Cloning And Expression Quantity Analysis Of Three WD40 Transcription Factors In Ginkgo Biloba L.

Ginkgo biloba L.are ancient precious plants,have important physiological activity and edibleness,the main physiological active components in that are flavonoids and terpene lactones.Flavonoids in Ginkgo biloba leaf have many important physiological activity,such as neutralizing free radicals,antioxidation,antineoplastic activity,treating cardiovascular disease and so on.Its medicinal value attracts extensive attention worldwide,how to improve its content has become a popular topic.The traditional cultivation has a long operation period and long-term responses,and genetic engineering technology is concentrated in the key genes of enzyme,which involved in flavonoid biosynthesis.However,the biosynthesis pathways of flavonoid are complex and not controlled by little key gene.Transcription factors can regulate multiple key genes of enzyme in flavonoid biosynthesis,to increase flavonoid production.So the study of transcription factors regulation is another effective means to improve the flavonoid pathway.Using the transcriptome database of Ginkgo biloba L.and RACE means,three GbWD40transcription factor genes were cloned,named GbWD401,GbWD402 and GbWD403.The physicochemical property and expression patterns of these genes investigated by bioinformatics and fluorescent quantitation,and analyse the subcellular localization.The main research contents and results are as follows:?1?The coding region of the GbWD401 was 2313 bp,and its deduced protein was770 amino acids,the protein molecular formula was C₃₇₄₅H₅₈₇₉N₁₀₅₉O₁₁₈₅S₃₀,relative molecular mass 85.66KD.GbWD401 transcription factor genes contained six conserved WD40 functional areas.Phylogenetic tree analysis showed that GbWD401was high identities to Amborella trichopoda WD40 and Nelumbo nucifera WD40.Real-time PCR analysis showed that,gene expression of GbWD401 in different ginkgo organs is tissue-specific,GbWD401 gene had no expression in roots,and the expression levels in the leaves were higher.The gene expression of GbWD401 almost not influenced by drought,had the greatest effect under UV and CCC treatment,but inhibited by GA₃ treatment.The plant expression vector PCAMBIA1304 connected with GbWD401 transcription factors was successfully constructed.Subcellular localization analysis found GbWD401 gene expressed in the nucleus.?2?The coding region of the GbWD402 was 1605 bp,and its deduced protein was534 amino acids,the protein molecular formula was C₂₅₁₈H₃₈₇₉N₇₀₅O₇₈₆S₁₈,relative molecular mass 57.18KD.GbWD402 transcription factor genes contained five conserved WD40 functional areas.Phylogenetic tree analysis showed that GbWD402was high identities to Amborella trichopoda WD40.Real-time PCR analysis showed that,gene expression of GbWD402 in different ginkgo organs is tissue-specific,GbWD402 gene had no expression in roots,and the expression levels in the leaves were higher.The gene expression of GbWD402 almost not influenced by drought,had the greatest effect under UV and CCC treatment,but inhibited by GA₃ treatment.The plant expression vector PCAMBIA1304 connected with GbWD402 transcription factors was successfully constructed.Subcellular localization analysis found GbWD402 gene expressed in the nucleus.?3?The coding region of the GbWD403 was 2406 bp,and its deduced protein was801 amino acids,the protein molecular formula was C₃₈₁₇H₆₁₄₂N₁₁₁₂O₁₁₇₄S₃₈,relative molecular mass 87.61KD.GbWD403 transcription factor genes contained four conserved WD40 functional areas.Phylogenetic tree analysis showed that GbWD403 was high identities to Amborella trichopoda WD40.Real-time PCR analysis showed that,gene expression of GbWD403 in different ginkgo organs is tissue-specific,GbWD403 gene had no expression in roots,and the expression levels in the leaves were higher.The gene expression of GbWD403 almost not influenced by drought,had the greatest effect under UV and CCC treatment,but inhibited by GA₃treatment.The plant expression vector PCAMBIA1304 connected with GbWD403transcription factors was successfully constructed.Subcellular localization analysis found GbWD403 gene expressed in the nucleus.