Studies On Functional Study Of PhSUT4 Gene In Phyllostachys Pubescens

As the initial product of photosynthesis,sucrose is mainly transported to different tissues and organs through sugar transporter SUTs to promote plant growth and development.Bamboo has a high growth rate and need obtain a plenty of sucrose from the mother plants to support their high grow rates.Therefore,the development of bamboo shoots is inevitably associated to the rapid partitioning of carbon and the accumulation of enzymes related to sucrose transport.Eventhough SUT genes have been isolated from a wide variety of plant species,the bamboo SUT gene have never been cloned.In this study,we cloned one sucrose transporter cDNA from Phyllostachys pubescens.Through bioinformatics analysis,tissue expression pattern analysis,subcellular localization and promoter analysis,combined with transgenic function verification,the role of bamboo SUT in the sugar transport,photosynthesis and secondary growth was studied,which laid a foundation for the improvement of bamboo quality by genetic engineering technology in the future.The main findings are as follows:（1）Isolation and characterization of PhSUT4 form Phyllostachys pubescens.This study successfully cloned a sugar transporter gene and nominated as PhSUT4 gene.The PhSUT4 gene is 1791 bp in length and encodes 596 amino acids,belonging to the SUT2 group.The phylogenetic tree showed that it was clustered together with rice OsSUT4,and the comparative analysis of amino acid sequence also found that it had a high similarity with rice OsSUT4 sequence.The PhSUT4 protein has 10 structurally conserved transmembrane domains and four phosphorylated serine sites.The expression pattern analyzed by qRT-PCR showed that the PhSUT4 gene was highly expressed in the vascular-rich parts（such as the veins of the leaf,the lateral branches and the middle of the 100cm shoots）.With the increase of photosynthesis,the expression of PhSUT4 gene increased,but it was also limited.After PEG₄₀₀₀ and MeJA induction treatment,the expression level of PhSUT4increased sharply in the 12 hours,and then gradually stabilized.The subcellular localization showed that PhSUT4 gene was localized to the cell membrane.These findings suggested PhSUT4 gene might be responsible for the loading and unloading process of sucrose in the phloem.（2）Analysis of the PhSUT4 promoterThe promoter fragment of PhSUT4 was obtained which was 1956 bp in length.And The amplified promoter fragmentwas cloned into pBI-mcs vector to produce PhpSUT4 construct and then transferred into tobacco.The obtained transgenic tobacco was identified by GUS staining and showed that the promoter was mainly expressed in roots and leaves.After MeJA treatment,the promoter was expressied in roots,stems and veins of leaves of positive tobacco at the same time.This indicates that the promoter PhpSUT4 mainly activates the PhSUT4 to participate in the loading of sucrose in the phloem of the leaf and the unloading process of the phloem in the root organ.When the external environment changed,the endogenous SUT4 gene might be fully mobilized in the phloem such as veins and stems to withstand harsh environments.（3）Functional analysis of overexpressing PhSUT4 gene tobaccoIn this study,three high-level transgenic tobacco-positive plants were successfully obtained by DNA and RNA double-level verification.The expression levels of exogenous PhSUT4 gene in these three positive plants showed different degrees of increase,especially in Line3 and Line4,followed by Line2.The biomass of transgenic tobacco increased significantly,including stem diameter,stem height,leaf area,floral organs and seed size.At the same time,the chlorophyll content in the mature leaves of the transgenic plants was significantly increased compared with that of the control.The photosynthesis of the transgenic plants was increased by 2-3 folds compared with that of the control plants,and the soluble sugar content and cellulose content increased correspondingly 2-3 folds,while the lignin content was relatively reduced.Tissue section analysis showed that the width of the xylem in the stem of the transgenic tobacco was narrowed,and the cell wall of the layered cells and parenchyma cells increased and thickened,respectively.（4）Gene gun and Agrobacterium agrobacterium combined induction method for genetic transformation in bamboo Chimonobambusa opienensis Hsueh et YiThe immature seeds of Chimonobambusa opienensis Hsueh et Yi germinated were used as materials to carry out genetic transformation by gene gun method and Agrobacterium synergistic induction method.Resistance screening and DNA and RNA levels were used for validation and three high expression of PhSUT4 gene were obtained.