Study On The Function Of MiR156 Related To The Occurrence Of Paulownia Witches’ Broom

Paulownia is a species of Scrophulariaceae,which has strong adaptability and wide distribution.The promotion and planting of Paulownia can bring huge economic and ecological benefits.However,Paulownia is susceptible to be infected by phytoplasma in the growth process,which leads to the occurrence of witches’ broom,such as branches,yel owing and shrinking of leaves,shortening of internodes,and death of branches,which will eventually cause plant death.This has led to serious economic losses in many paulownia planting areas.The phytoplasma is difficult to be cultured in vitro.The early science and technology workers have carried out a lot of researches on the biological direction related to the occurrence of Paulownia witches’ broom(Pa WB),and the molecular regulation mechanism of Paulownia witches’ broom.has been preliminarily explored.Previous studies have shown that mi RNA plays important regulatory role in the growth and development of plants.Therefore,in-depth study of mi RNAs plays an important role in revealing the regulation of functional genes.In this study,Paulownia fortunei was used as material to study mi R156 related to the occurrence of paulownia witches’ broom found in our laboratory.The target gene of paulownia mi R156f-5p which was specifically related to the occurrence of paulownia witches’ broom was identified by degradome sequencing.After analysis,the positive protein interacting with its target gene SPL6 was screened by yeast two-hybrid technique,so as to further explore the expression and regulation mechanism of mi R156f-5p-SPL6 module in the occurrence process of paulownia witches’ broom.The results of the study are summarized as follows:1.Paulownia mi R156f-5p(Pau-mi R156f-5p)has a certain relationship with Gma-mi R156 b involved in the regulation of Glycine max(Linn.)Merr plant structure.In this study,a phylogenetic tree was constructed by bioinformatics analysis of the model plant Glycine max(Linn.)Merr and the mi R156 family of Paulownia,Theobroma cacao,and Vitis vinifera L.plants with witches’ broom.The analysis showed that the mi R156 gene family have a highly conservative among different species.Conservatively,Pau-mi R156f-5p has a certain relationship with Gma-mi R156 b,which regulates plant structure in Glycine max(Linn.)Merr.Therefore,further analysis of mi R156f-5p differentially expressed during the occurrence of Paulownia witches’ broom.The results showed that the precursor sequence could form a perfect secondary stem-loop structure,and the mature sequence was located on the arm of the 5’ end of the hairpin structure,which was consistent with the mi RNA precursor sequence characteristics.2.Pau SPL6 is a target gene of mi R156f-5p specifically related to the occurrence of Paulownia witches’ broom.The seven target genes of mi R156f-5p were identified by the Target Finder software and degradome sequencing technology,and the expression levels of differentially expressed target genes in the diseased and healthy seedlings were further combined with the previous transcriptome sequencing.The q RT-PCR experiment verified the accuracy of the previous sequencing results.In addition,the expression pattern between Paulownia mi R156f-5p and its target gene is negatively regulated.Bioinformatics analysis showed that the SPL gene family in different species had a SBP conserved domain,which are highly conserved in different species.The results of homology analysis indicated that Pau SPL6 was the main target gene in response to the occurrence of Paulownia witches’ broom.3.Fifteen proteins that interacted with Pau SPL6 were initially screened.In this experiment,a yeast two-hybrid c DNA library of Paulownia was constructed,and the self-activation and toxicity detection experiments of bait protein were also carried out.The results showed that the bait expression vector itself has no transcriptional activity,can be successfully expressed in yeast,and has no toxicity to yeast cel s,and can be used for screening yeast two-hybrid c DNA library.172 positive clones were screened by three high stringency screenings,and 15 positive proteins interacting with Pau SPL6 protein were obtained by sequencing analysis.