| Wolfberry (Lycium Lin.) is family Solanaceae, an important both for food and medicine of plant resources in China. It is significantly tolerant to drought and cold and has extensive adaptability, is a pioneer species of saline soil. China has rich in wolfberry germplasm resources, but only cultivation physiology, genetic breeding and processing produce of Lycium bararum L.among abundant wolfberry resources were numerously and deeply studied, and its value in both for food and medicine was deep understanding. Only Lycium barbarum L.of medicinal plants was collected in Chinese Pharmacopoeia. Wolfberry fruits contained abundance of sugars, and content and composition of sugars not only determined basic materials supply in fruits during wolfberry fruits quality development, but also affected substrates in many secondary metabolites and active substance synthesis. At present, the mechanism of wolfberry fruits sugar accumulation and molecular regulation was not clear. In this study, fruits shape change, sugar contents, transcriptome sequencing, genes cloning and expression in eight different genotypes wolfberries were determined during five stages of fruit development. The characteristics of sugar accumulation in different genotypes wolfberries fruits were compared, and differential expression genes (DEGs) associated with sugar metabolism during fruits development were analysed. Five DEGs associated with fructose metabolism were screened out, and cloning and expression of three DEGs were analysed. It is provided a theoretical base for illustrating molecular regulation of wolfberry fruits quality formation. The main conclusions are as follows:(1) Wolfberry fruits were divided into two growing types according to changing on fruit weight, fruit length and major size. Two rapid growth stages belonged to the first type (double "S"), and early rapid growth stage and late rapid growth stage, Lycium barbarum L. is the first type. One rapid growth stage belonged to the second type (single "S"), Lycium ruthenicum Murr. is the second type. The length diameter of fruit was significant elongation in four genotypes wolfberries (Lycium barbarum, Lycium chinense var. potaninii, Lycium yunnanense, Lycium barbarum Linn. var. auranticarpum×Lycium barbarum) in early stage of fruit development, then the major diameter of fruit was significant thickly. The length diameter of fruit was significant elongation in Lycium chinense Mill. during the whole growth stages, and the equivalent of growth of fruit length diameter was major diameter in Lycium ruthenicum Murr.(2) Total sugar, LBP, fructose, glucose, sucrose, erythritol, arabinose, rhamnose, fucosylation, galactose, xylose and sorbose were detected in mature wolfberry fruits by using GB and gas phase chromatography (GC) methods, and the content of total sugar, LBP, fructose, glucose, sucrose were higher than 1 mg/g FW. The sugar content had large differences in eight different genotypes wolfberries, among which, more kinds of sugar and higher sugar content in Lycium barbarum L. were found, less kinds of sugar and lower sugar content in Lycium ruthenicum Murr.. Sugar accumulation of wolfberry fruits were yet divided into two types according to changing on sugar components and content during the growth stages. The first type was hexose type as a representative of Lycium barbarum L, and the main sugars were fructose and glucose at fruit mature period. The second type was fructose type as a representative of Lycium ruthenicum Murr., the main sugars were fructose at fruit mature period.(3) Three development stages (green fruit phase, immature fruit phase, mature phase) of wolfberry fruit were transcriptome sequencing by runing on the Illumina HiseqTM 2500 sequencing platform. Transcriptome data collected 50.90Gb clean data, and assembled 126,332 transcripts and 87,698 unigenes, average length were 947 bp and 797 bp. Unigenes were used for BLAST sesrches and annotation against the NCBI Nr database,38,978 unigenes were annotated to NR protein database,38,891 unigenes were annotated to Trembl database,31,761 unigenes were annotated to the GO database,6,120 unigenes annotated to K.O database; 31,761 unigenes were also aligned by BLAST to 58 function groups GO database,6,120 unigenes were annotated to 275 pathways.(4) Through analysis DEGs between three fruit development stages, there were 10,369 DEGs during fruit development stages of 14-30 day, of which 4733 were up-regulated,5636 were down-regulated. During fruit development stages of 14-35 day, there were 11644 DEGs, of which 5795 were up-regulated,5849 were down-regulated. During fruit development stages of 30-35 day, there were 7272 DEGs, of which 3939 were up-regulated,3333 were down-regulated.33 DEGs were selected from significantly enriched DEGs in the pathways, which involved in four pathways of sugar metabolism.The research showed that LbAl, LbSS, LbXI, LbPFK and LbSPS were key genes in wolfberry fructose metabolism.(5) The full-length cDNA sequence of LbAI, LbSS and LbSPS from the fruits of wolfberry were cloned by the technology of rapid amplification (RACE), which were 2190 bp,2741 bp and 3198bp, and GenBank accession numbers were KM191309, KM191310 and KM 191308, respectively. The protein sequence of three genes had the highest homology with Solanum tuberosum and Solamun lycopersicum. The expression level of LbAl, LbSS and LbSPS in fruits had happen during fruit development stages, and had great difference; The expression level of LbAl and LbSPS in flowers had higher, and the both in roots had lower, while the expression level of LbSS in roots had higher, and its in leaves had lower. Three genes expression level gradually decreased with changing of fruit colors from red and yellow to black, and Lycium ruthenicum Murr. was the lowest; The expression level of three genes in fruits were decreased after NaCl treatment, and genes expression level steadily decreased with the increasing of NaCl stress. The correlation analysis showed that LbAI expression level was significantly correlated with fructose content, which supposed that LbAI may play a key role in wolfberry fructose accumulation... |
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