Functional Analysis Of Cotton (Gossypium Hirsutum)GhPIP1;4 On The Response To Plant Drought And High Salt Stress

With the global warming,shortage of water resources and soil salinization become more and more serious,resulting in the drop of the average production of major crops in the world by as much as 50%.Therefore,it is of great significance to explore functional genes involved in stress resistance and apply them in genetic breeding to improve crop yield and quality.Cotton(Gossypium hirsutum)is one of the important economic crops of China,which plays important roles in the national economy.However,harsh environments such as drought and high salt severely restrict cotton production and quality.It is well known that plant aquaporins not only participate in the transport of water,but also play an important role in the stress response of plants.In present study,we isolated and identified a cotton plasma membrane aquaporin gene named GhPIPl;4.And then we studied the expression pattern of GhPIP1;4 and the activity of its promoter.At the same time,the overexpression and RNAi vectors of GhPIP1;4 were constructed and transformed into arabidopsis thaliana and cotton,respectively.Multiple lines of transgenic plants were obtained and were used to investigate the function of GhPIP1;4 in plant response to stress and drought stress.The main results are as following:1.GhPIP1;4 is preferentially expressed in cotton roots.To study the expression pattern of GhPIP1;4,total RNAs of cotton roots,leaves,and other tissues were extracted and used in Northern blot analysis.The result showed that GhPIP1;4 was preferentially expressed in cotton roots.Moderate hybridization signal was also detected in hypocotyls and petals.To investigate whether the expression of GhPIP1;4 is regulated by root development,cotton roots at different developmental stages and partitioning the roots were collected and used in Northern blot analysis.The result showed that the transcripts of GhPIP1;4 were mainly concentrated in young roots,especially in the tips of young roots.To further check the fine expression site of GhPIP1;4 in the root,in situ hybridization experiment was performed.The results showed that the hybridization signal mainly appeared in the vascular tissue of the cotton root tip.In summary,the GhPIP1;4 gene is predominantly expressed in cotton roots and it is presumed that it may participate in root water transport.2.Activity analysis of GhPIP1;4’s promoter.To investigate the activity of GhPIPl;4’s promoter,the 1.2 kb upstream sequence of this gene was cloned and inserted into pBI101 plasmid.And then the constructed pBI101-GhPIP1;4p;:GUS fusion expression vector was transformed into Arabidopsis.Multiple transgenic lines were obtained which express GhPIP1;4p::GUS stably.We found that the GUS signal was mainly distributed in the roots of Arabidopsis thaliana through GUS histochemical staining analysis.GUS signals were also observed in the main veins of rosette and cauline leaves,but no GUS signal was detected in the organs such as stems,capsules,and flowers.3.Identification of over-express GhPIP1;4 transgenic arabidopsis and analysis of salt and drought toleranceIn order to study the function of GhPIP1;4 its ORF frangment was inserted into the pBI121 plasmid to construct its overexpression vector.Then the pBI121-35S::GhPIP1;4 vector was transformed into arabidopsis and multiple transgenic lines stably expressing GhPIP1;4 were obtained.The plants with higher expression levels of GhPIP1;4 were selected out and treated with NaCl,mannitol,and drought,their phenotypes were observed,as well as their physiological indicators(such as SOD,H2O2,POD,MDA,proline,chlorophyll,etc.)were determined.Comparing with wide type plants,transgenic plants exhibited significant growth advantages and tolerance to high salinity and drought stress.It indicated that GhPIP1;4 was invloved in response to salt and drought stress of plants.4.Identification of GhPIPl;4-RNAi transgenic cotton and analysis of salt and drought toleranceIn order to further study the function of GhPIP1;4 in cotton,we selected the 3’-UTR sequence with a strong sequence specificity of GhPIP1;4 to construct a pBI121-35S::GhPIP1;4-RNAi vector.After transformation into cotton,the positive transgenic lines with significantly decreased expression levels of GhPIP1;4 were selected out by PCR and RT-PCR for stress treatment.These plants were treated with mannitol,NaCl,and drought,and their phenotypes were observed,and the physiological indicators(such as SOD,H2O2,POD,MDA,proline,chlorophyll,etc.)of the plants were measured.Compared with wild type plants,it was found that the transgenic cotton showed obvious growth disadvantage and certain susceptibility to high salt,drought and osmotic stress.It can be concluded that the silence of GhPIP1;4 gene increase the sensitivity of plants to drought and salt stress.In a word,cotton GhPIP1;4 is expressed dominantly in cotton roots.Overexpression of this gene may significantly increase salt tolerance and drought resistance of plants.Conversely,using RNAi technology to decrease the expression level of this gene,the transgenic plants become more sensitive to high salt and drought stress.These data strongly suggest that GhPIP1;4 may be involved in plant response to drought and salt stress.