| Cotton is one of the important economic crops in our country.Cotton fiber is also the main raw material of the textile industry.Xinjiang cotton is not only a crop with high economic value,but also a support industry of the prosperity of farmers.The stable development of the cotton industry will not only promote the unity and development of various ethnic groups,but also contribute to the long-term stability of our country.Due to the influence of geographical location,Xinjiang already lacks water resources.However,as the global climate continues to change in recent years,Xinjiang’s natural precipitation has been further affected,resulting in frequent droughts,which have seriously affected the production and fiber quality of cotton in our country.Therefore,it is necessary to excavate the drought-tolerant genes of cotton and cultivate new drought-tolerant cotton varieties through molecular breeding,thereby enhancing the tolerance of cotton to drought stress and promoting the development of cotton industry.The WRKY family is one of the largest transcription factor families related to drought resistance in plants,and plays an important role in the process of plants resisting drought stress.In this experiment,a WRKY transcription factor gene GhWRKY70 was identified from Gossypium hirsutum L.,and its molecular mechanism of drought resistance was studied by gene silencing GhWRKY70 in G.hirsutum and heterologous overexpression in Arabidopsis thaliana.The key results are followed:(1)The full length cDNA of transcription factor gene GhWRKY70 was 828 bp,and GhWRKY70 protein consists of 275 amino acids.It has no signal peptide,no transmembrane structure,contains multiple phosphorylation sites and is a hydrophilic protein.Analysis and alignment of amino acid sequences and the subcellular localization of GhWRKY70 revealed that GhWRKY70 is a typical class Ⅲ WRKY transcription factor specifically expressed in nucleus.(2)The expression pattern of GhWRKY70 was analyzed.First,the expression pattern of GhWRKY70 in the drought-tolerant variety S08 and common variety C312 treated with PEG simulated drought stress,followed by the specific expression pattern of GhWRKY70 in different tissues and organs in G.hirsutum cv.C312.The results showed that the expression level of GhWRKY70 gene was induced by drought stress,and there was no significant change in S08,but significant change in C312 and high expression in leaves.(3)Using virus-induced gene silencing(VIGS)to silence GhWRKY70 in G.hirsutum and GhWRKY70 interference lines were obtained.Through the observation of their phenotype,the observation of stomatal opening and the measurement of related physiological indicators,we found that under drought stress,the interfered lines were more sensitive to drought stress,with larger stomatal opening,faster leaf water loss rate and greater plant damage compared to control plants.This indicated that silencing GhWRKY70 could reduce the tolerance of cotton to drought stress.(4)The transgenic A.thaliana and transgenic cotton embryogenic callus overexpressing GhWRKY70 was obtained by Agrobacterium-mediated genetic transformation technology.Through observation of the phenotype of transgenic Arabidopsis,measurement of stomatal opening,physiological indicators related to drought resistance,and expression level of genes related to drought stress,we found that under drought stress,the transgenic Arabidopsis was not sensitive to drought,the stomata closed early,the higher expression levels of the genes related to drought stress and less damage to plants.In addition,Arabidopsis leaves were treated with abscisic acid(ABA),and it was found that the closure of transgenic Arabidopsis stomata was more sensitive to ABA and the expression of ABA synthesis-related genes in transgenic Arabidopsis was higher.The results further indicated that GhWRKY70 played a positive regulatory role in the process of plant response to drought stress,which was dependent on ABA.In summary,this study identified a class Ⅲ WRKY transcription factor gene GhWRKY70,from G.hirsutum.The expression of this transcription factor gene was induced by drought stress,and it is highly expressed in leaves with obvious tissue and organ specificity.Subcellular localization showed that GhWRKY70 was specifically expressed in the nucleus.Through VIGS and Agrobacterium-mediated genetic transformation techniques,GhWRKY70-silenced cotton and overexpressed transgenic Arabidopsis were obtained,respectively.The phenotypes,physiological indicators and genes of the GhWRKY70-silenced lines and overexpressed lines were studied under drought stress.The results showed that GhWRKY70 plays a positive regulator to resist drought stress when plants are subjected to drought stress.The drought-tolerant mechanism of GhWRKY70 and the transgenic cotton embryogenic callus obtained in this study provided the basis for breeding drought-tolerant cotton varieties. |
