Cloning And Activity Analysis Of A Salt-Related Transcription Repressor OsZIM8 Promoter In Rice At Seedling Stage

In recent years,due to the effects of climate warming and irrational agricultural technology measures,the degree of soil salinization has been intensifying and the area has been expanding,which seriously affected the development of agricultural production in China.Rice planted in saline-alkali soil is one of the effective measures to utilize and improve saline-alkali soil.However,rice is extremely sensitive to salt stress at seedling and panicle differentiation stages,and there is a great influence on the development of rice production in saline soil.Therefore,studying the salt tolerance mechanism of rice and improving the salt tolerance of rice seedlings are of great significance for improving and making full use of the saline-alkali soil resources.In previous studies,a transcription represser OsZIM8 associated with salt tolerance at seedling stage was identified by fine mapping.On this basis,the promoter of this gene was cloned,and the bioinformatics analysis was carried out.The promoter expression fragment was transiently expressed.The analysis and alignment of the OsZIM8 promoter sequence and genotype analysis of japonica rice germplasm resources will lay the foundation for the functional identification of the OsZIM8 gene.The main findings are as follows:1.According to the genomic sequence of the cultivated rice variety Nipponbare,the OsZIM8 promoter was predicted to be 2453 bp in length.Bioinformatics analysis showed that the promoter contains the core promoter elements TATA-box,CAAT-box and enhancer,and there are a large number of other cis-acting elements,including photoresponsive elements,ABA response elements,drought stress response elements,the cryoresponsive element,the pathogen response element,the elemental response element,the salt inducing element,the MeJA response element,the wound response element,and the salicylic acid response element.The results indicated that the OsZIM8 promoter may be an inducible promoter,which is induced by response to various stresses.It may conditionally regulate the expression level of the OsZIM8 gene.2.The full length(2453bp)of OsZIM8 promoter and the deletion fragments of 353bp,853bp and 1353bp were obtained by PCR cloning using genomic DNA of Nipponbare as template.Recombinant expression vectors of the 353bp,853bp,1353bp and 2453bp OsZIM8 promoter deletion fragments and GUS reporter gene were constructed(pBWA(V)HG-OsZIM8-I,pBWA(V)HG-OsZIM8-II,pBWA(V)HG-OsZIM8-?,pBWA(V)HG-OsZIM8-IV).The Agrobacterium tumefaciens-mediated transformation of Nipponbare callus was carried out,and the transient expression level of GUS gene was detected by staining under transgenic callus under normal growth conditions and 125 mM NaCl stress.The results showed that under normal growth conditions,different OsZIM8 promoter deletion fragments were all activated,but there were some differences in the activity level between these four fragments.The 853 bp promoter fragment showed the strongest activity.The OsZIM8 promoter fragment of different lengths could respond to salt stress and when under salt stress,the expression activity of 1353 bp fragment was significantly decreased,while the OsZIM8 full length promoter activity level was increased.It is suggested that there may be enhancement elements in the 353-853bp fragment,and there may be stress-related inhibitory elements in 853?1353bp,which affect the expression of GUS gene.3.The salt tolerance of 90 japonica rice germplasm resources were evaluated.Five different sites were found between the promoters of OsZIM8 gene from different japonica rice germplasm resources by sequencing.They were SNP1,SNP2,Indell,Indel2,Indel3.Divided these 90 japonica rice germplasm into 6 types according to the different genotypes.Among them,The salt tolerant score of CC---(SNP2,SNP1,Indel3,Indel2,Indell from left to right)was significantly higher than other five types.