Study On The Difference Of FRO2 Gene Expression In The Citrus Rootstock Of Trifoliata Orange(Poncirus Trifoliata L.Raf.) And Zhique(Citrus Wilsonii Tanaka)

In China,Chongqing,Sichuan,Southern Shaanxi,Danjiang Reservoir and other major citrus-producing areas,alkaline soils with limestone as the soil matrix,iron deficiency induced yellowing is common in citrus orchards,which seriously affects the yield and quality of citrus.Common methods used to overcome the iron deficiency not only have little effect,but also easily cause pollution to the environment.As we all know,citrus is propagated by grafting,and rootstocks have significant effects in nutrient absorption and utilization.Therefore,screening or breeding iron-tolerant rootstocks is considered to be one of the most economical strategies to solve the iron deficiency caused yellowing.We previously found that the Satsuma mandarin grafted on Zhique rootstock is resistant to iron deficiency in field condition,which was further confirmed by physiological experiments in green house,but the mechanism is unclear.Citrus is a dicotyledonous plant and belongs to mechanism I plant considering the iron absorption mechanism.The root system of the Mechanism I plant could complete the iron absorption through three processes of iron acidification,reduction and transport.In this study,the Fe-deficiency-tolerant rootstock Zhique and the Fe-deficiency-sensitive rootstock trifoliate orange were used as materials to explore the function of the citrus ferric chelate reducatase FRO2 gene.The expression of FRO2 and the difference in FRO2 promoter activity between two rootstocks under iron restriction were compared.By studying the FRO2 function,we aim to understand the reduction mechanism of iron absorption in citrus,and thus provides a theoretical basis for the screening and cultivation of iron efficient rootstock germplasms.The main results of this work are as follows:1.The open reading frames of Trifoliate orange and Zhique FRO2 are both 2157 bp,encoding 718 amino acids,but there are 19 amino acid sequence differences between the two rootstocks.2.The gene function complementary experiment by using Arabidopsis mutants proved that the FRO2 gene of citrus can restore the phenotype of the Arabidopsis mutant fro2-1 to the wild-type level under the condition of iron deficiency,and maintain the ironhomeostasis of Arabidopsis.3.Under the hydroponic iron deficiency treatment,the expression trend of FRO2 gene first increased and then decreased in the seedlings of Trifoliate orange and Zhique,and both reached the peak on the 10 th day of the treatment.However,the expression of FRO2 gene in Trifoliate orange and Zhique is different.On the 10 th day of iron deficiency,the expression of Trifoliate orange FRO2 gene was significantly higher than Zhique,which was 1.86 times that of Zhique.On the 15 th day of iron deficiency,the expression of Zhique FRO2 gene was significantly higher than Trifoliate orange,which was 1.63 times that of Trifoliate orange.4.On the 7th day of iron deficiency treatment,the results of Promoter::GUS showed that the activity of Trifoliate orange FRO2 promoter was stronger than that of Zhique,but the colorimetric experiment of high iron reductase showed that the secretion of ferric chelate reducatase in the roots of Zhique was higher than that of Trifoliate orange.There may be a post-transcriptional level of gene regulation that leads to differences in the secretion of ferric chelate reducatase from the Zhique and Trifoliate orange.5.The Trifoliate orange and Zhique FRO2 promoters were predicted to have a total of 25 functional elements with the same type,mainly involving light response,abiotic stress,plant hormone response elements and meristem-specific expression,etc.The number of each element was partially different.In addition,the Trifoliate orange has 2unique functional,namely anti-oxidation-related elements ARE and light-responsive elements TCT-motif.The Zhique has 8 unique functional elements,among which the element ERE involved in ethylene reaction and the element chs-CMA1 a related to light response are located in the Zhique promoter MITE.The difference in the cis-acting elements of the promoter may be related to the difference in the transcription level of the Trifoliate orange and Zhique.