Crops ›› 2016, Vol. 32 ›› Issue (3): 37-44.doi: 10.16035/j.issn.1001-7283.2016.03.008

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Isolation of a Alkali Stress Responsive Gene GsCHX19 and Transformation into Medicago sativa L．

Yang Hao,Zhu Yanming   

  1. Key Laboratory of Agricultural Biological Functional Genes,Northeast Agricultural University,Harbin 150030,Heilongjiang,China
  • Received:2016-03-10 Revised:2016-04-15 Online:2016-06-15 Published:2018-08-26
  • Contact: Yanming Zhu

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Abstract:

GsCHX19, a cation/H + antiporter gene, has been identified as a putative bicarbonate stress responsive gene in previous studies, through bioinformatic analysis of transcriptome sequencing data of the Glycine soja G07256 under alkali stress. In this study, the full length coding region of GsCHX19 gene was cloned and constructed into the plant expression vector pCAMBIA330035Su. The recombinant plasmid was then transferred into the cotyledon nodes of Medicago sative by the way of agrobacterium-mediated genetic transformation method. And 20 resistant seedlings were obtained after screening on the medium supplement with 0.5mg/L glufosinate-ammonium. By detecting selection marker of bar gene and PCR test, and then RT-PCR, 11 plants were further confirmed as transgenic plants. The real-time PCR results indicated that GsCHX19 showed different expression levels in different transgenic plants. All the results showed that the GsCHX19 was transferred into alfalfa and successfully expressed. The transgenic plants will provide important experimental materials for development of saline-alkali tolerant alfalfa cultivars.

Key words: Cation/H + antiporter, GsCHX19, Glycine soja, Alfalfa, Transformation

Fig.1

GsCHX19 alkali stress transcriptome gene expression"

Fig.2

GsCHX19 gene full-length was amplified by PCR M:Nucleic acid molecular weight standards DL5000; 1:Deionized water as negative control; 2:PCR products"

Fig.3

Construction of plant expression vector of pCAMBIA330035Su-GsCHX19"

Fig.4

Plant expression vectors pCAMBIA3300-GsCHX19 plasmid identified by PCR M:Nucleic acid molecular weight standards DL5000; 1:Positive control(PCR productsof GsCHX19); 2:Deionized water as negative control; 3-5:Positive transformants"

Fig.5

pCAMBIA330035Su-GsCHX19 plant expression vector transformed into Agrobacterium PCR identification results M:Nucleic acid molecular weight standards DL5000; +:Positive control (pCAMBIA330035Su-GsCHX19 plasmid); –:Deionized water as negaive control; 1-6:Positive transformants"

Fig.6

PCR detection results of tansformed GsCHX19 gene resistance plants M:Nucleic acid molecular weight standards DL8000; +:Positive control (pCAMBIA330035Su-GsCHX19 plasmid ); -:Non-transgenic plants;1-10:Transformed GsCHX19 resistant plants"

Fig.7

RT-PCR detection of alfalfa of transformed GsCHX19 gene WT:Non-Transgenic alfalfa; 1-5:Transformed GsCHX19 resistant plants"

Fig.8

Real-time PCR detection of alfalfa of transformed GsCHX19 gene"

[1] 张立全, 张凤英 , 哈斯阿古拉.紫花苜蓿耐盐性研究进展. 草业学报, 2012,6(12):296-305.
[2] 唐立郦, 朱延明, 才华 , 等.GsZFP1基因植物表达载体构建及对苜蓿的遗传转化.作物杂志,2012(4):41-44.
doi: 10.3969/j.issn.1001-7283.2012.04.009
[3] 施莉娟, 许雷, 温洋 , 等. 新疆大叶紫花苜蓿两种再生体系的比较. 东北农业大学学报, 2010,41(4):94-97.
doi: 10.3969/j.issn.1005-9369.2010.04.020
[4] Manohar M, Shigaki T, Hirschi K D . Plant cation/H+ exchangers (CAXs):biological functions and genetic manipulations . Plant Biology(Stuttg), 2011,13(4):561-569.
doi: 10.1111/j.1438-8677.2011.00466.x pmid: 21668596
[5] Zhang Y, Peng X, Chai T , et al. Structure and function of tonoplast cation/H+ antiporters in plant:a review . Chinese Journal of Biotechnology, 2011,27(4):546-560.
doi: 10.3724/SP.J.1011.2011.00462 pmid: 21847988
[6] Reguera M, Bassil E, Blumwald E . Intracellular NHX-type cation/H+ antiporters in plants . Molecular Plant, 2014,7(2):261-263.
doi: 10.1093/mp/sst091 pmid: 23956073
[7] Bao A K, Du B Q, Touil L , et al. Co-expression of tonoplast cation/H+ antiporter and H-pyrophosphatase from xerophyte Zygophyllum xanthoxylum improves alfalfa plant growth under salinity,drought and field conditions . Plant Biotechnology Journal, 2016,14(3):964-975.
doi: 10.1111/pbi.2016.14.issue-3
[8] Song C P, Guo Y, Qiu Q S , et al. A probable Na+(K+)/H+ exchanger on the chloroplast envelope functions in pH homeostasis and chloroplast development in Arabidopsis thaliana . Proceedings of the National Academy of Science of the United States of America, 2004,101(27):10211-10216.
doi: 10.1073/pnas.0403709101
[9] Cellier F, Conejero G, Ricaud L , et al. Characterization of AtCHX17,a member of the cation/H+ exchangers,CHX family,from Arabidopsis thaliana suggests a role in K+ homeostasis . Plant Journal, 2004,39(6):834-846.
doi: 10.1111/tpj.2004.39.issue-6
[10] Chanroj S, Lu Y, Padmanaban S , et al. Plant-specific cation/H+ exchanger 17 and its homologs are endomembrane K+ transporters with roles in protein sorting . Journal of Biological Chemistry, 2011,286(39):33931-33941.
doi: 10.1074/jbc.M111.252650
[11] Evans A R, Hall D, Pritchard J , et al. The roles of the cation transporters CHX21 and CHX23 in the development of Arabidopsis thaliana. Journal of Experimental Botany, 2012,63(1):59-67.
doi: 10.1093/jxb/err271 pmid: 21976771
[12] Wang X Q, Shen X, Yun-mian H E, et al . An optimized freeze-thaw method for transformation of Agrobacterium tumefaciens EHA105 and LBA4404. Pharmaceutical Biotechnology, 2011,18(5):382-386.
[13] 郑先哲, 王建英, 董航飞 . 干燥条件对苜蓿品质指标的影响. 东北农业大学学报, 2009,40(6):101-105.
doi: 10.3969/j.issn.1005-9369.2009.06.020
[14] 李文桂, 陈雅棠 . 转基因苜蓿的研究进展. 热带医学杂志, 2009,9(10):1204-1208.
[15] DuanMu H, Wang Y, Bai X , et al. Wild soybean roots depend on specific transcription factors and oxidation reduction related genes in response to alkaline stress. Functional & Integrative Genomics, 2015,15(6):651-660.
[16] Sze H, Padmanaban S, Cellier F , et al. Expression patterns of a novel AtCHX gene family highlight potential roles in osmotic adjustment and K+ homeostasis in pollen development . Plant Physiology, 2004,136(1):2532-2547.
doi: 10.1104/pp.104.046003
[17] Padmanaban S, Chanroj S, Kwak J M , et al. Participation of endomem-brane cation/H+ exchanger AtCHX20 in osmoregulation of guard cells . Plant Physiology, 2007,144(1):82-93.
doi: 10.1104/pp.106.092155
[18] Pardo J M, Cubero B, Leidi E O , et al. Alkali cation exchangers: roles in cellular homeostasis and stress tolerance. Journal of Experimental Botany, 2006,57(5):1181-1199.
doi: 10.1093/jxb/erj114 pmid: 16513813
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