Identification And Functional Analysis On The Family Of Cytokinin Response Regulators (RRs) In Vernicia Fordii

Vernicia fordii is an important industrial oil tree species unique to my country.The oil extracted from its seeds is called tung oil,which is widely used in industrial production.Fewer female flowers and abnormal development of a single tree of tung tree are one of the main reasons for the low seed setting rate.Therefore,studying the development of female flowers of tung tree is of great significance to increase yield.In order to clarify the molecular mechanism of the female flower development of tung tree,this study used the flower buds of sanniantong treated with exogenous hormone 6-BA and without treatment,at different developmental stages as materials,based on tung tree genome and transcriptome data,Carried out the cloning and functional study of cytokinin response regulator(Response Regulators,RRs)family genes in tung tree.The main findings are as follows:1.Identification of RR gene family in tung tree.Using bioinformatics methods,28 members of the tung tree RR family were analyzed and identified from the tung tree genome database.The family’s encoded protein consists of 118-801 amino acids,with a molecular weight of 13.14-87.09 kDa and an isoelectric point of 4.91.Between-9.65.The chromosome location shows that 27 genes are located on 10 chromosomes,and they are unevenly distributed.According to the homologous evolution relationship,the VfRRs gene family can be divided into four subclasses:A,B,C,and PRR.The members of each subclass show high consistency in gene structure and conserved domain characteristics.This further supports the reliability of homologous evolution relationship clustering.2.Analysis of gene expression pattern of RR family in tung tree.Transcriptome data and qRT-PCR technology were used to explore the expression patterns of the flower buds,fruits and different tissues of tung tree at different developmental stages.The VfRRs genes are mainly expressed in the roots and stems of tung tree.The VfRRs genes are mainly expressed in the fruit.It is expressed in the first three stages of development(10,15,20 WAF),among which VfRR17a,VfRR2b,and VfRR10 genes are expressed at higher levels,indicating that they may be involved in the process of cell division and nutrient accumulation in the fruit.In 4 different development stages Among the flowers,B-type VfRR10,VfRR11a/b/c and VfRR12 and C-type VfRR22a have higher expression levels in flowers,indicating that these genes play a regulatory role in the development of tung tree flowers.Using qRT-PCR technology to analyze the expression patterns of flower buds at different developmental stages after and without treatment with the exogenous hormone 6-BA,the results showed that the expression pattern of VfRRs gene in untreated flower buds was basically consistent with the transcriptome data.This further illustrates the reliability of the transcriptome data.In the flower buds after 6-BA treatment,the expression levels of VfRR17a of type A and VfRR22a of type C were significantly up-regulated before treatment 30d,which indicates that VfRR17a and VfRR22a are affected by 6-BA.The induction of pistil may play an important role in the development of pistil.The 6-BA treatment failed to change the transcription level of class B VfRRs,indicating that class B VfRRs are not induced by 6-BA.3.Cloning of RR gene and analysis of protein subcellular location in tung tree.Five VfRRs were obtained by PCR cloning technology,and they were VfRR2b,VfRR10,VfRR12,VfRR17a,VfRR22a;by constructing a plant fluorescent protein fusion expression vector,using Agrobacterium to infect tobacco leaves for transient expression for tobacco subcellular localization analysis,It shows that VfRR2b,VfRR10,and VfRR12 of type B are located in the nucleus,and it is clear that VfRRs protein of type B has the nuclear localization characteristics of transcription factors.VfRR17a is located in the cytoplasm,it is speculated that there may be an unknown pathway involved in the regulation of flower development.VfRR22a is located in the endoplasmic reticulum,suggesting that VfRRs protein may also have cytokinins The function of the receptor.4.Analysis of heterologous expression of RR family genes in tung tree.By constructing a plant overexpression vector and transferring it into wild-type Col-0 Arabidopsis and American tobacco,VfRR17a/22a has harvested T3 generation and tested the gene expression level of T3 Arabidopsis positive seedlings.The results showed that the expression levels of Arabidopsis lines with VfRR17a/22a gene overexpression were significantly higher than those of wild-type lines.VfRR2b/10/12 harvested T2 generation positive transgenic plants,transgenic Arabidopsis plants have not been clearly observed phenotype.GUS test results of transgenic tobacco plants showed that VfRRs genes were mainly expressed in roots and leaves,and the expression in roots was mainly concentrated in the root tips,indicating that this may be related to the synthesis of cytokinins in the root tips.The genetic transformation of Arabidopsis and tobacco laid an important foundation for elucidating the function of tung tree RRs family in female flower development.