Biotechnology Bulletin ›› 2021, Vol. 37 ›› Issue (12): 13-21.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0497
Previous Articles Next Articles
SHI Xin-yue(), SHANG Xiao-yao, ZHOU Ling-fang, ZHANG Tie-jun, CHAO Yue-hui()
Received:
2021-04-15
Online:
2021-12-26
Published:
2022-01-19
Contact:
CHAO Yue-hui
E-mail:shi_xinyue@163.com;chaoyuehui@bjfu.edu.cn
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 |
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 |
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.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
[1] |
Jofuku KD, den Boer BG, Van Montagu M, et al. Control of Arabidopsis flower and seed development by the homeotic gene APETALA2[J]. Plant Cell, 1994, 6(9):1211-1225.
pmid: 7919989 |
[2] |
Zhao L, Luo QL, Yang CL, et al. A RAV-like transcription factor controls photosynjournal and senescence in soybean[J]. Planta, 2008, 227(6):1389-1399.
doi: 10.1007/s00425-008-0711-7 pmid: 18297307 |
[3] |
Chung MY, Vrebalov J, Alba R, et al. A tomato(Solanum lycopersicum)APETALA2/ERF gene, SlAP2a, is a negative regulator of fruit ripening[J]. Plant J, 2010, 64(6):936-947.
doi: 10.1111/tpj.2010.64.issue-6 URL |
[4] | 马勇, 陈秀莉, 张红霞, 等. 乙烯应答因子在果实发育方面的作用[J]. 北方园艺, 2017(8):195-200. |
Ma Y, Chen XL, Zhang HX, et al. Roles of ethylene response factors in fruit development[J]. North Hortic, 2017(8):195-200. | |
[5] |
Zhang ZJ, Zhang HW, Quan RD, et al. Transcriptional regulation of the ethylene response factor LeERF2 in the expression of ethylene biosynjournal genes controls ethylene production in tomato and tobacco[J]. Plant Physiol, 2009, 150(1):365-377.
doi: 10.1016/S0176-1617(97)80135-6 URL |
[6] |
Zhang G, Chen M, Chen X, et al. Phylogeny, gene structures, and expression patterns of the ERF gene family in soybean(Glycine max L.)[J]. J Exp Bot, 2008, 59(15):4095-4107.
doi: 10.1093/jxb/ern248 URL |
[7] |
Aya K, Hobo T, Sato-Izawa K, et al. A novel AP2-type transcription factor, SMALL ORGAN SIZE1, controls organ size downstream of an auxin signaling pathway[J]. Plant Cell Physiol, 2014, 55(5):897-912.
doi: 10.1093/pcp/pcu023 URL |
[8] |
Rashotte AM, Mason MG, Hutchison CE, et al. A subset of Arabidopsis AP2 transcription factors mediates cytokinin responses in concert with a two-component pathway[J]. PNAS, 2006, 103(29):11081-11085.
pmid: 16832061 |
[9] | 张燕婉, 叶珏, 时那, 等. 蛋白质免疫印迹技术的实验研究[J]. 实验技术与管理, 2008, 25(10):35-37. |
Zhang YW, Ye J, Shi N, et al. Experimental research on protein immunoblot assay[J]. Exp Technol Manag, 2008, 25(10):35-37. | |
[10] | 李晓军, 秦浚川, 武建国. 蛋白印迹技术研究进展[J]. 临床检验杂志, 2004, 22(3):227-229. |
Li XJ, Qin JC, Wu JG. Advances in western blot technology[J]. J Clin Lab Sci, 2004, 22(3):227-229. | |
[11] |
Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T)method[J]. Nat Protoc, 2008, 3(6):1101-1108.
pmid: 18546601 |
[12] |
Zhao J, Li G, Yi GX, et al. Comparison between conventional indirect competitive enzyme-linked immunosorbent assay(icELISA)and simplified icELISA for small molecules[J]. Anal Chim Acta, 2006, 571(1):79-85.
doi: 10.1016/j.aca.2006.04.060 URL |
[13] |
Yang J, Zhang J, Wang Z, et al. Hormonal changes in the grains of rice subjected to water stress during grain filling[J]. Plant Physiol, 2001, 127(1):315-323.
pmid: 11553759 |
[14] | 武羿君, 徐庆国. 2, 4-D和6-BA对高羊茅愈伤组织诱导的影响[J]. 作物研究, 2012, 26(S1):21-24. |
Wu YJ, Xu QG. Effects of 2, 4-D and 6-BA on callus induction of tall fescue[J]. Crop Res, 2012, 26(S1):21-24. | |
[15] | 郭畅, 杨微, 林贵美, 等. ZT、KT和2, 4-D对香蕉胚性愈伤组织诱导的影响[J]. 现代农业科技, 2013(8):57-58, 61. |
Guo C, Yang W, Lin GM, et al. Effect of ZT, KT and 2, 4-D on induction of embryogenic callus from banana(Musa spp. )[J]. Mod Agric Sci Technol, 2013(8):57-58, 61. | |
[16] | 牛山, 韩清芳, 贾志宽. 不同处理对苜蓿扦插生根能力的影响[J]. 西北农业学报, 2007, 16(1):149-152. |
Niu S, Han QF, Jia ZK. Effects of different treatments on rooting capacity of Medicago sativa[J]. Acta Agric Boreali Occidentalis Sin, 2007, 16(1):149-152. | |
[17] | 蒙海涛, 韩清芳, 贾志宽, 等. 水培紫花苜蓿的茎段生根过程中内源激素含量变化[J]. 植物生理学通讯, 2008, 44(3):498-500. |
Meng HT, Han QF, Jia ZK, et al. Changes of the endogenous hormone contents in alfalfa stem segments during the rooting process under solution culture[J]. Plant Physiol Commun, 2008, 44(3):498-500. | |
[18] | 杜凯青, 张众, 张珊, 等. 生长素对苜蓿扦插的影响[J]. 草原与草业, 2019, 31(3):47-51. |
Du KQ, Zhang Z, Zhang S, et al. Effect of auxin on alfalfa cutting[J]. Grassland Prataculture, 2019, 31(3):47-51. | |
[19] | 马斯琦, 刘万峰, 吕林, 等. 烟草中脱落酸的相关研究进展[J]. 中国烟草科学, 2020, 41(1):94-100. |
Ma SQ, Liu WF, Lyu L, et al. Review on abscisic acid(ABA)in tobacco[J]. Chin Tob Sci, 2020, 41(1):94-100. | |
[20] | Lim PO, Nam HG. The molecular and genetic control of leaf senescence and longevity in Arabidopsis[M]// Current Topics in Developmental Biology. Amsterdam:Elsevier, 2005:49-83. |
[21] | 李琦. 赤霉素对植物生长影响的研究进展[J]. 农家参谋, 2018(5):86. |
Li Q. Research progress of gibberellin on plant growth[J]. Peasant Staff, 2018(5):86. | |
[22] |
高秀华, 傅向东. 赤霉素信号转导及其调控植物生长发育的研究进展[J]. 生物技术通报, 2018, 34(7):1-13.
doi: 10.13560/j.cnki.biotech.bull.1985.2018-0447 |
Gao XH, Fu XD. Research progress for the gibberellin signaling and action on plant growth and development[J]. Biotechnol Bull, 2018, 34(7):1-13. |
[1] | LI Jia-le, LIN Sheng-hao, XU Wen-tao. Construction of an Ultra-sensitive Colorimetric Biosensor for Insect Resistance Genes Based on Loop-mediated Isothermal Amplification [J]. Biotechnology Bulletin, 2022, 38(8): 69-76. |
[2] | ZHANG Ya-han, ZHU Li-xia, HU Jing, ZHU Ya-jing, ZHANG Xue-jing, CAO Ye-zhong. Opportunities and Challenges of Glyphosate in the Application of Biotechnology Breeding in China [J]. Biotechnology Bulletin, 2022, 38(11): 1-9. |
[3] | WANG Qi-wen, LI Pan, Pan Cui-yun, HAN Fen-xia. Effect of Ethylene Glycol on the Expression of Exogenous Genes in Vivo [J]. Biotechnology Bulletin, 2019, 35(4): 64-68. |
[4] | LIU Cha, HAN Li-hong, WANG Hai-bo, GAO Yong, TANG Li-zhou. Research Advances on Plant Thaumatin-like Protein Family [J]. Biotechnology Bulletin, 2018, 34(3): 9-17. |
[5] | Jing Zhaobin, Lei Yushan, Li Yongwu. Biotechnology and Kiwifruit Breeding in China [J]. Biotechnology Bulletin, 2015, 31(7): 1-10. |
[6] | He Qian, Wang Yanping, Chen Yuzhen, Lu Cunfu. Ectopic-overexpression of AmHsa32 from Ammopiptanthus mongolicus Promoted Heat Tolerance in Nicotiana benthamiana [J]. Biotechnology Bulletin, 2015, 31(5): 100-105. |
[7] | Lu Baorong. Analysis of Fitness Effect and Its Application in Assessing Environmental Risk Caused by Transgene Flow [J]. Biotechnology Bulletin, 2015, 31(4): 7-16. |
[8] | Wang Hefei, Liu Dongjun. Transgenic Technology: Establishment of Animal Models and Treatment of Diabetes Mellitus [J]. Biotechnology Bulletin, 2015, 31(10): 89-98. |
[9] | Lu Rui, Song Shaozheng, Qi Zhengqiang, Ge Xin, Shao Bin, Cheng Yong. Preparation of Recombinant Human Plasminogen Activator in Rabbit Mammary Gland and Detection of Expressed Products [J]. Biotechnology Bulletin, 2015, 31(10): 216-221. |
[10] | Li Ji, Lu Xu, Huang Tiandai, Hua Yuwei, Huang Huasun. Optimization of Digoxigenin Based Southern Blot for Transgenic Hevea brasiliensis Analysis [J]. Biotechnology Bulletin, 2014, 0(8): 76-81. |
[11] | Dong Ying, Hu Hongxia, Wang Wei, Tian Zhaohui. Construction of Transgenic Goldfish Based on Tol2 Transposable Element [J]. Biotechnology Bulletin, 2014, 0(5): 122-128. |
[12] | Gan Yimei, Zhang Shuzhen, Zeng Fanyun, Feng Cuilian, Yang Benpeng. Advance in Sugarcane Transgenic Breeding [J]. Biotechnology Bulletin, 2013, 0(3): 1-9. |
[13] | Chen Sijie, Zhang Hefei, Zhang Cuizhen, Peng Gang. Fluorescence-activated Cell Sorting(FACS)of Fluorescently Tagged Cells from Transgenic Zebrafish Larvae for Gene Expression Analysis [J]. Biotechnology Bulletin, 2013, 0(11): 112-116. |
[14] | Liang Hongwei Chen Faju Wang Yubing Li Fenglan, Lu Hai. Expression of Human Glucagon-like Peptide-1 in Transgenic Tobaccos [J]. Biotechnology Bulletin, 2013, 0(1): 139-143. |
Viewed | ||||||
Full text |
|
|||||
Abstract |
|
|||||