Genome-wide Analysis Of The MYB Gene Family In Tung Tree(Vernicia Fordii)

Tung tree is an important woody oil tree species in China.The oil extracted from the seeds of tung tree is called tung oil.Tung oil is an excellent dry vegetable oil,which is widely used in industrial production.The ratio of female to male was low,resulting in low yield per plant.It is important to study the development of tung tree flower to increase the yield of tung tree.MYB transcription factor is one of the largest family of transcription factors in plants,widely involved in the regulation of plant growth and development,metabolism,biological stress and non-biological stress,hormone response.This study used the flowers and seeds of different developmental stages of 'hunan putaotong' which is the main cultivation varieties of tung tree in Hunan Province as material.On the basis of the genome sequencing database of tung tree in our laboratory,we completed the following works:identification of the MYB transcription factor family of tung tree,expression patterns of MYB transcription factor family,cloning and functional validation studies of 2 MYB transcription factor members and a male sterility gene.The main research results are as follows:1.Identification of MYB transcription factor family from tung tree.The amino acid sequences of MYB transcription factors were retrieved from the annotation files of the genome sequencing database of tung tree in our laboratory,and were submitted to two online software Pfam and SMART for verification.At least 149 MYB transcription factors were confirmed to exist in the genome of tung tree.According to the characteristics of each MYB transcription factor domain,149 MYB transcription factors were divided into 5 categories:MYB-related(53),R2R3-MYB(91),3R-MYB(3),4R-MYB(1),5R-MYB(1).The functional prediction indicated that R2R3-MYB transcription factors may be involved in the regulation of male reproductive organ development,secondary metabolism,stress and other physiological processes.2.Cloning and expression pattern analysis of MYB transcription factor members from tung tree.Two MYB transcription factors,named VfMYB21 and VfMYB103,were isolated and cloned by RT-PCR amplification using male flowers of'hunan putaotong' as materials.Through the RNA-seq technology studied the expression pattern of VfMYB21 gene and VfMYB103 gene in 8 different development periods of flowers and 5 different development period of the seeds,and qRT-PCR technology studied the expression pattern of VfMYB21 gene and VfMYB103 gene in 20 different development periods of flowers and 8 different development period of seeds.The result show that:VfMYB21 gene is mainly expressed in flowers 1 day before flowering and 2 days before flowering,VfMYB103 gene is mainly expressed in flowers 30 days before flowering,two genes in 13 different development period of seeds were not expressed.3.Cloning and expression pattern analysis of a male sterility gene from tung tree.A male sterility gene was cloned from the male flowers of 'hunan putaotong' was named VfMS1.Through the analysis of the expression pattern of VfMSl gene by RNA-seq technology and qRT-PCR technology,it can be seen that the expression pattern of VfMS1 gene and MYB transcription factor member VfYB103 is similar,specifically expressed in flowers,and mainly expressed in flowers 30 days before flowering,not expressed in seeds of 13 different developmental stages.4.The subcellular localization of MYB21,MYB 103 and MS1 proteins of tung tree.DAPI was used to stain the nucleus and agrobacterium infects the instantaneous expression of tobacco leaves for subcellular localization analysis.The results showed that VfMYB21,ffMYB103 and VfMS1 proteins were located in the nucleus.5.The research of interactive relations between VfMYB103 and VfMS1 proteins.Yeast expression vector pGBKT7-VfMYB103 and pGADT7-VfMS1 were successfully constructed,and they were co-transformed into yeast strain AH 109.The results were verified by medium culture and the experiment of x-gal color reaction,the interaction between VfMYB103 and VfMS1 protein was confirmed.Therefore,VfMYB103 and VffMS1 proteins may be involved in the regulation of the development of tung flower.6.Transgenic plants with overexpression of VfMYB21 gene were obtained.Plant overexpression vector pCAMBIA 1300-35S-VfMYB21 was constructed by homologous recombination method.TO generation transgenic arabidopsis seeds were harvested and T1 generation transgenic arabidopsis positive plants were obtained after screening.