| Tung tree(Vernicia fordii)is a unique industrial oil tree in China,with seed kernel oil content up to 70%.The oil extracted from its seeds is called tung oil,which is a high-quality dry oil in industrial oil.Tung tree is mainly a unisexual flowering plant,and the number of female flowers is the key reason to determine the oil yield.Secondly,the female flowers are accompanied by stamen structure in the early development,the stamen structure needs to degenerate to form normal female flowers,otherwise it will form sterile hermaphrodite flowers.Therefore,it is important to increase the number of female flowers and ensure the normal development of female flowers to increase the yield of tung tree.In order to clarify the molecular mechanism of female flower development,the group screened out the Cytokinin Response Regulator VfRR22,which plays an important role in female flower development,based on the genomic data of tung tree.In this study,the biological function of VfRR22 was investigated by using gene cloning,quantitative real-time PCR,yeast two-hybrid interaction protein screening technique and transgenic technique using’Putao’ tung as the research material.It provides a scientific basis for illustrating the molecular regulation mechanism of VfRR22 in flower development of tung tree,and also provides an important genetic resource for revealing the female flower development mechanism of tung tree.The main findings are as follows:(1)The expression pattern of VfRR22 in different periods of floral development was found.Using the inflorescence buds of ’Putao tung’ at different periods and female and male flowers at different developmental stages after treatment with exogenous 6-BA as materials,we found that the expression of VfRR22 in female and male flowers at different flowering stages of tung tree was highest at 30 DBF(Days before The expression of VfRR22 was highest in female and male flowers at 30 DBF(Days before flowering)and decreased at 10 DBF and 1 DBF,indicating that VfRR22 played a role in the early development of female and male flowers of tung tree;VfRR22 expression was up-regulated before 12 h of exogenous 6-BA treatment,indicating its early positive response to the regulation of exogenous 6-BA;subcellular localization showed that the protein was localized in the nucleus The subcellular localization showed that the protein was localized to the nucleus and regulated in the nucleus.(2)Screening of the reciprocal proteins of the VfRR22.Using ’Putao’ tung inflorescence buds at different developmental stages as material,a yeast two-hybrid library was constructed and 132 candidate intercross proteins were initially screened using yeast two-hybrid technology,and seven intercross proteins were finally screened by point-to-point experimental validation,namely:meristem-specific expression protein KIN 10,mitochondrial synthesis-related protein NDUFB9,ERF2,GAPT,CABP,XBAT31,and ALE2.CABP and XBAT31 are all involved in the signalling response to the phytohormone ethylene.(3)The biological functions of the VfRR22 were clarified.Based on the sequence of the VfRR22,we constructed plant heterologous expression vectors and transferred them into wild-type Col-0 Arabidopsis and American safflower tobacco by using the floral tube passage method and leaf disk method,respectively.It was found that after exogenous 6-BA treatment,VfRR22 transgenic Arabidopsis showed significantly shorter inflorescence length,significantly earlier flowering time,slightly shorter pod spacing and slightly reduced number of pods.The floral organs of VfRR22 transgenic tobacco showed shortened filaments,depressed stigmas,and slow pollen tube growth rate,and the transgenic tobacco had smaller fruit pods,fewer seeds,and difficulty in natural fruiting.These results indicate that the VfRR22 plays an important role in flower development and lays a heavy foundation for the investigation of the mechanism of tung tree female flower development. |
PDF Full Text Request