Cloning Of Transcription Factor VvMYBA6 And VvMYB12 In Vitis Vinifera L. And Functional Identification Of Genes In Arabidopsis Thaliana

Flavonoids is one of the most important plant pigments,which plays a key role in human health protection.In addition,it is also one of complex traits,which is associated with the temperature,humidity,light quality and cultivation practices.In order to provide interest information of genetic and molecular regulation mechanism of flavonoids biosynthesis,this review describes related genes in flavonoids biosynthesis in plants.Abiotic stresses,such as drought,salinity,chemical toxicity,and oxidative stress have enormous impact on agriculture production and result in the deterioration of the ecological environment.Plants respond and adapt to abiotic stresses by a variety of biochemical and physiological mechanisms during growth and development,which requires for precise regulation of functional genes expression.Therefore,the review of resistance genes is important for exploring the molecular mechanism of plant development,improving agricultural traits and ecological environment.According to the research of model plant Arabidopsis thaliana,we studied the expression characterization of these genes and expect that it will be used in the genetic engineering breeding and regulate gene expression.The main results are as follows:1.Two kinds of transcription factor MYB from Vitis Vinifera L.,VvMYBA6 and VvMYB12 were synthesized with the method of PTDS,and their functions were identified in Arabidopsis thaliana.The full-length cDNA of VvMYBA6 has a 738 bp open reading frame(ORF)and encode 245 amino acids,with a molecular mass of 28.09 KDa and a pI of 9.24.Sequence analysis via BLAST、MEGA 6.0 and GenBank shows that VvMYBA6 has a high identify and close relationship with genes related to flavonoids biosynthesis.Real-time quantitative PCR analysis showed that the transcript level of VvMYBA6 was highest in the berry skin of Vitis vinifera and the leaf of transgenic Arabidopsis plants,which was positive correlated with flavonoids accumulation in different organs of Vitis vinifera L.and Arabidopsis thaliana.These results demonstrated that transcription factor VvMYBA6 might be involved in flavonoids biosynthesis.2.Compared with WT,VvMYBA6 transgenic Arabidopsis plants accumulated higher content of flavonoids;HPLC showed that compared with WT,higher content of cyanidin,petunidin and pelargonidin were accumulated in VvMYBA6 transgeninc Arabidopsis plants,especially cyaniding.Under salt stress,compared with WT,VvMYBA6 transgenic plants showed significantly enhanced tolerance against salt stress.qRT-PCR analysis demonstrated that VvMYBA6 expressing has significantly up-regulated expression level of genes involved in flavonoids biosynthesis,ABA biosynthesis,proline biosynthesis and ROS scavenging system,which led to higher content of flavonoids,ABA,proline and LEA and higher activities of superoxide dismutase(SOD),peroxidase(POD)and catalase(CAT),as well as reducing the content of H2O2 and malonaldehyde(MDA)so as to avoid cell membrane damage.In conclusion,VvMYBA6 could be used as an important transcription factor in the promotion of salt tolerance in plants through up-regulating biosynthesis of flavonoids,ABA and proline,and ROS scavenging system.3.Under 24 mg/L 2,4-DNT stress,seed germination of VvMYB12 transgenic Arabidopsis plants was significantly higher than WT;the growth condition and survival rate of transgenic plants were much better than WT under 2,4-DNT stress;HPLC analysis showed that compared with WT,Arabidopsis plants expressing VvMYB12 absorbed higher content of 2,4-DNT and kept healthy.Real-Time quantitative PCR(qRT-PCR)analysis showed that expression of VvMYB12 up-regulated expression level of genes involved in flavonoids biosynthesis and abiotic stress resistance,which resulted in higher content of flavonoids,ABA,LEA and proline,reducing the content of H2O2 and malonaldehyde(MDA),as well as higher activities of superoxide dismutase(SOD)and peroxidase(POD)and catalase(CAT),so as to enhance the antioxidant capacity of plants.In conclusion,VvMYB12 could enhance tolerance of plants against 2,4-DNT through up-regulating biosynthesis of flavonoids,ABA,proline and ROS scavenging system,which provides a new way for the use of phytoremediation technology to remove environmental pollution.