Cloning and Transformation of MsAP2 Gene in Medicago sativa

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

Abstract

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

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.

Figures/Tables 15

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

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

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

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

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

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

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

Fig.6 RT-PCR detection of transgenic M. sativa M:DNA marker DL2000 Plus II. 1-4: Transgenic alfalfa. W: Wild type

Fig.7 Protein detection of transgenic M. sativa M: Protein molecular weight standard. 1-4: Transgenic alfalfa. W: Wild type

Fig.8 Relative expression of MsAP2 gene of transgenic M. sativa Different letters indicate significant difference at 0.05 level, the same below

Fig.9 Plant hormone contents of transgenic M. sativa

Fig.10 Comparison of transgenic and wild-type plants

Fig.11 MsAP2 transgenic plants and wild-type plants

Fig.12 Comparison of transgenic and wild-type leaves

Fig.13 Comparison of transgenic and wild-type roots

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Cloning and Transformation of MsAP₂ Gene in Medicago sativa

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