Functional Study Of SUS5/SUS6/SUS7 Gene In Bambusa Emeiensis

The essential features of bamboo are the rapid growth,short production cycle and difficult to bloom.As an indispensable renewable resource,bamboo has a broad prospect of development and application.Bambusa emeiensis belongs to Gramineae and is a typical clustered bamboo.Bambusa emeiensis has high economic value because of its large biomass and high cellulose content.Sucrose synthase(SUS,EC2.4.1.13)is a multifunctional protein involved in the regulation of carbon sink strength,cellulose biosynthesis,carbon distribution and stress response in plants.However,the function of SUS in bamboo fiber development and abiotic stress is lacking.Previously,we cloned BeSUS5,BeSUS6 and BeSUS7genes and made a preliminary study.Now we further verified the functions of the three genes through subcellular localization analysis,promoter analysis and genetic transformation into Populus tomentosa,tobacco and bamboo.The role of BeSUS5-7 in the regulation of cellulose synthesis and response to high salt and drought stress was analyzed.The main results are as follows:(1)BeSUS5:GFP,BeSUS6:GFP,BeSUS7:GFP fusion proteins driven by CaMV 35S promoter were constructed respectively,and transformed into onion epidermal cells to investigate the subcellular localization of BeSUS5-7 in vivo.The subcellular localization analysis showed that BeSUS5 is targeted to the membrane,while BeSUS6 and BeSUS7 protein were mainly located on cell membrane and cytoplasm.(2)The promoter of BeSUS5 was obtained by genome walking.Bioinformatics analysis showed that there were abundant light response elements,abscisic acid response elements,MeJA response elements and low temperature response elements on the BeSUS5 promoter.BeSUS5promoter was transformed into tobacco,and stable expression plants were screened.Gus staining showed that the cotyledon and culms of transgenic plants were stained deeply.Under 100 mM NaCl treatment,Gus staining showed that BepSUS5 was expressed not only in the cotyledon,but also in the root.(3)The transgenic lines of poplar overexpressing the bamboo BeSUS5 gene were obtained and the further functional analysis were conducted.We found that overexpression of BeSUS5 enhanced the activity of SUS,and significantly promoted the growth of plants,especially the xylem growth.In BeSUS5 transgenic plants,the total soluble sugar content(TSS)and starch content were decreased in leaves,while the cellulose content was increased in stems,which indicating that overexpression of BeSUS5 might promote non-structural carbohydrates(NSC)partitioning to structure carbohydrates(SC)in plants.Consistent with these results,the expression of cellulose biosynthesis?related genes,such as CesA7 and KOR,were induced up-regulation.As a result,transgenic poplars displayed not only an increase in cell wall thickness and cell wall crystallinity,but also altered the fiber phenotype in the stem.(4)Overexpression BeSUS5,BeSUS6 and BeSUS7 gene into tobacco,and the transgenic plants with high expression were successfully obtained,It was found that the content of soluble sugar in transgenic tobacco was significantly higher than that in WT,but there was no significant difference in plant height compared with WT.After drought treatment,DAB and NBT histochemical staining indicating that the transgenic plants accumulated less ROS content after drought stress.In addition,the proline content of transgenic plants was higher than that of WT,while the MDA content was lower than that of WT,which indicated that BeSUS5-7 could improve the osmotic regulation ability of plants under drought stress and reduce the damage degree of cell membrane.In addition,the relative expression of SOD(superoxide dismutase,SOD)and CAT(Catalase,CAT)genes in BeSUS5-7transgenic plants was significantly increased compared with WT.After salt treatment,DAB and NBT staining,Chlorophyll fluorescence analysis and MDA content analysis showed that the transgenic plants not only accumulated less ROS content but also had higher photosynthetic efficiency than those of WT.The proline content of the transgenic plants was higher than that of the wild type,but the malondialdehyde content of the transgenic plants was lower than that of the wild type.qRT-PCR showed that the relative expression levels of SOD,APX(Ascorbate peroxidase),DERBIA(Dehydration.Responsive.Element.Binding.protrin),NHX(Na~+/H~+.Antiporter),CAT and SOS(Salt overly sensitive)genes in transgenic plants were significantly higher than that in WT.The above results showed that BeSUS5-7 gene might reduce ROS accumulation and improve the tolerance of transgenic plants under salt and drought stress by affecting the expression of antioxidant system gene.(5)Overexpression of BeSUS5 and BeSUS6 gene into Chimonobambusa quadrangularis(Fenzi)Makino,18 and 21 strains transgenic bamboo seedlings were obtained respectively.In terms of plant growth,the growth height of transgenic plants is higher than that of wild plants,and other physiological information needs to be studied.