对表达量 不同字母代表在0.05水平上显著,下同
Fig.8 Relative expression of MsAP2 gene of transgenic M. sativa Different letters indicate significant difference at 0.05 level, the same below
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