Biological Response And Regulatory Mechanism Of MiRNAs In Different Wheat Genotypes To Water Stress

As one of the most important abiotic stress,drought has a great impact on the growth and development of crops.According to the statistics,43% of the cultivated land is arid and semi-arid area.Therefore,most of the countries are facing to the problem of water crisis,which is also a problem in our country.Wheat is one of the most important crops around the world.More than 35% population is living mostly on the wheat.The demand of wheat is increasing as the population growing.However,most of the wheat in our country is planted in the arid and semi-arid area.The lost of wheat production because of lack of water might be more than the amount of all the other factors.That's why intense research of the drought resistance mechanism becoming so important.As we know,mi RNA plays an important role in the regulation of the growth,development and adversity stress of crops.After being exposed to the drought stress,crops will induce or inhibit expression of some mi RNA and cause a series changes on the morphological characteristics,physiology and biochemistry,which shows a comprehensive characteristics against the drought stress.Therefore,going into the drought resistance mechanism and indicate the characteristic of drought resistance based on the crops themselves has an important meaning on improving the ability of drought resistance.This article selects two representative wheat genotypes,Hanxan10 which is drought tolerant and Zhengyin1 which is drought susceptible,using the hydroponics method to study the changes of phenotype,physiology and cell and the resistance mechanism caused by mi RNA between different wheat genotypes.The main results are as follows:1.The growth and development of roots and leaves showed obvious differences in two wheat genotypes after water stress.Compared with the drought-tolerant cultivar,drought-susceptible cultivar shows a more severe dehydration stress symptoms.Such as a heavier wilting,a great reduction of roots length and number.In the same time,water stress can increase the concentration of the soluble protein in the leaves among different genotypes which can increase the root vigor but reduce the relative water content of leaves,chlorophyll content and the dry matter accumulation.The drought-susceptible cultivar has a more obvious reduction comparing with the drought-tolerant cultivar.This shows the water stress has an effect on the growth among the wheat of different genotypes and the effect on the drought-susceptible cultivar is heavier than the drought-tolerant cultivar.2.Water stress can increase the content of ABA in the roots and leaves and decrease the content of GA between different wheat genotypes.Among the drought-tolerant cultivar,the content of IAA increasing rapidly at the beginning of water stress.By stress time increasing,the content of IAA shows a downward trend.The content of IAA in drought-susceptible cultivar is always obviously lower than the CK.The content of ZR in different wheat genotypes is similar to the IAA.The change of IAA and ZR in different wheat genotypes indicates that at the beginning of the water stress the drought-tolerant cultivar can stimulate the growth of roots and leaves by accumulation of the IAA and ZR,which helps absorb the water and reduce the harm from water stress.3.The changes of cell differentiation were obvious in the leaf vascular tissue of different genotypes wheat under the condition of water stress.After water stress 12 h or 72 h,the cells of leaf xylem and phloem in drought-tolerant cultivar increased significantly,while the number of cells reduced significantly in drought-susceptible cultivar,especially after 72 h of dehydration stress,the number of cells in drought-susceptible cultivar decreased more significantly.These results indicated that drought-tolerant cultivar will strength the development of vascular tissue and improve the water efficiency by increasing the number of cells of leaf xylem and phloem in the process of water stress.Besides,water stress may lead the closure of stoma in different genotypes wheat,in terms of the degree of closure,the stomas of drought-tolerant cultivar were completely closed,while there were subtle gaps in the stomas of drought-susceptible cultivar,this can be attributed to the fast accumulation of ABA in root system of drought-tolerant cultivar in the initial stage of stress.4.Water stress can improve the contents of H2O2 and MDA and the production rate of O2-,but at many time points after stress,the increased range in drought-susceptible cultivar is more obvious than that in drought-tolerant cultivar.Besides,the activity of SOD?POD and CAT in different genotypes wheat increased first and decreased afterwards.But after 72~96h of water stress,the activity of antioxidase in drought-susceptible cultivar was significantly lowered compared to drought-tolerant cultivar.The result of further fluorescent quantitation shows that the relative expression of enzyme of Cu/Zn SOD?POD and CAT in drought-tolerant cultivar is significantly higher than that of drought-susceptible cultivar.These results indicate that the performance difference of different genotypes wheat in the resistance of water stress may be related to the antioxidant properties of themselves.5.With the method of deep-sequencing method,four small RNA libraries are built(C1,well-watered Hanxuan 10;T1,dehydration-stressed Hanxuan 10;C2,well-watered Zhengyin 1;T2,dehydration-stressed Zhengyin 1).The results showed that 367 mi RNAs in differential expression were identified in different genotypes wheat after water stress.Through the further comparative analysis,233 mi RNAs among the results in two genotypes wheat were both up-regulated expression,while 10 mi RNAs showed the tendency of down-regulated expression.It's interesting that 13 mi RNAs in two genotypes wheat exhibited opposite expression patterns after water stress.These mi RNAs were only down-regulated expression in drought-tolerant cultivar,while they showed the tendency of up-regulated expression in drought-susceptible cultivar.In addition,there also were 111 mi RNAs which had predominant expression only in one genotype wheat.6.Analyzing and verifying the expression level of some representative mi RNAs by q PCR and Northern blot,The result showed that the expression level of most mi RNAs keep consistent with deep-sequencing method.Besides,based on the method of bioinformatics,predicting the target genes of all differentially expressed mi RNAs,and commenting their functions.The biological functions of differentially expressed mi RNAs involves response to stress,metabolic process,regulation of transcription,growth and development,signal transduction,etc.The further research by q PCR verified the expression pattern of 12 representative target genes,and the result showed that there were negative correlation between the expression level of target genes and its corresponding mi RNAs,this provided evidence that mi RNAs may be involved directly or indirectly in the resistance of water stress by regulating the expression of their target genes.7.Combined with the changes of phenotype,physiology and cells of water-stressed wheat,analyzing the relationship between morphological and physiological characters and relevant mi RNAs and its corresponding target genes,then put forward the possible drought defense mechanism that mediated by mi RNAs for water-stressed wheat seedlings.