Preliminary Study On The Roles Of Ginkgo GbDFR Genes In Regulating Stress-Induced Anthocyanin Synthesis And Auxin Transport

Dihydroflavonol 4-reductase(DFR)is the first key enzyme involved in the synthesis of anthocyanins downstream of plants flavonoid synthesis pathway,DFR plays a vital role in the regulation of flavonoid synthesis and the accumulation of anthocyanins.In the previous research,we used the Arabidopsis At DFR gene as a reference,and discovered three new DFR family members,named GbDFR4,GbDFR5 and GbDFR6.Then preliminary analysis the functions of their encoded proteins.But their functions in Ginkgo biloba stress resistance and growth are still unclear.The main research contents and results are as follows:1.We applied salicylic acid(SA),UV-B and Na Cl exogenously to ginkgo seedlings under stress,and investigated the changes in the expression of ginkgo flavonoids,anthocyanins and the three GbDFR genes.The results showed that all three exogenous factors can affect the flavonoids and anthocyanin and the expression of three genes,indicating that Ginkgo biloba may adjust the ratio of flavonoids and anthocyanins by changing the expression of GbDFR gene,so that Ginkgo can respond to various adversity stresses better.2.The function of the three GbDFR transgenic tobacco T1 generations obtained by the leaf disc transformation method was verified,and the regulatory effect of the Ginkgo DFR family on the synthesis and accumulation of tobacco anthocyanins was investigated.The results showed that compared with the wild type,the three transgenic tobacco plants had higher anthocyanin content and darker flower color,confirming that GbDFR has a regulatory function on anthocyanin synthesis.3.The southern blot experiment was used to verify the independent line of GbDFR6 transgenic plants.The results showed that two of the three GbDFR6 transgenic tobacco strains tested were independent strains.4.It was found that GbDFR6 transgenic tobacco plants showed abnormal auxin-related phenotypes.The morphological comparison of leaves,roots,flowers,fruit pods,seeds and whole plants of wild-type and transgenic tobacco.The results showed that GbDFR6 transgenic tobacco showed late flowering,a higher proportion of twisted leaves,fewer lateral roots and a large number of seeds with abnormal embryo development.It is preliminarily inferred that the auxin transport of the genetically modified tobacco is abnormal.5.Exogenously applied auxin was applied to transgenic tobacco pods and NPA was applied to wild-type tobacco to examine the relationship between abnormal phenotypes of transgenic tobacco and auxin.The results showed that the abnormally developed seeds of genetically modified tobacco after exogenous application of auxin recovered partially;while the fruit pods of wild-type tobacco after NPA was applied,they appeared similar phenotypes to genetically modified tobacco,so it is speculated that the reason for the abnormally developed genetically modified tobacco seeds It may be abnormal auxin transport.However,further measurement results show that there is no significant difference in auxin content between transgenic and wild-type tobacco seeds,and the specific reasons need to be explored.In summary,our newly discovered three genes of the GbDFR family may not only participate in the distribution of anthocyanins and flavonoids in the process of Ginkgo biloba stress resistance,but also may have the function of regulating auxin transport,resulting in the special expression of transgenic tobacco type.