Cloning And Editing Effect Of Populus Tomentosa Sitosterol Glyeosyltransferase Gene

Populus tomentosa is a native poplar tree variety with high economic value.It is an important tree species for industrial timber,afforestation and ecological protection in China.Cellulose is very important for the wood processing,papermaking,textile and chemical industries,while cellulose synthesis is the most important biochemical process in plant cells.The function of sitosterol glycosyltransferase is to bind the membrane to glycosylate specific receptors to produce sitosterol-glucoside.In the process of cellulose molecule synthesis,sitosterol-glucoside is used as a primer to form glucan chains in the form of β-1,4-glycosidic bonds using UDP-glucose as the substrate under the action of cellulose synthase.Therefore,sitosterol glycosyltransferase is crucial for the synthesis of plant fibers.In this study,Populus tomentosa was used as the test material.Real-time PCR was used to clone the SGT gene family members for subsequent bioinformatics and gene expression analysis.Further use of the gene editing technology CRISPR/Cas9 to create a new mutant material and study its function.The research results lay a foundation for further analysis of the function of SGT gene in cellulose synthesis of P.tomentosa.It provides a new way to explore the genetic improvement of forest tree and is of great significance to the genetic improvement of P.tomentosa.The main researches are as follows:1.The PtSGT1,PtSGT3 and PtSGT4 genes in the PtSGT gene family of Populus tomentosa were cloned and the conserved domain analysis of the PtSGT gene might be related to glycosyltransferase and UDP-glucuronide transfer;The results of real-time quantitative PCR showed that all the genes in the PtSGT gene family had the highest expression in the stem.It was speculated that the PtSGT gene may play a more important role in stem growth.2.8 single gene target vectors and 4 dual gene target vectors were constructed using CRISPR/Cas technology,and transformed into P.tomentosa using leaf disk transformation method.Obtained 51 PCR-detected of P.tomentosa.Two transgenic lines 8②-Ⅰ and 6-B with targets 8② and 6 in the PtSGTl and PtSGT4 genes were finally tested.3.Changes in sugar metabolism show that in the 8②-Ⅰ transgenic lines,there was no significant difference in the glucose in the stems compared with the wild type and in the 6-B transgenic lines,the glucose in stems increased significantly.4.The results of xylem sections of wild-type and transgenic 8②-Ⅰ and 6-B of Populus tomentosa showed that the xylem cells of the wild-type were closely arranged and the cells were smaller.The xylem cells of the transgenic 8②-Ⅰ and 6-B were disorderly and loosely arranged,and the cells were larger than the wild type.The wild-type xylem cells were thicker than the xylem cells of the transgenic 8②-Ⅰ and 6-B.The shape of the parenchyma cells in the wild-type pulp was disorderly.The shape of the parenchyma cells in the transgenic 8②-Ⅰand 6-B pulps was a more regular polygon and the arrangement was also relatively neat.