Transformation and Expression Pattern Analysis of Gene MtSAG113 from Medicago truncatula

doi:10.13560/j.cnki.biotech.bull.1985.2020-0481

Abstract

Abstract:

Senescence Associated Gene 113（SAG113）genes belong to PP2Cc super family and they are involved in plant senescence. This work aims to analyze the expression characteristics of the gene MtSAG113 of Medicago truncatula and to explore the function of the MtSAG113. This gene was cloned from M. truncatula. Using Nicotiana tabacum L. and M. truncatula R108 as experimental materials,the Agrobacterium-mediated leaf disc method was used to transform tobacco to obtain transgenic plants with resistance. The expression of MtSAG113 from M. truncatula was detected under induction of stress and exogenous hormones. The results showed that 5 transgenic plants successfully expressed in tobacco were obtained by PCR detection and the transgenic plants presented the phenotype of shortness and premature leaf senescence compared with the wild ones. The expressions of MtSAG113 in the senescence leaves were much higher than those in non-senescence leaves. In untreated leaves,the expression of this gene gradually increased with time and the gene was significantly up-regulated expressed under drought stresses. The expressions were significantly up-regulated under salt stresses and reached the peak when suffered for 12 h. The gene was particularly sensitive to 6-BA induction,and the expression increased significantly with time increasing,reached a peak at 4 h,which was about 1 000 times higher than those untreated. Treated with ABA and IAA,MtSAG113 exhibited a similar expression pattern,the expression increased from 4 h,reached a peak at 12 h. The subcellular localization results demonstrated that the gene was located in the nucleus. Above results indicate that MtSAG113 may play a certain role in senescence regulation in M. truncatula,and high salinity,drought and exogenous hormones regulate the expression of MtSAG113,which provides a basis for further function analysis of MtSAG113.

Key words: MtSAG113 gene, Medicago truncatula, tobacco, stress, exogenous hormone

LI Shu-wen, LI Yin-rui-zhi, DONG Di, WANG Meng-di, CHAO Yue-hui, HAN Lie-bao. Transformation and Expression Pattern Analysis of Gene MtSAG113 from Medicago truncatula[J]. Biotechnology Bulletin, 2022, 38(1): 108-114.

Figures/Tables 12

Fig.1 PCR detection of transgenic plants M ：DNA marker DL 2000 plus II; 1：WT; 2-8：regeneration plant

Fig.2 RT-PCR detection of transgenic tobacco M ：DNA marker DL 2000 plus Ⅱ; 1：negative control; 2-6：transgenic tobacco

Fig.3 Phenotype of transgenic tobacco 1: Wild type tobacco; 2-4: transgenic tobacco

Fig.4 Expression analysis of MtsAG113 gene in Medicago truncatula leaves

Fig.5 Comparison of mature and senescent leaves of Medicago truncatula 1: Mature leaves; 2: senescent leaves

Fig.6 Expression analysis of MtsAG113 gene in Medicago truncatula under untreated conditions

Fig.7 Drought-stress expression of MtsAG113 gene in Medicago truncatula

Fig.8 Expression of MtsAG113 gene in Medicago truncatula under salt stress

Fig.9 Effects of 6-BA induction on MtsAG113 gene expres-sion in Medicago truncatula

Fig.10 Effects of IAA induction on MtsAG113 gene expre-ssion in Medicago truncatula

Fig.11 Effects of ABA induction on MtsAG113 gene expre-ssion in Medicago truncatula

Fig.12 Results of subcellular localization

References 21

[1]	曹宏, 章会玲, 盖琼辉, 等. 22个紫花苜蓿品种的引种试验和生产性能综合评价[J]. 草业学报, 2011, 20(6):219-229.
	Cao H, Zhang HL, Gai QH, et al. Introduction test and comprehensive evaluation of production performance of 22 alfalfa varieties[J]. Acta Prataculturae Sinica, 2011, 20(6):219-229.
[2]	连燚, 冷暖, 李殷睿智, 等. 蒺藜苜蓿MtGT1基因克隆、表达及亚细胞定位分析[J]. 分子植物育种, 2019, 17(13):4241-4248.
	Lian Y, Leng N, Li YRZ, et al. Cloning, expression and subcellular localization analysis of MtGT1 gene of Medicago truncatula[J]. Molecular Plant Breeding, 2019, 17(13):4241-4248.
[3]	刘建利, 云天运, 曹君迈, 等. 酸处理对蒺藜状苜蓿种子萌发的影响[J]. 种子, 2008, 27(9):87-88.
	Liu JL, Yun TY, Cao JM, et al. The effect of acid treatment on the germination of alfalfa truncatula seeds[J]. Seeds, 2008, 27(9):87-88.
[4]	Kang Y, Li M, Sinharoy S, et al. A snapshot of functional genetic studies in Medicago truncatula[J]. Frontiers in Plant Science, 2016, 7:1175. doi: 10.3389/fpls.2016.01175 pmid: 27555857
[5]	Endre G, Kereszt A, Kevei, Zoltán, et al. A receptor kinase gene regulating symbiotic nodule development[J]. Nature(London), 2002, 417(6892):962-966. doi: 10.1038/nature00842 URL
[6]	Stacey G, Libault M, Brechenmacher L, et al. Genetics and functional genomics of legume nodulation[J]. Current Opinion in Plant Biology, 2006, 9(2):110-121. pmid: 16458572
[7]	王建勇, 姚晓华, 张志斌. 植物叶片衰老机理与调控研究进展[J]. 安徽农业科学, 2011, 39(31):19036-19038.
	Wang JY, Yao XH, Zhang ZB. Research progress on the mechanism and regulation of plant leaf senescence[J]. Journal of Anhui Agricultural Sciences, 2011, 39(31):19036-19038.
[8]	Breeze E, Harrison E, Mchattie S, et al. High-resolution temporal profiling of transcripts during Arabidopsis leaf senescence reveals a distinct chronology of processes and regulation[J]. The Plant Cell, 2011, 23(3):873-894. doi: 10.1105/tpc.111.083345 pmid: 21447789
[9]	王立, 张晓薇. 矮牵牛转SAG12-IPT基因的研究[J]. 重庆理工大学学报:自然科学, 2014, 28(9):62-65, 116.
	Wang L, Zhang XW. The transfer of SAG12-IPT gene from petunia[J]. Journal of Chongqing University of Technology(Natural Science), 2014, 28(9):62-65, 116.
[10]	Bustin S. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays[J]. Journal of Molecular Endocrinology, 2000, 25(2):169-193. pmid: 11013345
[11]	Ginzinger DG. Gene quantification using real-time quantitative PCR:An emerging technology hits the mainstream[J]. Experimental Hematology, 2002, 30(6):503-512. pmid: 12063017
[12]	Hoekema A, Hirsch PR, Hooykaas PJJ, et al. A binary plant vector strategy based on separation of vir-and T-region of the Agrobacterium tumefaciens Ti-plasmid[J]. Nature, 1983, 303(5913):179-180. doi: 10.1038/303179a0 URL
[13]	宋吉轩, 干旱胁迫下植物生长调节剂对羊草生长及生理特性的影响与转录组分析[D]. 重庆:西南大学, 2017.
	Song JX, Effects of plant growth regulators on growth and physiological characteristics of Leymus chinensis under drought stress and transcriptome analysis[D]. Chongqing:Southwest University, 2017.
[14]	樊海潮, 顾万荣, 尉菊萍, 等. 植物生长调节剂增强玉米抗倒伏能力的机制[J]. 江苏农业学报, 2017, 33(2):253-262.
	Fan HC, Gu WR, Wei JP, et al. Mechanisms of plant growth regulators enhancing corn lodging resistance[J]. Journal of Jiangsu Agricultural Sciences, 2017, 33(2):253-262.
[15]	Sillanpää M, Kontunen-Soppela S, Luomala EM, et al. Expression of senescence-associated genes in the leaves of silver birch(Betula pendula)[J]. Tree Physiology, 2005, 25(9):1161-1172. pmid: 15996959
[16]	Jing HC, Sturre MJ, et al. Arabidopsis onset of leaf death mutants identify a regulatory pathway controlling leaf senescence[J]. Plant Journal, 2002, 32(1):51-63. doi: 10.1046/j.1365-313X.2002.01400.x URL
[17]	Lim PO, Kim HJ, Hong GN. Leaf Senescence[J]. Annual Review of Plant Biology, 2007, 58(1):115-136. doi: 10.1146/arplant.2007.58.issue-1 URL
[18]	张兰, 滕珂, 尹淑霞. 草地早熟禾叶绿素酶1基因 PpCHL 1的克隆和表达分析[J]. 中国草地学报, 2016, 38(4):1-7.
	Zhang L, Teng K, Yin SX. Cloning and expression analysis of chlorophyllase 1 gene PpCHL from Poa pratensis L.[J]. Chinese Journal of Grassland, 2016, 38(4):1-7.
[19]	Singh S, Singh A, Nandi AK. The rice OsSAG12-2 gene codes for a functional protease that negatively regulates stress-induced cell death[J]. Journal of Biosciences, 2016, 41(3):445-453. doi: 10.1007/s12038-016-9626-9 URL
[20]	Chen HJ, Lin ZW, Huang GJ, et al. Sweet potato calmodulin SPCAM is involved in salt stress-mediated leaf senescence, H₂O₂ elevation and senescence-associated gene expression[J]. Journal of Plant Physiology, 2012, 169(18):1892-1902. doi: 10.1016/j.jplph.2012.08.004 URL
[21]	Zhang KW, Xia XY, Zhang YY, et al. An ABA-regulated and Golgi-localized protein phosphatase controls water loss during leaf senescence in Arabidopsis[J]. Plant Journal, 2012, 69(4):667-678. doi: 10.1111/tpj.2012.69.issue-4 URL