Functional Analysis Of A Small GTP-Binding Protein Gene GhROPs In Drought Resistance Of Cotton (Gossypium Hirsutum L.)

Cotton is an important agricultural economic crop in our country,and drought is the primary abiotic factor restricting the yield and fiber quality of cotton.Traditional cross-breeding cannot meet the actual needs of production and development.Therefore,mining drought-related genes in cotton and exploring the molecular mechanism of drought tolerance are of great significance to the innovation of cotton stress-resistant germplasm resources.As a plant-specific Rho-like small G protein,ROP protein plays the role of a“molecular switch”in plants,regulating the growth and development of plants and the stress response to adversity stress.In this study,the biological function of Gh ROPs gene in drought resistance of cotton was initially clarified by using VIGS technology,combined with the determination of relevant physiological and biochemical indicators.The main results were as follows:（1）Using upland cotton Xinluzao 22 as material,the expression patterns of 5 Gh ROPs（GhROP1,Gh ROP3,Gh ROP4,Gh ROP6 and Gh ROP8）genes were probed under 4 different tissues（root,stem,true leaf and cotyledon）and 4 different stresses（drought,high salt,low and high temperature）treatments using q RT-PCR.The results showed that 5 Gh ROPs genes were expressed in 4 different tissues,and had different degrees of response to 4 different stresses including drought.（2）Using upland cotton Xinluzao 26 as material,construction of silencing vectors for 5 Gh ROPs genes,and their biological functions in the drought-tolerance response of cotton were preliminarily explored by VIGS technology.The results showed that after inhibiting the expression of GhROP1,Gh ROP3,Gh ROP4,and Gh ROP8 genes,the drought tolerance of the plants was significantly improved.At the same time,the water loss rate（WLR）of the isolated leaves of the silenced plants was significantly reduced.The contents of amino acid（Pro）and chlorophyll（Chl）and the activities of 3 antioxidant enzymes were significantly higher than those of the negative control,and the contents of malondialdehyde（MDA）and hydrogen peroxide（H₂O₂）were significantly decreased;After inhibiting the expression of Gh ROP6gene,the drought resistance of plants did not change significantly.（3）GhROP1,Gh ROP3,Gh ROP4,and Gh ROP8 genes all responded to exogenous ABA and IAA treatment,q RT-PCR was used to detect the expression changes of abscisic acid（ABA）and auxin（IAA）signaling pathway-related genes in plants after VIGS treatment.The results showed that the expression levels of ABA synthesis and pathway-related genes in silenced plants were significantly increased,while the expression levels of metabolism-related gene Gh CYP707A were significantly decreased;the expression levels of IAA synthesis and pathway-related genes in silenced plants remained unchanged except for a few significantly lower than the negative control plants.（4）Constructed overexpression vectors of 5 Gh ROPs genes,and wild-type Arabidopsis thaliana was transformed by inflorescence dipping method.The results of GUS staining and RT-PCR showed that T₁transgenic positive plants of GhROP1 and Gh ROP4 had been obtained.The expression of 5 Gh ROPs genes all have certain tissue specificity and respond to abiotic stress such as drought;GhROP1,Gh ROP3,Gh ROP4 and Gh ROP8 genes may act as negative regulators in cotton drought resistance by participating in the negative regulation of ABA signaling pathway and the positive regulation of IAA signaling pathway;Gh ROP6 gene may not be involved in mediating the drought resistance of cotton,and its biological function remains to be further explored;Overexpression vectors of 5Gh ROPs genes were successfully constructed and transformed into wild-type Arabidopsis thaliana,which provided a material basis for further exploration of their biological functions using heterologous overexpression transgenic technology.