Cloning And Functional Comparison Of Iron Transporter IRT1 And Its Promoter In Trifoliate Orange And Ziyang Xiangcheng

Iron(Fe)deficiency is common in citrus plants that grow on alkaline or slightly alkaline soil,especially those grafted on trifoliate orange [Poncirus trifoliata(L.)Raf.] rootstocks.At present,the underlying reason regarding to trifoliate orange is susceptible to Fe deficiency stress and the molecular biological studies on absorption and transport of Fe in citrus are scarce.It has been well documented that IRT1(iron-regulated transporter 1),as a transporter,plays an important role in the absorption of Fe by plant roots,and its transcription expression level is regulated by upstream transcription factors.Previously,we had proved that trifoliate orange Pt IRT1 showed strong Fe absorption ability in yeast,but its transcriptional expression level was obviously lower than that of Ziyang xiangcheng(Citrus junos cv.Ziyang xiangcheng),a rootstock tolerant to Fe deficiency.Therefore,we speculate that although Pt IRT1 is a high Fe absorptive transporter,low expression level in trifoliate orange might limit its Fe absorption efficiency and lead to Fe deficiency in trifoliate orange.In order to confirm our hypothesis,and to study the cause of low expression of Pt IRT1,trifoliate orange(senesitive to Fe deficiency)and Ziyang xiangcheng(tolerant to Fe deficiency)were used as materials,and IRT1 gene and its promoter were isolated and functional comparison in these two rootstocks.In addition,interaction of IRT1 promoter and FIT transcription factor was confirmed via yeast one-hybridization.This study is a continuation and in-depth study of previous work,which can not only help answer scientific questions in citrus production practice,but also provide a new theoretical basis for citrus related fields and lay a foundation for the final solution of citrus Fe deficiency.In this study,IRT1 gene and FIT transcription factor were first isolated from hydroponic seedlings of trifoliate orange and Ziyang xiangcheng,respectively,and their expression levels were determined in different tissues and under iron deficiency conditions.In addition,IRT1 promoter were isolated from two rootstocks and fused with GUS to transform Arabidopsis thaliana for analyzing promoter activity.Yeast onehybridization was performed to verify the interaction of IRT1 promoter and FIT transcription factor.The main results are as follows:1.Cloning and sequence comparison of IRT1 gene and FIT transcription factor in trifoliate orange and Ziyang xiangcheng.The ORF full-length of IRT1 and FIT were cloned from two rootstocks,respectively,and they were named Pt IRT1 and Pt FIT for trifoliate orange,and Cj IRT1 and Cj FIT for Ziyang xiangcheng.Sequencing results showed that ORF length of both Pt IRT1 and Cj IRT1 were 1061 bp,and there were three nucleotides and one amino acid differences between Pt IRT1 and Cj IRT1(99.43% similarity).The ORF length of both Pt FIT and Cj FIT were 941 bp,but there were 17 nucleotides and 10 amino acids differences between Pt FIT and Cj FIT(96.81% similarity).2.Expression patterns of PtIRT1,CjIRT1,PtFIT and CjFIT.Under normal condition, expression levels of PtIRT1,CjIRT1,PtFIT and CjFIT were very low in stem and leaf,but significantly higher in root.In the condition of iron deficiency,the expressions of Pt IRT1,Cj IRT1,Pt FIT and Cj FIT in the root were significantly upregulated.After 10 d of iron deficiency,the up-regulated levels of Cj IRT1 and Cj FIT in the root were significantly higher than those in Pt IRT1 and Pt FIT.3.Cloning and sequence analysis of IRT1 promoter of trifoliate orange and Ziyang xiangcheng.2022 bp and 1966 bp of promoter fragments before the transcription initiation site of IRT1 gene were cloned from trifoliate orange and Ziyang xiangcheng,respectively,and the sequence similarity of the two promoters was 91.54%.Component prediction analysis on Plant CARE showed that the Pt IRT1 promoter and Cj IRT1 promoter have 19 types of functional components respectively,including the core component that makes up the promoter and the environmental response component.The core components are CAAT-box,TATA-box,etc.,and the environmental response components include optical response components and hormone response components,like Box 4,I-box,TATC-box,TCA-element,ABRE,etc.Two components in Pt IRT1 and Cj IRT1 promoter are different.The TC-rich repeats and LTR components in Pt IRT1 promoter are involved in response to stress and low temperature respectively,and the AAAC-motif and GT1-motif components in Cj IRT1 promoter are involved in response to light.4.Genetic transformation and activity analysis of IRT1 promoter in Arabidopsis thaliana.The upstream promoter fragments of IRT1 transcription initiation site of trifoliate orange and Ziyang xiangcheng with a length of 1500 bp,1000bp,500 bp and 300 bp were cloned.These fragments were connected to GUS fusion expression vector and then genetically transformed into Arabidopsis thaliana.After three generations of culture,homozygous plants containing target fragments were selected.GUS staining was performed on these plants after normal and iron-deficient culture to analyze the activity of different promoter segments.The results showed that under normal culture conditions,the seedlings of Arabidopsis thaliana with different fragments were stained to a certain extent.Among them,all seedlings transferred into the 1500 bp fragment will be stained blue,and the degree of staining is relatively deep.Plants transferred into 1000 bp,500bp and 300 bp segments will have a small amount of staining on leaves and roots.In the condition of iron deficiency,the whole plants with Pt IRT1 promoter 1500 bp,1000bp and Cj IRT1 promoter 1500 bp,1000bp and 500 bp were stained deeply.The results showed that IRT1 promoter was activated by iron deficiency stress.5.Yeast one-hybridization to verify the interaction between FIT transcription factor and IRT1 promoter.Build contain prey plasmid of transcription factors,and cloned the length of 1000 bp Pt IRT1,Cj IRT1 promoter fragments into the bait vector,putting the bait vector into yeast,after background expression level detection,select the appropriate Ab A concentration,then transformation prey vector into the yeast,and inoculated in medium with appropriate Ab A concentration.If the transgenic yeast can grow normally,that means transcription factors FIT and Pt IRT1,Cj IRT1 promoter exist interaction,and lays a foundation for subsequent screening of functional elements in the promoter that interact with FIT.