Mechanism Of Efficient Accumulation Of Pectin In Premna Puberula Pamp. Leaves

Premna puberula Pamp.,a deciduous shrub widely cultivated in China,belongs to Verbenaceae.The green bean curd made from its leaves is elegant in color and has a strong special fragrance,which is particularly concerned for its abundant pectin.Because of this,in recent years,the cultivation scale is expanding gradually,and the development and utilization of its leaves has also achieved great results.However,there is little scientific research on the synthesis and efficient accumulation of pectin in the plants,especially in leaves.Therefore,take P.puberula.as materials,the paper studied the different development period of pectin synthesis,accumulation of high levels of physical reasons and possible biochemical pathways,the relationship between pectin structure and efficient accumulation.Transcriptome and proteome methods were used to predict the metabolic pathways leading to the efficient accumulation of pectin in leaves and to analyze the gene families with significant differences.The effects of overexpressed key genes on the synthesis and accumulation of pectin in transgenic plants were studied to lay a theoretical foundation for revealing the mechanism of the accumulation of high pectin content in the medicinal and edible plant resources of this genus.On this basis,it can provide material conditions for improving pectin levels in plants by means of genetic engineering.The main research results are as follows:1.The physiological basis for the efficient accumulation of pectin in the leaves of P.puberulaThrough the determination of photosynthetic characteristics,carbohydrate and cell wall composition of leaves,pectin content in different tissues and leaves at different developmental stages,and the possible synthesis pathway.The main reasons for high pectin content in the leaves were as follows: 1)During the development of P.puberula,the accumulation time of pectin was earlier,and a certain amount of pectin had been accumulated before the leaves were expanded,and the accumulation showed a trend of increasing and then decreasing;2)The activity of the galactosyl transferase(GAUT)remained high level during the development,which could make the leaves continuously synthesize pectin.In addition,there were glucuronate pathway,galactose pathway and inositol pathway.The existence of multiple pathways ensured that more substances could be used as substrates to synthesize pectin through multiple channels;3)The activities of pectinesterase(PE),pectinlyase(PL)and polygalacturonase(PG)remained relatively low during development,so pectin was rarely degraded and accumulated continuously;4)The soluble sugar content,starch content and pectin content were significantly correlated,suggesting that soluble sugar could promote the formation and accumulation of pectin.In addition,glucose and fructose were the main compounds in the phloem of P.puberula,and sucrose was not detected during the development.Combined with the activity of sucrose synthase,the transport rate of sucrose in phloem was high after long-distance transport through sugar transporter.2.The relationship between structure and efficient accumulation of pectin in leaves of P.puberulaThe relationship between pectin structure and the efficient accumulation of pectin in the leaves of P.puberula was studied by studying the pectin content in the leaves and the properties and structures of the leaves and commercial pectin(apple pectin and citrus pectin).The results showed that: the pectin content in the leaves of P.puberula was high with abundant arabinose(20.25%)and galactose side chain.The methyl esterification degree(DM)of three pectin polysaccharides were higher than the acetylation degree(DA)(DA of P.puberula pectin,apple pectin and citrus pectin were 12.5,5.2 and 2.3,respectively),showing that galacturonic acid(Gal A)residues of methylation level was higher than that of acetylation level.This explained the reason why the activity of pectin esterase was relatively stable during the development of P.puberula: the high degree of esterification inhibited the breaking of the long chain of pectin galacturonic acid,so pectin was less degraded during the development of leaves of P.puberula,which was beneficial to the accumulation of pectin.3.Pectin metabolism pathways and its regulation in the leaves of P.puberulaThe metabolic pathway of pectin accumulation in the leaves of P.puberula was analyzed by means of transcriptome and proteomics in two stages(Pp＿L and Pp＿H)with significant difference in pectin content.Transcriptomic and proteomic methods were used to analyze the molecular regulatory pathways of pectin accumulation in the leaves of P.puberula and 74534 unigenes with an average length of 840 bp were assembled by transcriptome sequencing,and 17425 differentially expressed genes(DEGs)were found,of which 8049 genes were up-regulated and 9376 genes were down-regulated.Then GO and KEGG analysis of DEGs showed that DEGs were mainly enriched in carbohydrate metabolism,lipid metabolism,energy metabolism,catabolism and degradation processes.A total of 3956 proteins were identified and quantified by proteome sequencing,including 946 differential proteins(624 up-regulated proteins and 322down-regulated proteins).GO and KEGG analysis of differential proteins revealed that the proteins with significant differences were mainly concentrated in: pentose phosphate pathway,pentose and glucuronic acid transformation,phenylalanine metabolism,amino sugar and nucleotide sugar metabolism.The results displayed that enzymes associated with sucrose biosynthesis,glycosyltransferase,sugar transporters and pectin degradation enzyme genes were all involved in the pectin synthesis and accumulation process of P.puberula leaves.4.Functional analysis of Pp SWEET15 gene in P.puberulaIn order to further explore the mechanism of the efficient accumulation of pectin,a total of 15 genes were identified from the SWEET gene family of P.puberula,which were divided into 4 subfamilies.Every subfamily had similar gene structure and relatively conserved domains.Most of these genes had the ability to transport sucrose,glucose or fructose;The ORF region of Pp SWEET15 gene was 700 bp,encoding 231 aa,and was located in the endoplasmic reticulum.The gene participated in the sucrose and glucose transport in the leaves of P.puberula,and indirectly increased the content of UDP-Gal A,the synthesized substrate of pectin,and promoted the efficient accumulation of pectin in the leaves of P.puberula.From the above description,it can be inferred that sucrose was transformed into UDP-Glc under the action of hexokinase and invertase,Pp SWEET15 gene promoted sucrose and glucose transport,and there were glucuronate pathway and galactose pathway,and the expression of PE,PL and PG were inhibited,and the degradation of pectin was delayed.All of them promoted the efficient accumulation of pectin in the leaves of P.puberula.