Study On The Mechanism Of VcHEC Gene In Dormancy Release Of Blueberry Flower Bud

The fruit yield of blueberry(Vaccinium Spp.)is closely related to the quality of flower bud dormancy release,and the pistil,as an important reproductive organ in flower bud,can affects the quality of flower bud,but also influences the fruit development in future,but there are few studies on the molecular mechanism of blueberry flower bud dormancy.As an important member of b HLH transcription factor family,HEC gene plays an important role in the development of pistil.In this study,blueberry flower buds were used as experimental materials,in the process of different types of flower buds dormancy release,morphological anatomy,physiology and molecular biology methods were used to explore the pattern of pistil development,changes of hormone contents and the expression patterns of VcHEC gene in different types of flower buds dormancy release.Exploring the gene function and network regulation mechanism by means of genetic transformation and yeast one-hybrid.This study can enrich the biological function of VcHEC gene.At the same time,it contributes a new idea to the study of dormancy release mechanism.Specific research results are as follows:(1)The flower bud of ’O’Neal ’blueberry was in the early stage of morphological differentiation on June 30,the scales and flower primordia with incomplete differentiation could be seen.On July 14,it was in the stage of inflorescence differentiation,and the central flower primordium began to sprout obviously.On July 29,it was in the stage of stamen and pistil differentiation,and the formed terminal flower and lateral flower could be seen.With the accumulation of cold capacity,the sprouting rate of flower buds increased significantly during the release of endo-dormancy.It was in the period of endo-dormancy during 0-8 days of cold treatment,and the endo-dormancy was released within 12 days of cold treatment,at which time the chilling requirement accumulated to 288 C.U,and then entered the stage of ecodormancy.By measuring five indexes of pistil morphological changes during the endodormancy release,it was found that with the release of endo-dormancy,the stigma width,style length,pistil length,flower longitudinal diameter and flower transverse diameter increased significantly,and the content of IAA in flower bud also increased significantly,which was the same as the trend of pistil growth and development,suggesting that the change of pistil size during endo-dormancy release may be related to IAA content.(2)Through phylogenetic analysis,it was found that CUFF.5776.1 is a homologous gene of HEC gene in Arabidopsis thaliana,named VcHEC gene,and carried out a series of bioinformatics analysis.The expression characteristics of VcHEC gene during morphological differentiation,physiological dormancy release,endo-dormancy release and ecodormancy release of flower buds were studied by q RT-PCR.The results showed that there were significant differences in the expression of VcHEC gene during the above processes,and the expression pattern of auxin transport gene Vc PIN3 during physiological dormancy release,endo-dormancy release and ecodormancy release was the same as VcHEC.It is suggested that the involvement of VcHEC gene in the process of dormancy release of blueberry flower buds may be related to auxin transport.(3)The biological function of VcHEC gene was analyzed by genetic transformation of Arabidopsis thaliana.The results showed that without 4℃ treatment before sowing,it took at least 60 h for wild type Arabidopsis seeds to germinate,but only 48 hours for transgenic seeds,and the overall germination rate of seeds was accelerated after cold treatment at 4℃for 1 day before sowing.After cold treatment for 2 days,the seed germination rate was greatly accelerated,but the germination rate of transgenic seeds was always higher than wild type.It is suggested that VcHEC gene can promote the seed germination of Arabidopsis thaliana,and the longer the accumulation of chiling,the higher the efficiency of promoting seed germination.Arabidopsis thaliana plants with overexpression of VcHEC gene showed early flowering and the number of rosette leaves was more than that of wild type.The main stem of transgenic Arabidopsis thaliana was thicker and its capsule were longer than those of wild type.(4)There are some light response elements such as G-box and ACE in the promoter of VcHEC gene and some action elements related to hormone response,such as TCA-element(salicylic acid response related to),ABRE(abscisic acid response)and P-box(gibberellin response related),indicating that the expression of VcHEC gene may be induced and regulated by light and hormones.Transient expression of different deleted fragments in tobacco showed that with the deletion of the 5 ’end sequence,the GUS staining of tobacco leaves injected with p1 VcHEC(-1775 bp ～-1 bp),p2 VcHEC(-1165 bp ～-1 bp)and p3 VcHEC(-694 bp ～-1 bp)decreased gradually,and the promoter activity decreased gradually,but the GUS staining intensity of the full-length pro VcHEC(-2272 bp ～-1 bp)of VcHEC promoter was significantly lower than that of p1 VcHEC without 497 bp.In transgenic Arabidopsis thaliana with pro VcHEC promoter,it was observed that pro VcHEC could drive the expression of GUS gene in stems,leaves and petals,but the activity of GUS protein was low,which was consistent with the transient expression of tobacco,so it was speculated that the cis-acting element between upstream gene and VcHEC promoter-2272 bp ～-1775 bp fragment was combined,thus inhibiting the expression of GUS gene.(5)The VcHEC-p Ab Ai bait expression vector was constructed by yeast one-hybrid experiment,and the bait vector was transformed into yeast.The c DNA AD fusion expression library was screened by self-activation detection,and 12 proteins or transcription factors that could bind to VcHEC-H1 promoter sequence were screened.These include squamosal promoter-binding-Like(SPL)transcription factors,prephenate dehydratase,GDSL esterase/lipase,seed ripening regulated protein,fucose gyrase,60 s ribosomal proteins,uncharacterized protein,wound-induced protein and annexin,etc.The binding between them and VcHEC-H1 promoter was confirmed by gyration verification.Through the analysis of the existing transcriptome data,it is found that the change of Vc SPL gene expression is negatively correlated with VcHEC,and it is speculated that it may be the key factor to inhibit VcHEC.The specific regulatory relationship between VcHEC gene with the above 12 genes needs to be further explored.