Functional Characterization Of VcMYB And VcHSP17.7 And Establishment Of Leaf Regeneration System Of Blueberry

Blueberry,as an economically important fruit crop with recognized nutritional characteristics,is benefit for human health and has led to an increase in consumer demand over the last several years.Blueberry plants are susceptible to drought,low temperature,salt and other stresses in their production,which limit the production and cultivated area.The steadily increasing economic importance of blueberry has led to gaining a better understanding of the stress-tolerant mechanism of the blueberry crop in order to extend the growing areas.MYB transcription factors and small heat shock proteins play crucial roles in plant responses to various stresses.The objectives of this study were to provide theoretical basis and lay the foundation for stress resistance breeding of blueberry.A small heat shock protein(sHSP)gene and MYB transcription factor related to proanthocyanidin biosynthesis were isolated from Vaccinium corymbosum L.The promoters of the MYB and sHSP gene were also obtained.The characteristics and functions of the two genes and their promoters were analyzed.Adventitious shoot regeneration system from leaves of'Premier' blueberry cultivar was established.The main results are as follows:1.The cDNA sequence of VcMYB was obtained from V corymbosum by reverse transcription polymerase chain reaction(RT-PCR)and Rapid Amplification of cDNA Ends(RACE).The tertiary structure of VcMYB contains Myb-DNA-binding region.The VcMYB protein was predicted in the nucleus.The sequence of VcMYB share high homology with VuMYB,and it was close to VuMYB.2.Subcellular localization of the VcMYB protein in Nicotiana benthamiana leaves was carried out,the result revealed that VcMYB localized in the nucleus.3.The transcript was detected in all tested blueberry organs,including fruits,leaves and stems.The transcript levels were strongest in young fruits compared with other developmental stage fruits.4.The full-VcMYB coding sequence was inserted into the 3302Y to generate binary fusion construct 35S::VcMYB.Agrobacterium GV3101 transformed with plasmid 35S::VcMYB was used to infect Arabidopsis.The results showed that the transgenic plants response LED light treatment,the transcript levels of VcMYB was significantly elevated.5.The promoter sequence of VcMYB was cloned by the methods of FPNI-PCR.The promoter sequence was inserted into the pCAMBLA1391Z to generate binary fusion construct VcMYBpro::GUS.Agrobacterium GV301 transformed with the plasmid was used to infect Arabidopsis.The result of GUS histochemical staining showed that GUS reporter gene expression was driven by VcMYB promoter in different tissues and organs of transgenic plants,including rosette leaf,flower,silique and root.The transgenic plants could response to abiotic stress,the espression of the GUS gene could be induced by ABA(abscisic acid),low temperature,light treatment.6.VcHSP17.7,a sHSP gene was isolated from V.corymbosum,which has an ORF of 483bp,encoding 160 amino acids.The tertiary structure of VcHSP17.7 protein contains a-crystallin domain(ACD).The VcHSP17.7 protein was predicted in the cytosol.The sequence alignment analysis and phylogenetic analysis suggested that it belongs to cytosolic CII sHSP subfamily.7.Subcellular localization of the VcHSP17.7 protein in N.benthamiana leaves was carried out,the result revealed that VcHSP17.7 localized in the cytosol.8.The transcript was detected in all tested blueberry organs,including fruits,leaves,opening flowers and stems.The transcript level was higher in fruits at the late developmental stage.VcHSP17.7 gene expression was significantly induced by heat in leaves.The expression of the VcHSP17.7 gene in blueberry fruit was induced by heat shock treatment and gradually increased during subsequent low temperature storage.VcHSP17.7 might be involved in the chilling tolerance of blueberry fruit caused by heat shock treatment.9.Ttransgenic Arabidopsis plants overexpressing VcHSP17.7 decreased the heat resistance during germination,young seedling and adult plants period under heat stress condition.Heterologous expression of the VcHSP17.7 gene in Arabidopsis thaliana,whcih enhanced salt-tolerance of transgenic Arabidopsis young seedling.10.The promoter sequence of VcHSP17.7 was cloned.The sequence analysis of VcHSP17.7 promoter showed that it contains some cis-acting element involved in low-temperature,defense and stress,and light responsiveness et al.11.The coding sequence of VcHSP17.7 was inserted into the pEASY-E1 to generate binary fusion construct.And then the recombinant plasmid was transformed into Escherichia coli.The results displayed that the fusion protein were expressed under the induction of IPTG.Most of the fusion protein was produced in the form of inclusion body,and a small part was soluble protein.12.The young shoots of blueberry cultivars were used as explants,the test-tube plantlets were obtained.Adventitious shoot regeneration system from leaves of 'Premier' blueberry cultivar was established.Leaf explants excised from 6 week-old shoots newly developed from the cultures,were placed onto the adventitious shoot regeneration medium MWPM+4.0 mg/L ZT.The Leaf explants with adventitious shoot were transferred to MWPM plus 1.0 mg/L ZT for continued for 30 d,and then the elongated shoots were selected for rooting.The optimum medium for rooting was 1/2MWPM+1.5 mg/L IBA+0.5 mg/L NAA.