关键词: 陆地棉, 盐胁迫, GhPEAMT1, 基因克隆, 基因沉默

Abstract:

【Objective】Phosphoethanolamine-N-methyltransferase (PEAMT) is the key enzyme in the synthesis of plant phosphocholine, which is the precursor of choline, a glycine betaine synthesis substrate that can enhance plant resistance. The biological function of GhPEAMT1 gene in response to salt stress in upland cotton was studied by cloning, expression pattern analysis and functional verification, so as to provide gene resources for breeding salt tolerant cotton varieties.【Method】According to the screening of transcriptome sequencing data, GhPEAMT1 gene was selected as a salt-tolerant candidate gene. The target gene was amplified by polymerase chain reaction (PCR). Gene structure characteristics predict protein relative molecular mass and evolutionary relationship were analyzed by bioinformatics; Salt-tolerant cotton variety Earlistable 7 and salt-sensitive cotton genotype Nandanbadidahua were treated with 200 mmol·L ^-1 NaCl solution, and cotton leaves and roots were taken for fluorescence quantitative PCR (qRT-PCR) to analyze the expression characteristics in tissues. Subcellular location vector was constructed for transient transformation of tobacco to determine the location of protein in the cell. The gene overexpression vector was constructed, and Arabidopsis thaliana was transformed by inflorescence infection method, and the germination rate and taproot length of transgenic Arabidopsis under salt stress were analyzed. Virus induced gene silencing (VIGS) technology was used on Earlistable 7 to silence the target gene and the efficiency of gene silencing was verified by real-time fluorescence quantitative analysis of the cotton leaves. Then the enzyme activities of catalase (CAT) and glutathione peroxidase (GPX) in the leaves of cotton were measured. 【Result】Two homologous genes, GhPEAMT1A and GhPEAMT1D, were cloned and the CDS lengths were 1 488 bp and 1 485 bp, respectively. The phylogenetic tree showed that GhPEAMT1 had the closest genetic relationship with the Sea island cotton, and had the higher homology with Cocoa compared with other species. Under salt stress conditions, the expression levels of GhPEAMT1A gene and GhPEAMT1D gene in leaves and roots of salt-tolerant Earlistable 7 were significantly higher than that of salt-sensitive Nandanbadidahua. Subcellular localization shows that the gene is located in the cytoplasm. Overexpression of this gene for Arabidopsis thaliana found that the germination rate of transgenic Arabidopsis thaliana was significantly higher than that of wild-type on MS medium of 100 and 150 mmol·L ^-1 NaCl. The taproot root length of transgenic Arabidopsis was significantly longer than that of wild-type on the MS medium of 50 mmol·L ^-1 and 100 mmol·L^-1 NaCl. After treatment with 40 g·L^-1 NaCl solution for 24 h, the cotton leaves with successfully silenced GhPEAMT1A and GhPEAMT1D gene were more wilting than those of CK and TRV2:00 injections. After salt stress, the TRV2::GhPEAMT1A cotton plants were reduced in catalase (CAT) and glutathione peroxidase (GPX) content in comparison with the unloaded injected cotton plants. 【Conclusion】Arabidopsis overexpression and VIGS experiments show that the GhPEAMT1 gene can respond to salt stress and play a positive regulatory role.

Key words: Upland cotton, salt stress, GhPEAMT1 gene, gene cloning, gene silencing