紫花苜蓿转录因子基因MsAP2的克隆及转化

doi:10.13560/j.cnki.biotech.bull.1985.2021-0497

摘要/Abstract

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。

关键词: 紫花苜蓿, AP₂/ERF转录因子, MsAP₂, 转基因

Abstract:

Alfalfa（Medicago sativa）is an important leguminous forage plant. Studying the function of MsAP₂ provides theoretical guidance and material basis for exploring the signal transduction network of MsAP₂,and also provides some reference for alfalfa biotechnology breeding technology. The MsAP₂ was cloned by RT-PCR,and the 3302-3flag-AP₂ plant expression vector was constructed by DNA recombination technology. The M. sativa was transformed by Agrobacterium-mediated method,and the regenerated plants were identified by transgene detection,gene expression analysis,determination of endogenous hormones（abscisic acid,cytokinin,auxin and gibberellin）and phenotype identification. The results showed that the MsAP₂ transgenic M. sativa plants were successfully obtained. Compared with the wild type,the MsAP₂ gene expression and hormone levels of transgenic plants were significantly changed. The transgenic plants demonstrated premature senescence,the leaf shape and size changed,the root growth was inhibited and the number of branches changed.

Key words: Medicago sativa L., AP₂/ERF transcription factor, MsAP₂ gene, transgene

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.

图/表 15

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 MsAP₂gene sequence
MsAP₂-r	GCAACTACACTATGAGCTCTAGATT	获得MsAP₂基因序列 Obtain MsAP₂gene sequence
M13f	CGCCAGGGTTTTCCCAGTCACGAC	检测克隆载体 Detection of clone vector
M13r	AGCGGATAACAATTTCACACAGGA	检测克隆载体 Detection of clone vector
3302-AP₂-f	GAACACGGGGGACTCTTGACATGGCTTCAACCACCAAAGA	构建表达载体 Construction of expression vector
3302-AP₂-r	TGTAATCCAGATCTACCATGCTAGATTAGAGTGCAATCAT	构建表达载体 Construction of expression vector
3302-f	TGACGCACAATCCCACTATCCTT	检测表达载体 Detection of expression vector
Nos181-r	CGTATTAAATGTAATTGCGGGAC	检测表达载体 Detection of expression vector
AP₂-RT-f	GGCTTCAACCACCAAAGACTCAG	基因表达分析 Gene expression analysis
AP₂-RT-r	CACTCCCTTGTACCGCTTCCATA	基因表达分析 Gene expression analysis
actin-f	TGATCTGGCTGGTCGTGACCTTA	扩增内参 Amplified internal reference
actin-r	ATGCCTGCTGCTTCCATTCCTAT	扩增内参 Amplified internal reference

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1 MsAP2的克隆

Fig.1 Cloning of MsAP2M:DNA marker DL2000 PlusⅡ. 1:MsAP2

图2 3302-3flag-AP2转大肠杆菌

Fig. 2 3302-3flag-AP2 transferred to E. coli M:DNA marker DL2000 PlusⅡ.1-5:3302-3flag-AP2

图3 3302-3flag-AP2转农杆菌

Fig.3 3302-3flag-AP2 transferred to Agrobacterium M:DNA marker DL2000 PlusⅡ. 1-5:3302-3flag-AP2

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

图9 转基因紫花苜蓿激素含量

Fig.9 Plant hormone contents of transgenic M. sativa

图10 转基因植株与野生型对比

Fig.10 Comparison of transgenic and wild-type plants

图11 转MsAP2植株与野生型植株

Fig.11 MsAP2 transgenic plants and wild-type plants

图12 转基因植株与野生型叶片对比

Fig.12 Comparison of transgenic and wild-type leaves

图13 转基因植株与野生型根系对比

Fig.13 Comparison of transgenic and wild-type roots

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紫花苜蓿转录因子基因MsAP₂的克隆及转化

Cloning and Transformation of MsAP₂ Gene in Medicago sativa

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