Verification Of Genetic Transformation Function Of Transcription Factor CsMYB77 And Their Interaction Gene Excavation

The annual supply of fresh citrus fruits and the enrichment of early and late maturing citrus varieties are one of the key research contents in China's citrus industry.Because of the long childhood and the low efficiency of conventional breeding methods,it has become an important breeding method to carry out citrus ripening breeding by molecular technology.Therefore,it is of great significance and application value to analyze the molecular mechanism of citrus fruit ripening regulation for the development of citrus industry.The transcription factor Cs MYB77(Csg23950)was genetically transformed into Tomato by predecessors,and it was preliminarily determined that the gene had the function of delaying the ripening of fruit.On the basis of this study,q RT-PCR analysis,double molecule fluorescence complementary and double luciferase interaction experiments were carried out to confirm the downstream target gene of the transcription factor Cs MYB77,which further explained the sub mechanism of the transcription factor regulating citrus fruit ripening and provided a theoretical basis for citrus ripening breeding.The main results are as follows:1.Phenotype observation and statistics were carried out on the T3 generation of Cs MYB77 transgenic tomato line.The fruit color breaking period was 3-5 days later than that of WT and control group.After 3 generations of observation,the delayed fruit ripening character of Cs MYB77 transgenic tomato line was stable.The expression of the ARF family gene in tomato was analyzed,and it was found that the gene Sl ARF6 was related to fruit color breaking;in addition,the gene Sl ARF6 and Sl ARF14 were up-regulated significantly.2.Cs MYB77-OE,Cs MYB77-CRISPR and PBI121 were genetically transformed into kumquat.Among them,6 positive seedlings were obtained from overexpression lines,with a positive rate of about 5.6%;3 positive seedlings were obtained from CRISPR lines,and 2 positive seedlings were obtained from PBI121,with a positive rate of 16.6% and15.4%,respectively.The phenotype of T0 generation of Cs MYB77 overexpression line was observed.The results showed that compared with WT,the overexpression line of Cs MYB77 had the characters of plant dwarfing,late flowering and late fruiting.The resultsof q RT-PCR showed that the expression of Fh MYB77 was 12-16 times higher than that of WT,and the expression of fharf6 and fharf14 were significantly different.In the Cs MYB77-CRISPR and wild type(WT)Satsuma,the target genes Fh MYB77,Fh ARF6,Fh ARF8 and Fh ARF18 were down-regulated in varying degrees.3.The double luciferase interaction between Cs MYB77 and Cs ARF6,Cs ARF17 was carried out.The results showed that there was no significant difference between the experimental group and the control group,that is,there was no interaction between Cs MYB77 and these two genes.The trans acting elements of 19 genes of Citrus ARF family were predicted.It was found that the promoter regions of Cs ARF5,Cs ARF8 and Cs ARF18 contained MBS1.Fh MYB77 and Fh ARF5,Fh ARF8,as well as Fh ARF18 were tested with double luciferase respectively.The results showed that Fh MYB77 and Fh ARF8 interacted with each other.Fh MYB77 may regulate the ripening of citrus fruit by binding to the promoter of Fh ARF8 gene...