| Lycium barbarum L.is a perennial woody plant that is an important medicinal and food homologous plant resource in China.The medicinal value,color,flavor and taste of Lycium barbarum fruit are affected by the content and types of sugar in the fruit.The key metabolism to the quality of Lycium barbarum fruit is sugar accumulation,and sucrose metabolism is an important part of sugar accumulation.The molecular mechanism is still unclear,so in this study,the "Ningqi No.1" Lycium barbarum was used as a material to construct an SSH library to obtain differential gene expression profiles of fruit;the Lycium barbarum neutral invertase(NI)gene was cloned and biologically informatics and analysis of spatial and temporal expression patterns;and using "Ningqi No.1" and"Ningqi No.3"Lycium barbarum fruits at different developmental stages as materials,the sugar content and key enzyme genes of sucrose metabolism(Lb SAI,Lb NI,Lb SPS and Lb SuSy)gene expression and enzyme activity changes were studied to confirm that there are varieties of key enzyme genes of sucrose metabolism;at the same time,genetic transformation and molecular functions of the two genes Lb SAI and Lb SPS were further analyzed.The specific research results are as follows:1.The SSH library of Lycium barbarum fruit was successfully constructed using SSH technology.A total of 1066 EST sequences were obtained by sequencing including 1,051 high-quality EST sequences and the recombination rate was 98.59%.There were 654 single genes in the sequence,of which 37.78%were redundant.There were 654 single genes obtained,with an average length of 548.07 bp.586 ORF sequences were obtained,and the average length of ORF was 324.88 bp.These genes are involved in the regulation of physiological processes including amino acid and nucleotide metabolism,defense mechanisms,energy metabolism,transcriptional regulation,cytoskeleton,signal transduction,lipid transport and metabolism,intracellular transport,secretion and vesicle transport.A gene database was provided for future research on the molecular mechanism of Lycium barbarum.2.RACE technology was used to clone the Lycium barbarum NI gene.The Lb NI(GenBank accession number is KR026955)gene open reading frame 1653 bp,encoding 545 amino acids.Lb NI protein secondary structure prediction found that the irregular coiled structure accounted for 39.63%;the α-helix accounted for 36.51%;the extended chain structure accounted for 18.53%;the β-turn was the least,only 5.32%.Bioinformatics analysis showed that the Lb NI protein contains the conserved domain of the glycosidase 100 superfamily,which is very similar to NI proteins such as tomato and tobacco,and has no acidic invertase conserved domains NDPNG/A,FRDP and WECP.The Lb NI gene is expressed at all stages of Lycium barbarum fruit formation,and there are diferences in organs and tissues.The expression level in leaves and peels is high,indicating that neutral invertase is involved in various developmental processes of Lycium barbarum.The change of Lb NI enzyme activity is consistent with the gene expression pattern,indicates that there is no secondary regulation mechanism for the expression of Lb NI,and the transcription level regulation of Lb NI is one of the important links in regulating the quality of the Lycium barbarum fruit.3.Analysis of sugar content,gene expression and enzyme activity of different medlar varieties showed thatAnalysis of sugar content,key enzyme gene expression and enzyme activity of different Lycium barbarum varieties showed that "Ningqi No.1" maintained high hexose(glucose and fructose)levels,while sucrose level was lower than "Ningqi No.3".During development of Lycium barbarum fruit,the expression of Lb SPS and Lb NI genes was consistent with the law of changes in enzyme activity,the expressions of Lb SAI and Lb SuSy genes were not consistent with the law of changes in enzyme activity The key gene expression of sucrose metabolism is different among Lycium barbarum varieties In this sense,NI and SPS in the development of Lycium barbarum fruit seem to play a major role in sugar metabolism.4.The Lb SPS and Lb SAI genes were transferred into Nicotiana tobacum cv.SamsunNN by Agrobacterium-mediated method,and positive transgenic plants were obtained.Positive transformation rates were 63.6%and 77.8%,respectively.Analysis of the gene expression of the positive plant leaves by qPCR detection revealed that the SAI expression in seven transgenic lines was higher than that of wild type.However,the SAI activity of the transgenic lines did not show a significant increase compared to the wild type.Analysis of seven positive plants revealed that the SPS expression at the transcription level increased,and the corresponding enzyme activity also increased.5.The Lb SPS and Lb SAI genes were transferred into "Ningqi No.1" wolfberry by Agrobacterium-mediated method,and positive callus and adventitious buds were obtained.Currently,no positive plants have been obtained.In addition,compared to EHA105 and GV3101 strains,Agrobacterium AGL1 strains are more likely to carry foreign genes into the leaves of Lycium barbarum.The function of Lb SPS and Lb SAI genes in Lycium barbarum needs further study.The research results provide a theoretical reference for a comprehensive understanding of the mechanism of sucrose metabolism genes during the development of Lycium barbarum fruits.It also provides accumulated research data and experimental materials for further revealing the molecular mechanism of sugar metabolism in Lycium barbarum fruits,and lays a foundation for the research function of Lycium barbarum transgene.It has important theoretical significance for further revealing the synthesis mechanism of Lycium barbarum polysaccharide. |
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