γ-Aminobutyric acid alleviates litchi thaumatin-like protein-induced inflammation and reduces gut microbial translocation

Previous research reported litchi thaumatin-like protein(LcTLP)could lead to inflammation,which is a factor causing the adverse reactions after excessive intake of litchi.As a main amino acid in litchi pulp,γ-aminobutyric acid(GABA)was found with anti-inflammatory effect.Therefore,this study aimed to investigate the effects of GABA on LcTLP-induced inflammation through RAW264.7 macrophages and C57BL mice models.In vitro study showed GABA could effectively regulate the level of inflammatory cytokines(interleukin(IL)-1β,IL-6,IL-10,and prostaglandin E2)and Ca2+in cells,and inhibit the phosphorylation of p65,IκB,p38,c-Jun N-terminal kinase(JNK)and extracellular signal-regulated kinase(ERK).These results indicate GABA alleviated inflammation through nuclear factor-κB and mitogen-activated protein kinase pathway signaling pathways.In vivo experiment was performed to verify the anti-inflammatory effect of GABA,and the results demonstrated that GABA reduced the inflammation and oxidative stress in the liver of LcTLP-treated mice,as it down-regulated the pro-inflammatory cytokines,malondialdehyde,aspartate transferase,and alanine transaminase.The relative expression of phosphorylated p38,JNK and ERK in mice liver with GABA treatment were reduced to 65%,39%and 80%of the control group,respectively.Furthermore,GABA treatment enriched probiotic bacteria and decreased pathogenic bacteria in mice gut,which reveals GABA could effectively reduce the translocation of gut microbiota.

荔枝(Litchichinensis Sonn.)花芽分化过程的细胞超微结构观察

应用透射电镜对荔枝茎尖生长锥由营养生长转变为生殖生长过程的细胞超微结构进行观察 ,结果表明 ,荔枝圆锥花序发育前期细胞排列紧密 ,细胞质浓 ,液泡小 ,细胞核大并占据着细胞大部分位置 ;细胞质内有丰富的线粒体和膜状系统 ,整个膜状系统内连细胞核 ,外接胞间连丝 ;细胞内囊泡大量形成、融合与迁移 .这些均显示出细胞内结构物质在此期经历着活跃的周转和合成代谢 ,细胞内部在基因的控制下有序地表达 ,为发育过程质的转变积累足够的信息和物质 .生长锥由尖长变为宽平的花序原基时 ,其组织细胞结构也发生了相应的变化 ,最显著的特征是在此过程中 ,有一些细胞发生了程序性死亡 .正在凋亡的细胞核染色质固缩、趋边、核膜破裂或皱缩、细胞质囊泡化并将降解的物质运到周围细胞 。

RAPD Analysis of Litchi chinensis Sonn.from Different Origins of China

[Objective] To lay the foundation for the classification and breeding of Litchi （Litchi chinensis Sonn.） through RAPD （Random Amplification Polymorphic DNA） analysis of the Litchi cultivars with different origins. [Method] 5 Litchi cultivars from Sichuan, Guangxi, Guangdong, Fujian and Hainan were used as experimental materials to extract genomic DNA by modified CTAB method. Twenty primers with well reproducibility were randomly selected to conduct RAPD amplification to analyze the polymorphism and genetic relationship of the five cultivars. [Result] The polymor- phism of the five Litchi cultivars was 61%, with the similarity coefficient of 0.571 4- 0.803 6. The similarity coefficient of those from Guangdong and Guangxi was the largest of 0.803 6, and from Sichuan and Hainan was the lowest of 0.571 4. The resolution and production of RAPD was significantly improved by appropriately pro- longing the ramp time from annealing to extension to 0.3℃/s. [Conclusion] This study provided theoretical basis for the classification and breeding of Litchi.

荔枝(Litchi chinensis)细胞培养的初步研究

用荔枝无菌苗作为外植体，在添加了ＡｇＮＯ３或蜂王浆的含１ｍｇ／Ｌ６－ＢＡ和１ｍｇ２，４－Ｄ的ＭＳ培养基中诱导出愈伤组织，在同样培养基中继代培养，得到了稳定的培养细胞系．这种细胞在含１ｍｇ／Ｌ６－ＢＡ＋２ｍｇ／Ｌ２，４－Ｄ的ＭＳ培养基中悬浮培养，培养周期２５ｄ．结果表明：维生素Ｃ和ＮａＣｌ在悬浮培养中有良好的防褐作用．

Agrobacterium tumefaciens-mediated transformation of embryogenic callus and CRISPR/Cas9-mediated genome editing in‘Feizixiao'litchi

Litchi(Litchi chinensis Sonn.)is a type of commercially prevalent subtropical and tropical fruit.Since litchi has a highly heterozygous genetic background and a long reproductive cycle,conventional breeding methods(such as hybridization)have limited ability to nurture new litchi cultivars.Here,an efficient and stable Agrobacterium tumefaciens-mediated genetic transformation of embryogenic callus was established in‘Feizixiao’litchi.Transgenic materials were verified using polymerase chain reaction(PCR)analysis,β-glucuronidase(GUS)assay,and green fluorescent protein(GFP)assay.To implement the technology of the Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)/associated protein 9(CRISPR/Cas9)technology in‘Feizixiao’litchi and verify the validity of these transformation systems,the litchi polyphenol oxidase gene(LcPPO,JF926153)was knocked out.Various categories of mutations,covering base insertions,deletions,and substitutions,were found in transgenic materials via sequence analysis.The transformation system achieved high feasibility and efficiency,and the system of CRISPR/Cas9 was successfully employed to edit genes in‘Feizixiao’litchi.This work provides an essential foundation for investigating the functions of genes and accelerating litchi genetic improvement.

Cloning and Sequence Analysis of Expansin Genes from Litchi chinensis Fruit

Using PCR degenerate primers, designed with reference to the sequences of the conserved amino acids of known expansins, to amplify cDNA fragments in litchi fruit by RT-PCR, two different cDNA fragments , named as Lc-Exp1 and Lc-Exp2 , were cloned. Lc-Exp1 and Lc-Exp2 was respectively composed of 531 bp encoding 177 amino acids and 537 bp encoding 179 amino acids. Eight cysteine residues and three tryp-tophan residues, which is supposed to be the characteristics of expansins, are conserved in both Lc-Exp1 and c-Exp2. In addition, the homology between the two expansins is 71. 6% at nucleotide acid sequences and 76.3% at amino acid sequences. The homology of Lc-Exp1 with Fa-Exp2 or Pp-Exp1 was 92.7% or 92.1%, but that of Lc-Exp2 with Fa-Exp2 or Pp-Exp1 was only 77. 4% or 76.3% at amino acid sequences.

Molecular cloning,characterization and expression profile of the sucrose synthase gene family in Litchi chinensis

Sucrose synthase(SUS,EC 2.4.1.13)is widely considered as a key enzyme involved in plant sucrose metabolism,and the gene family encoding different SUS isozymes has been identified and characterized in several plant species.However,to date scant information about the SUS genes is available in Litchi chinensis Sonn.Here,we identified five SUS genes in litchi.These Lc SUSs shared high levels of similarity in both nucleotide and amino acid sequences.Their gene structure,phylogenetic relationships,and expression profiles were characterized.Gene structure analysis indicated that the Lc SUSs have similar exon-intron structures.Phylogenetic analysis revealed that the five members could be classified into three groups(LcSUS1 and LcSUS2 in SUSⅡ,LcSUS4 and LcSUS5 in SUSⅢ,and LcSUS3 in SUSⅠ),demonstrating evolutionary conservation in the SUS family across litchi and other plant species.The expression levels of Lc SUSs were investigated via real-time PCR in various tissues and different developmental stages of aril.For tissues and organs,Lc SUSs exhibited distinct but partially redundant expression profiles in litchi,being predominantly expressed in young leaves(sink).During aril development,the expression pattern of LcSUS1 was consistent with the trend of sugar accumulation,indicating it may play important roles in determination of sink strength in aril.Moreover,transcript levels of LcSUS2,LcSUS4,and LcSUS5 varied between cultivars with different hexose/sucrose ratios,which may regulate the sugar composition in aril.Our results provide insights into physiological functions of SUS genes in litchi,especially roles in regulating sugar accumulation in aril.

Construction of high-density SNP genetic maps and QTL mapping for dwarf-related traits in Litchi chinensis Sonn

Litchi chinensis Sonn is widely cultivated in subtropical regions and has an important economic value.A high-density genetic map is a valuable tool for mapping quantitative trait loci(QTL)and marker-assisted breeding programs.In this study,a single nucleotide polymorphism(SNP)-based high-density linkage map was constructed by a genotyping-by-sequencing(GBS)protocol using an F1 population of 178 progenies between two commercial litchi cultivars,‘Ziniangxi’(dwarf)and‘Feizixiao’(vigorous).The genetic map consisted of 3027 SNP markers with a total length of 1711.97 cM in 15 linkage groups(LGs)and an average marker distance of 0.57 cM.Based on this high-density linkage map and three years of phenotyping,a total of 37 QTLs were detected for eight dwarf-related traits,including length of new branch(LNB),diameter of new branch(DNB),length of common petiole(LCP),diameter of common petiole(DCP),length of internode(LI),length of single leaf(LSL),width of single leaf(WSL),and plant height(PH).These QTLs could explain 8.0 to 14.7%(mean=9.7%)of the phenotypic variation.Among them,several QTL clusters were observed,particularly on LG04 and LG11,which might show enrichment for genes regulating the dwarf-related traits in litchi.There were 126 candidate genes identified within the QTL regions,55 of which are differentially expressed genes by RNA-seq analysis between‘Ziniangxi’and‘Feizixiao’.These DEGs were found to participate in the regulation of cell development,material transportation,signal transduction,and plant morphogenesis,so they might play important roles in regulating plant dwarf-related traits.The high-density genetic map and QTLs identification related to dwarf traits can provide a valuable genetic resource and a basis for marker-assisted selection and genomic studies of litchi.

Study on Effects of Organic Nutrient Solution on Amino Acid Contents in Litchi (Litchi chinensis Sonn.) Fruit

[Objectives] This study was conducted to investigate the effect of organic nutrient solution on litchi quality. [Methods] Different concentrations of organic nutrient solutions were sprayed on litchi leaves to study their effects on amino acid contents in the fruit of litchi cultivar "Qinzhouhongli". [Results] The results showed that the organic nutrient solution could significantly promote the growth of litchi fruit at various development stages and improve fruit quality by spraying the organic nutrient solution for 3 times with a dilution concentration of 500-1 500 times during fruit development(at an interval of 15 d) of litchi. It could increase the total essential amino acid, total semi-essential amino acid, lysine, phenylalanine, threonine, methionine, valine, leucine, histidine, arginine, aspartic acid, glycine, serine, proline, alanine and tyrosine contents. Spraying the organic nutrient solution with a dilution concentration of 1 000 times achieved the best effect of promoting the growth and improving quality of litchi fruit. [Conclusions] This study provides a theoretical basis for high-quality production and nutritional quality evaluation of litchi and the development of functional processed litchi products.

Effects of saponin from the seed of Litchi chinensis Sonn on TGF-β1, FN and SOCS-1 in renal tubular epithelial cells under high glucose

Objective:To investigate the effect of saponin from the seed of Litchi chinensis Sonn(SLS)on the growth and apoptosis of human kidney epithelial cells(HKC)cultured in high glucose.Methods:HKC were cultured in DMEM/F12 medium supplemented with 30 mmol/L glucose and treated with or without SLS.In the normal group,isometric DMEM/F12 medium with 5.5mmol/L glucose was added.The secretion of TGF-β1 and fibronectin(FN)were detected by ELISA.Cell apoptosis was detected by the method of Annexin V-FITC/PI double staining.Western blot was used to detect the level of suppressor of cytokine signaling-1(SOCS-1).Results:The result of ELISA showed that the secretion of TGF-β1 and FN was decreased in SLS groups compared with those in 30 mmol/L glucose treated group(P<0.05).There were more cells apoptosis in 30 mmol/L glucose treated group than that in the normal group(P<0.01).Compared with the 30 mmol/L glucose treated group,the apoptosis of HKC were significantly decreased in SLS groups(P<0.01).Western blot showed that the level of SOCS-1 in high glucose+SLS group was decreased(P<0.01),compared with the high glucose group.Conclusion:SLS can reduce the secretion of TGF-β1 and FN in HKC by reducing the deposition of extracellular matrix.SLS also significantly reduced the apoptosis of HKC by inhibiting the level of SOCS-1.These results suggest the roles of SLS in preventing the progress of glomerular sclerosis.

Synthesis of silver and gold nanoparticles from leaf of Litchi chinensis and its biological activities

Objective: To synthesize and isolate silver and gold nanoparticles from Litchi chinensis leaf methanolic extract, and to evaluate its comparative biological activities including muscles relaxant, analgesic, anti-inflammatory and antidiarrheal. Methods: The gold and silver nanoparticles were synthesized by dissolving methanolic extract in gold chloride and silver nitrate solution separately which were confirmed by colour change and UV-Vis spectroscopy, and pellets were collected through centrifugation. Biological activities of the extract were conducted on BALB/c mice through various standard methods and the data were subjected to One-way ANOVA. Results: The colorless gold chloride solution changed to purple soon after the addition of plant extract, demonstrating that the reaction took place and gold ions were reduced to gold nanoparticles, while colorless silver nitrate solution changed to light and dark brown that was indicative of silver nanoparticles. The muscles relaxant activity showed that silver nanoparticles were more effective than gold nanoparticles and methanolic extract in traction test. The analgesic activity showed that silver and gold nanoparticles showed highest percentage decrease in acetic acid induced writhing at the doses of 50, 100 and 150 mg/kg b.w. The highest anti-inflammatory activity was produced by gold nanoparticles followed by silver nanoparticles, while low activity was observed in methanolic leaf extract. Only the crude methanolic extract showed significant antidiarrheal activity as compared to the standard drug atropine sulphate, while antidiarrheal activities of gold and silver nanoparticles were non-significant. Conclusions: The present work concludes that isolated silver and gold nanoparticles from leaf methanolic extract shows strong muscles relaxant, analgesic and anti-inflammatory activities while crude methanolic extract possesses good antidiarrheal activity.

Study on Improving Litchi chinensis Production in Adverse Weather

Being sensitive to climatic conditions,the growth of Litchi chinensis（ lychee） can be greatly affected both in rainy warm winter which is conductive to winter shoot elongation,seriously affecting the formation of flower buds,and in rainy spring or cold late spring when lychee is flowering,seriously affecting pollination and fruit set. Thus,meteorological conditions play a decisive role in determining the distribution,growth and development,yield formation and quality of lychee. Based on meteorological conditions,some measures can be taken to reduce the impacts of adverse weather and take advantage of favorable weather conditions for the stable and high production of lychee. Therefore,the yield of lychee could be increased by improving the accuracy of weather forecasts and long-term cultivation and management techniques.

Biotransformation of Flavor Compositions During Fermentation of Litchi (Litchi Chinensis Sonn.) Fruits into Wine

The aim was to examine the biotransformation of chemical compounds during the fermentation of litchi wine.S.bccyanus BV818 was inoculated to litchi juice(Heiye)to initiate the fermentation.Acetic acid decreased dramatically,succinic acid and DL-malic acid increased sharply.Saturated free fatty acid increased,especially the concentration of the free fatty acid with long carbon chain(more than 10 carbons)increased significantly.The unique flavor compounds of fresh litchi including linalool,α-terpineol,β-citronellol and other terpenoids remained in the litchi wine were transformed to other aroma constituents,by which the primary litchi flavor was retained.The wine had a fruity flavor and delicate bouquet and had harmonious sourness and sweetness.The litchi'Heiye'was suitable for being fermented into litchi wine.

Expression of SAMDC Gene for Enhancing the Shelf Life for Improvement of Fruit Quality Using Biotechnological Approaches into Litchi (Litchi chinensis Sonn.) Cultivars

Polyamines play an important role in plant response to abiotic stress. S-adenosyl-1-methionine decarboxylase (SAMDC) is one of the key regulatory enzymes in the biosynthesis of polyamines. In order to better understand the effect of regulation of polyamine biosynthesis on the shelf life improvement of litchi fruit, SAMDC cDNA isolated from Datura stramonium cloned in pBI121 was introduced into litchi genome by means of Agrobacterium tumefaciens through zygote disc transformation. Transgene and its expression are confirmed by Southern and Northern blot analyses, respectively. Transgenic plants expressing Datura SAMDC produced 1.7- to 2.4-fold higher levels of spermidine and spermine than wildtype plants under normal environmental condition, which indicated that the transgenic litchi presented an enhanced polyamines synthesis compared to wildtype plants. Our results demonstrated clearly that increasing polyamine biosynthesis in plants may be a means of creating improved fruit shelf life germplasm.

Identification and Analysis on True or False Hybrids from Hybridization Populations of Litchi( Litchi chinensis Sonn.) Using EST-SSR Markers

In order to better distinguish true hybrids in litchi crossbreeding,F1generations of two litchi hybridized combinations namely that‘Xuehuaizi’בGuiwei’and‘Xuehuaizi’בJiaohesanyuehong’were used as materials to identify true hybrids,to construct mapping populations and to study the genetic diversities of the two populations with EST-SSR markers.The results showed that F1generations of the two hybrid populations could be identified with an identification rate of100%by the combination of four pair primers,respectively,and the 159 individual plants of the two populations were true hybrids.In addition,variations were exited in leaf morphology of the progenies of the two populations and bands of parents absented were occurred on genotype.The clustering analysis showed that 113 F1plants from the hybridized combination of‘Xuehuaizi’בGuiwei’were clustered into six categories（similarity coefficient was 0.68）,and 63.72%（72 plants）of which clustered into one group with male parent.The genetic distances between 32 plants（28.3%）and their parents were far which indicated that larger variation or recombinant appeared in these plants.Forty six hybrid progenies of the combination of‘Xuehuaizi’בJiaohesanyuehong’were divided into two categories when the similarity coefficient was 0.642 and most individual plants（60.87%）showed closer genetic relationship with female parent and partial maternal genetic tendency.It is concluded that EST-SSR markers are suitable to identify true hybrids of litchi.The construction of the two F1mapping populations has established basis for further genetic linkage mapping,meanwhile,has accumulated materials for cultivar improvement of litchi.

Physiological Changes during the Process of Pericarp Browning in the Postharvest Litchi

[Objective] Pericarp browning in the postharvest litchi significantly reduced its commercial value and limited the expanding of litchi markets. Physiological changes during the process of pericarp browning were determined in order to identify the underlying mechanisms. [Method] Matured Feizixiao fruits were stored at 25 ℃ and 70%±5% relative humidity. The physiological changes happened in pericarp during storage were tested at an 8-hour interval. [Result] The fruit of Feizixiao (Litchi chinensis Sonn. cv Feizixiao) turned completely brown within 72 h after being harvested under the experimental conditions. Sharp increase of the browning index occurred from 48 to 64 hours after harvest (HAH). With the browning of pericarp,water content of the whole fruit and pericarp decreased continuingly. In contrast,there were no significant changes in the water content of pulp during the same period. MDA content,pH value and relative leakage rate of pericarp were increased during storage. Most of pigment contents including anthocyanin,flavonoid,phenols,chlorophyll a and total chlorophyll decreased. POD activity was initially increased in 32 HAH and then decreased afterwards. PPO activity was decreased continuously,while the activities of catalase and superoxide dismutase exhibited the pattern of 'increasing-decreasing-increasing' as the storage time progressed. Correlation,stepwise regression and path analyses showed that water loss of pericarp was the major factor of pericarp browning. Principal and cluster analyses showed that there were two stages of pericarp browning during the course of litchi storage. [Conclusion] Water status of pericarp was the most important factor affecting pericarp browning. The pericarp browning happened by stages,which was mainly determined by the water loss of pericarp.

Effects of Chitosan and Salicylic Acid on Cold Resistance of Litchi under Low Temperature

This paper focused on the effect of spraying chitosan and salicylic acid to Litchi under low temperature stress conditions.The physiology and biochemistry of litchi were studied as well.Results showed that the appropriate concentration of chitosan and salicylic acid treatment could effectively reduce injury caused by low temperature to litchi,compared with water control,chlorophyll,proline,soluble protein content of litchi after treatment and the activity of protective enzyme increasing significantly.However,the accumulation of resistance could significantly be improved.Furthermore,when 1 000 mg/L chitosan combined with 50 mg/L salicylic acid,the litchi acquired the best cold resistance capability.

Effect of Paclobutrazol plus Ethephon Treatment on Endogenous Hormones and Carbon and Nitrogen Nutrients in Litchi Variety ‘Feizixiao'

[Objective] This study aimed to investigate the effect of spraying the mixture of paclobutrazol and ethephon on the endogenous hormones and carbon and nitrogen nutrients in litchi variety ＇Feizixiao＇.[Method] [Result] The results showed that foliar spraying of the mixture of paclobutrazol and ethephon could effectively inhibit litchi variety ＇Feizixiao＇ to produce winter shoots,promote the flower bud differentiation and improve flower formation rate; meanwhile,this treatment could also increase the contents of ABA and ZR,and the ratios of ABA/IAA,ABA/GA3,ZR/IAA,ZR/GA3,and decrease the contents of IAA and GA3; additionally,it could increase the contents of soluble sugar,starch and total nitrogen,and raise C/N ratio,thus improving the flower formation rate.[Conclusion] Foliar spraying of the mixture of paclobutrazol and ethephon is an effective pathway for solving warmth damage to litchi.

An Effective Method for Extracting Total RNA from Young Embryo of Seedless Litchi

A total RNA extraction method for young embryo of seedless litchi was introduced. CTAB, Phenol （saturated with water）, chloroform, Guanidine isothioeyanate were used as main extraction reagents. Polyphenolie compounds were removed effectively by added PVP into the extraction buffer solution. RNA was purified intensively by phenol, chloroform extraction, and ethanol deposition after deposited by LiCl. Both the results of formaldehyde denatured agarose gel eleetrophoresis and ultraviolet spectrophotometer analysis showed high integrity and purity of RNA. So the quality of extracted RNA could meet the demand of most molecular biology experiments that require higher quality RNA.

Relationship Between Litchi Embryo Abortion and Phenolic Inhibitors

Phenolic inhibitors were preliminarily isolated from the normal and abortive litchi (Litchi chinensis Sonn.) ovules with a series of solvents, and then separated and purified by thin-layer chromatography (TLC), further isolated and identified with GC-MS, and finally checked with standards. p-Hydroxybenzoic acid (p-HBA) was isolated and identified from litchi ovules in this experiment for the first time. Determination of its biological activity showed that p-HBA was a very strong growth inhibitory substance. In abortive ovules, p-HBA content and IAA oxidase activity were much higher; IAA content was much lower than those in normal ovules (P<0.01). It is suggested that p-HBA may be involved in the regulation of litchi embryo development; high content of p-HBA may accelerate IAA oxidation and affect the balance of growth promoting and inhibitory substances, causing embryo abortion.