Analysis Of Soluble Carbohydrate Composition In LgtC Transgenic Plants And Obtaining Of Transgenic Plants Containing Lactose Synthase Gene In Beta Vulgaris L.

Globotriose is a kind of oligosaccharides of great value.In E.coli,the synthesis of Globotriose from added lactose needs three key enzyme gene:(1) susA,encoding sucrose synthase(EC2.4.1.13);(2) galE,encoding UDP-galactose 4-epimerase(EC 5.1.3.2);(3) lgtC,encodingα-1,4-galactosyltransferase(EC 2.4.1.x).Globotriose can not be synthesized in plants,for the lack of both lgtC enzyme and the substrate of lactose.lgtC gene,which comes from Neisseria meningococcus(Neisseria meningitidis), catalyzes the transfer of a galactose residue from UDP-Gal to the end of lactose.In this study,the variation of carbohydrate composition was analyzed in transgenic line containing lgtC,to explore the mechanism of expression of lgtC on metabolism, growth,and development of transgenic plants.In order to determine the constituent and content of soluble carbohydrates in the leaves of transgenic sugar beet accurately,various carbohydrates in transgenic plants were separated and identified by Thin Layer Chromotography(TLC).Meanwhile,the content of sucrose,glucose and fructose were measured with HPLC-RID,and the content of UDP-Gal was measured with RP-HPLC combining with PDAD.Lactose synthase has two subunits:whey protein(α-lactalbumin),the regulatory subunit,encoded by gene LALBA;andβ-1,4-galactosyltransferase(EC 2.4.1.22),the catalytic subunit,encoded by Gal-T1 gene,which catalyzes the transfer of a galactose residue from UDP-Gal to the end of glucose for the synthesis of lactose.The objective of my work is to transfer both LALBA and Gal-T1 gene into multiple bud of beet containing lgtC to obtain transgenic lines with all these transgenes mentioned above, to explore the feasibility of using transgenic sugar beet to synthesize lactose and Globotriose.Analysis of carbohydrate composition of lgtC transgenic beet plantsIn this study,transgenic lines that overexpressed target gene,confirmed by RT-PCR, were chosen for determination of carbohydrate content,including total soluble carbohydrates,monosaccharides,disaccharides and nucleotide sugars.The results suggested that the contents of total soluble carbohydrates in sense transgenic lines were significantly higher than that of WT,either in leaves or roots.Accordingly,the levels of glucose,fructose and sucrose in these sense transgenic lines were all increased in considerable degrees.The content of UDP-Gal in the sense transgenic lines were all significantly(P≤0.05) lower than that of WT.However,the content of total soluble carbohydrates,as well as glucose,fructose and sucrose in the antisense line were not significantly different compared with WT.Moreover,transgenic lines showed normal growth and photosynthesis compared with WT,suggesting that transferring lgtC gene from prokaryotes into sugar beet did not affect the growth and photosynthesis.Obtaining beet containing lactose synthase geneLactose synthase genes(both LALBA and Gal-T1) were cloned from human cDNA library(Marathon-Ready cDNA),and recombined to a plant expression vector and transferred into sugar beet containing lgtC,and transgenic lines containing LALBA,Gal-T1 and lgtC were obtained.Firstly multiple bud induction and subculture system of four lgtC transgenic lines and WT were established.Of the four lines,Line 2 and 4 had higher lgtC enzyme activity and more vigorous proliferation of secondary buds.LALBA and Gal-T1were transferred to buds of Line 2 and 4 by Agrobacterium-mediated transformation.PCR results suggested that plants pyramiding LALBA,Gal-T1,and lgtC were obtained。The transgenes expression and the soluble carbohydrate composition in the transgenic plants were to be determined. In this study,transgenic lines containing lgtC gene and and transgenic plants pyramiding lgtC and lactose synthase gene from cDNA library were produced and the change of soluble carbohydrate composition in transgenic plants and the impact of genetic modification of carbohydrate metabolic pathways on carbohydrate accumulation and growth,development of transgenic plants were also studied.The preliminary research provided some productive and useful lines for further study on the controlling mechanism of carbohydrate metabolism in sugar beet cells,and shed light on the insight into the use of engineered sugar beet as a bioreactor for production of oligosaccharides of high value,such as Globotriose.