Cloning And Characterization Of GhYKT61 To Enhance Drought And Salt Tolerance In Plants

Abiotic stresses such as drought and salt severely affect the growth and development of plants,leading to a serious decline in crop yields.Soluble N-ethylmaleimide-sensitive factor attachment protein receptor(SNARE)is believed to be closely related to biological processes such as plant growth,development,cell homeostasis,and membrane fusion events in plants/crops.Many studies have shown that SNARE protein improves resistance to environmental stress by maintaining cell homeostasis.Drought and salt damage are the most important abiotic stresses in cotton production,which seriously affect its growth and development and yield formation.Although cotton is a pioneer crop tolerant to stress,cotton is mostly grown in arid and high-salt areas,and the intensified climate change in recent years has further aggravated the stress.Therefore,the use of agricultural biotechnology to improve the drought and salt tolerance of cotton and cultivate cotton varieties with high resistance to abiotic stress is of great significance for coping the climate change and ensuring stable and high yield of cotton.In this study,we used bioinformatics,biochemistry and molecular biology to clone the key R-SNARE gene Gh YKT61 from cotton in response to stress;using VIGS to interfere Gh YKT61 expression by drought and salt tolerance revealed significantly reduced in cotton.On the contrary,Gh YKT61 overexpressing plants showed significantly improved stress tolerance.The results are described precisely below.RNA-Seq analysis showed that cotton Gh YKT61(Gh?DO8G278300)gene expression was very significantly induced by drought and high salt stress,and this induced expression pattern showed a relatively high consistency in the vegetative growth and reproductive growth stages,suggesting that the Gh YKT61 gene is important genes for cotton drought and high salt response.Based on this,we cloned Gh YKT61,full-length coding region of upland cotton were amplified and ligated in Blunt zero vector by reverse transcription and PCR.Gh YKT61 contains 6 exons and 5 introns and encodes a typical VAMP-like protein,which is widely present in plants,and there are a large number of homologous genes in cotton.Further q RT-PCR analysis found that Gh?DO8G278300 is the most significant Gh YKT61 family member induced under adverse stress conditions.We use virus-induced gene silencing(VIGS)technology to interfere to the Gh YKT61 gene in cotton.The expression analysis found that the Gh YKT61 gene expression interference in cotton lines by varying degrees of down-regulation.The relative expression level of the Gh YKT61 gene in the VIGS cotton tissues showed significant down-regulation of three-fold compared to its expression in the non-silenced cotton seedlings and increased the sensitivity of silence plants to drought and salt tolerance by repressed ROS scavenging ability under drought or high-salt conditions.Correspondingly,VIGS interferes with key antioxidant enzymes in cotton,such as the activity of antioxidative enzymes,superoxide dismutase(SOD)and catalase(CAT),peroxidase(POD)and proline contents(Pro)were significantly declined in silence plants than in control normal plant.The above results indicate that VIGS plants suffer more extensive oxidative damage than normal control plants under stress conditions.Furthermore,construct the Gh YKT61 gene overexpression vector and genetically transform into tobacco.Two high-expressing strains OE-3 and OE-6 were screened by q RT-PCR analysis of positive transgenic tobacco.After 2 weeks of drought and high salt treatment in the greenhouse,the wild-type control showed severe wilting and dead phenotypes,while the wilting degree of the transgenic tobacco was significantly weaker than that of the wild-type control.Physiological index measurement results found that compared with the wild type,the relative water content(RWC),chlorophyll a and b contents of overexpression tobacco leaves under stress treatment conditions were significantly higher than that of the wild type control,and the antioxidant enzymes SOD,CAT,and POD activities and Pro content increased significantly.However,under normal growth conditions,the above physiological indicators are not significantly different between the over-expression line and the wild-type control.This marks the increasing scavenging ability of over-expressed tobacco plant.The above results indicate that the Gh YKT61 gene is an important functional gene in response to stress in cotton.Regulating the expression level of Gh YKT61 can significantly improve the resistance of plants to drought and salt stress.Therefore,Gh YKT61 can be used as an important genetic resource for cotton stress tolerance breeding.In the future,can deeply explore the biological function of Gh YKT61 gene in cotton stress tolerance response,clarify its molecular regulation mechanism,excavate excellent alleles in cotton resource materials,and use molecular breeding or genetic engineering to improve cotton strains and varieties resistance.