Cloning and Salt-tolerance Analysis of Stress Associated Protein Gene GhSAP8 in Gossypium hirsutum
Wang Yi-Xue1,2, Dong Yan-Hui1, Zhang Huan-Huan1, Hao Yao-Shan1, Sun Yi1, Wu Shen-Jie1,2,*
1 Biotechnology Research Center, Shanxi Academy of Agricultural Sciences, Taiyuan 030031, China; 2 Shanxi Key Laboratory of Cotton Germplasm Resources Utilization and Molecular Design Breeding, Yuncheng 044000, China
Abstract：Stress associated proteins (SAPs) are zinc-finger proteins containing A20 and (or) AN1 domain which are involved in abiotic stress responses in plants. In this study, a stress associated protein gene, GhSAP8 (GenBank No. XM_016813997.1), was isolated from upland cotton (Gossypium hirsutum) via homology cloning. The full-length sequence of GhSAP8 gene was 519 bp, encoding a protein of 172 amino acids with a relative molecular weight of 18.22 kD and an isoelectric point of 7.51. GhSAP8 contained 1 conserved A20 domain in the N-terminus and 1 AN1 domain in the C-terminus which was typical SAP protein domain combinations. The expression of GhSAP8 gene was identified in different tissues, and qRT-PCR detection showed that the highest expression occurred in seedling leaf and it was induced by treatment of salt which suggested that GhSAP8 gene might be involved in responses to salt stress. The GhSAP8 gene was constructed into the plant expression vector pCAMBIA1300 and transformed into Arabidopsis thaliana. Under the treatment with 150 mmol/L NaCl, the survival rate (53%~70%) of Arabidopsis with overexpressed GhSAP8 gene was significantly higher than that of the wild type (27%), indicating the overexpression of GhSAP8 gene could enhance the tolerance to salt stress. In addition, the expression of salt-stress responsive genes, such as AtP5CS1 (Δ1-pyrroline-5-carboxylate synthetase 1), AtRD29B (responsive to desiccation 29 B), AtSOS1 (salt overly sensitive 1) and AtNHX1 (sodium/hydrogen exchanger 1), were significantly higher in transgenic Arabidopsis than that in wild type under salt stress. However, only a few of transgenic Arabidopsis lines showed increased expression of AtP5CS1, AtRD29B and AtSOS1 in wild type under non-salt stress. The above results indicated that GhSAP8 gene might improve tolerance to salt stress in transgenic Arabidopsis by regulating the expression of salt-stress responsive genes. The present study could provide superior gene resource for cultivation of transgenic salt-tolerant cotton.
[1] 陈芸, 郑勇, 刘霞, 等. 2016. 过量表达棉花CBF2基因提高转基因拟南芥抗旱耐盐能力[J]. 植物科学学报, 34(6): 888-900. (Chen Y, Zheng Y, Liu X, et al.2016. Overexpression of the cotton CBF2 gene enhances salt and drought tolerance in Arabidopsis thaliana[J]. Plant Science Journal, 34(6): 888-900.) [2] 王彩香. 2011. 小麦抗逆相关基因TaABC1和TaSAP1/2的分离及功能分析[D]. 博士学位论文, 中国农业科学院, 导师: 景蕊莲, pp: 50-51. (Wang C X.2011. Isolation and functional analysis of stress-response genes TaABC1 and TaSAP1/2 from wheat (Triticum aestivum L.) [D]. Thesis for Ph.D., Chinese Academy of Agricultural Sciences, supervisor: Jing R L, pp: 50-51.) [3] 杨勇, 田新权, 刘会利, 等. 2017. 陆地棉NAC转录因子基因GhNAC6的克隆、表达和耐盐性分析[J]. 棉花学报, 29(2): 138-146. (Yang Y, Tian X Q, Liu H L, et al.2017. Cloning, expression and salt-tolerance analysis of the NAC transcription factor gene GhNAC6 in upland cotton[J]. Cotton Science, 29(2): 138-146.) [4] 杨召恩, 杨作仁, 刘坤, 等. 2013. 一个亚洲棉MYB家族新基因的克隆及特征分析[J]. 中国农业科学, 46(1): 195-204. (Yang Z E, Yang Z R, Liu K, et al.2013. Cloning and characterization of a noval gene of MYB family from Gossypium arboreum L.[J]. Scientia Agricultura Sinica, 46(1): 195-204.) [5] 喻树迅. 2013. 我国棉花生产现状与发展趋势[J]. 中国工程科学, 15(4): 9-13. (Yu S X.2013. Present situation and development trend of cotton production in China[J]. Engineering Science, 15(4): 9-13.) [6] 张安红, 王志安, 肖娟丽, 等. 2017. 棉花抗逆基因GhAREB4的克隆及功能分析[J]. 西北植物学报, 37(5): 857-863. (Zhang A H, Wang Z A, Xiao J L, et al.2017. Cloning and characterization of a transcription factor gene GhAREB4 in Gossypium hirsutum[J]. Acta Botanica Boreali-Occidentalia Sinica, 37(5): 857-863.) [7] Asish K P, Anath B D.2005. Salt tolerance and salinity effects on plants: A review[J]. Ecotoxicology and Environmental Safety, 60(3): 324-349. [8] Bahmani K, Noori S A S, Darbandi A I, et al.2015. Molecular mechanisms of plant salinity tolerance: A review[J]. Australian Journal of Crop Science, 9(4): 321-336. [9] Ben Saad R, Fabre D, Mieulet D, et al.2012. Expression of the Aeluropus littoralis AlSAP gene in rice confers broad tolerance to abiotic stresses through maintenance of photosynthesis[J], Plant Cell and Environment, 35(3): 626-643. [10] Ben Saad R, Zouari N, Ben Ramdhan W, et al.2010. Improved drought and salt stress tolerance in transgenic tobacco overexpressing a novel A20/AN1 zinc-finger 'AlSAP' gene isolated from the halophyte grass Aeluropus littoralis[J]. Plant Molecular Biology, 72(1-2): 171-190. [11] Charrier A, Lelievre E, Limami A M, et al.2013. Medicago truncatula stress associated protein 1 gene (MtSAP1) overexpression confers tolerance to abiotic stress and impacts proline accumulation in transgenic tobacco[J]. Journal of Plant Physiology, 170(9): 874-877. [12] Charrier A, Planchet E, Cerveau D, et al.2012. Overexpression of a Medicago truncatula stress-associated protein gene (MtSAP1) leads to nitric oxide accumulation and confers osmotic and salt stress tolerance in transgenic tobacco[J]. Planta, 236(2): 567-577. [13] Clough S J, Bent A F.1998. Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana[J]. Plant Journal, 16(6): 735-743. [14] Giri J, Dansana P K, Kothari K S, et al.2013. SAPs as novel regulators of abiotic stress response in plants[J]. Bioessays, 35(7): 639-648. [15] Hasegawa P M, Bressan R A, Zhu J K, et al.2000. Plant cellular and molecular responses to high salinity[J]. Annual Review of Plant Physiology and Plant Molecular Biology, 51: 463-499. [16] Hozain M, Abdelmageed H, Lee J, et al.2012. Expression of AtSAP5 in cotton up-regulates putative stress-responsive genes and improves the tolerance to rapidly developing water deficit and moderate heat stress[J]. Journal of Plant Physiology, 169(13): 1261-1270. [17] Jain M, Khurana P, Tyagi A K, et al.2008. Genome-wide analysis of intronless genes in rice and Arabidopsis[J]. Functional and Integrative Genomics, 8(1): 69-78.
