| As a high-grade natural food additive and health product,pectin is widely used in food,medicine,textile and other industries.Premna puberula is a dual-purpose plant for medicine and food.Its leaves are rich in pectin and have high edible value and economic value.Galactosyltransferase(GAUT)is one of the key enzymes involved in pectin synthesis.It plays an important role in pectin synthesis pathway.It catalyzes the addition of galacturonic acid group(D-Gal A)to the extended end of galacturonic acid glycan chain to synthesize homogalacturonan(HG),and participates in the synthesis of three pectin polysaccharides at the same time.The expression abundance of GAUT will affect the biosynthesis of pectin,and then affect the structure of plant cell wall.Based on the transcriptome data of P puberula,this study screened its internal reference genes,cloned the galactosyltransferase gene(Pp GAUT),analyzed the bioinformatics and expression characteristics,and explored the changes of pectin content and enzyme content of transgenic tobacco,in order to lay a foundation for further revealing the pectin biosynthesis pathway and regulation of P puberula.The main results are as follows:1.Based on the transcriptome data of P puberula,combined with software,the expression stability of six candidate internal reference genes in different organs and treatments of P puberula was comprehensively analyzed.The results showed that the expression of 18 S r RNA and ACT was the most stable in different tissues of P puberula;UBC、18S r RNA and EF-1could be selected as internal reference genes under dark culture conditions;ACT、EF-1 and UBC can be used as internal reference genes under exogenous ethylene treatment.Under simulated drought treatment,the sixth gene needs to be introduced for correction.In addition,the expression stability of ACT and EF-1 in all samples is good,which can be used for fluorescence quantitative expression analysis.2.The complete ORF of Pp GAUT gene is 1704 bp,encoding 567 amino acids.The molecular weight of the encoded protein is 65308.94 D and the isoelectric point is 9.38.The secondary structure of Pp GAUT is mainly a-helix,in which the α helix is 51.32%;subcellular localization predicted that Pp GAUT protein was located in Golgi apparatus and belonged to transmembrane hydrophilic protein.3.The results of real-time quantitative PCR showed that Pp GAUT was stably expressed in all organs of P puberula,and the expression in leaves was higher than that in stems and roots;The expression of Pp GAUT in different developmental stages increased first and then decreased with time,and reached the highest in July.4.pBI121-Pp GAUT binary expression vector was constructed,and the transgenic tobacco was obtained by agrobacterium mediated leaf disk method.Compared with the wild type,the activity of GAUT enzyme in transgenic tobacco plants were higher than those in the wild type.At the same time,overexpression of GAUT gene increased the accumulation of pectin. |
