Species-specific regulatory pathways of small RNAs play sophisticated roles in flower development in Dimocarpus longan Lour.

Flower development plays vital role in horticultural plants.Post-transcriptional regulation via small RNAs is important for plant flower development.To uncover post-transcriptional regulatory networks during the flower development in Dimocarpus longan Lour.‘Shixia’,an economically important fruit crop in subtropical regions,we collected and analyzed sRNA deep-sequencing datasets and degradome libraries Apart from identifying miRNAs and phased siRNA generating loci(PHAS loci),120 hairpin loci,producing abundant sRNAs,were identified by in-house protocols.Our results suggested that 56 miRNA-target pairs,2221-nt-PHAS loci,and 111 hairpin loci are involved in posttranscriptional gene silencing during longan reproductive development.Lineage-specific or species-specific post-transcriptional regulatory modules have been unveiled,including miR482-PHAS and miRN15.miR482-PHAS might be involved in longan flower development beyond their conserved roles in plant defense,and miRN15 is a novel miRNA likely associated with a hairpin locus(HPL-056)to regulate strigolactone receptor gene DWARF14(D14)and the biogenesis of phasiRNAs from D14.These small RNAs are enriched in flower buds,suggesting they are likely involved in post-transcriptional regulatory networks essential for longan flower development via the strigolactone signaling pathway.

Phytochemical constituents and biological activities of longan (Dimocarpus longan Lour.) fruit: a review

Longan(Dimocarpus longan Lour.),as an edible fruit and traditional Chinese medicine,has been consumed for thousands of years.Longan pulp has abundant nutritional phytochemicals such as protein,carbohydrate,vitamin C,polysaccharides,polyphenols,which shows multiple biological activities including antioxidant,immunomodulatory and antitumor effects.Longan pericarp also demonstrates biological activities because of its rich content of polysaccharides and polyphenols.This review summarizes the bioactive compounds and bioactivities of longan pulp and aims to provide comprehensive information for future development of longan as a functional health food.

Genome-wide analysis of the CCCH zinc finger family in longan:Characteristic identification and expression profiles in Dimocarpus longan Lour

CCCH(C3 H) Zinc finger(Znf) transcription factors(TFs), as a novel type of Znf gene, regulate the expression of genes by binding to their mRNAs and play important roles in plant growth and development and abiotic stress resistance.Longan(Dimocarpous longan) is a tropical/subtropical fruit tree of great economic importance in Southeast Asia.However, genomic information on C3 H and their functions in longan are still unknown. In this study, a comprehensive analysis of the longan C3 H(DlC3 H) gene family was carried out. A total of 49 DlC3 H genes in three clades were identified from the longan genome database. Characteristics of the genes were analyzed with respect to gene structure,motif composition, phylogenetic tree and potential functions. The analysis of alternative splicing(AS) events suggested that AS events in DlC3 H genes were related to the transformation from longan non-embryonic to embryonic cultures.Promoter analysis indicated that most of the DlC3 H genes included cis-acting elements associated with hormones and stresses responses. Quantitative real-time PCR(qRT-PCR) analysis indicated that 26 of the 49 DlC3 Hs, which possess methyl jasmonate(MeJA) and abscisic acid(ABA) responsive cis-acting elements, showed differential expression patterns under treatment with ABA, MeJA and their endogenous inhibitors, suggesting that DlC3 Hs might be involved in the ABA and MeJA signaling pathways. The expression profiles of 17 of the 49 DlC3 Hs in non-embryonic callus and three tissues of embryonic cultures showed that only five of the 17 DlC3 Hs had the same expression trends as the FPKM trends in transcriptome data;the expression levels of DlC3 H07/14/16/36/49 in embryogenic callus and DlC3 H04/38 in globular embryos were high, suggesting that they have different functions in embryonic development. Further, we verified that DlC3 H01/03/05/11/19/39 were regulated by sRNAs by a modified 5’ RLM-RACE method. This study provides the first systematic analysis of C3 H genes in longan, and found that C3 H genes may be involved in hormone and stress responses, and somatic embryogenesis. Our preliminary investigation may provide clues to further studies on the characteristics and functions of this family in longan.

龙眼DlSWEET1基因的克隆、表达及功能分析

【目的】SWEET(sugars will eventually be exported transporters)是一类参与植物生长发育多个过程的糖转运蛋白,分析DlSWEET1基因在龙眼不同组织和处理下的表达,探究其在果实糖积累中的功能。【方法】以龙眼松风本果实为材料,克隆DlSWEET1基因,采用实时荧光定量PCR分析DlSWEET1在龙眼不同组织器官的表达以及在激素、冷、热、干旱胁迫下的表达模式。通过亚细胞定位、糖转运活性分析及草莓瞬时转化研究DlSWEET1基因的功能。【结果】DlSWEET1基因开放阅读框(ORF)全长为750 bp,编码249个氨基酸,包含一个PQ-loop保守结构域和蛋白典型保守结构域MtN3_slv。DlSWEET1在龙眼根、茎、叶、果肉等组织中均有不同程度的表达,在叶中的表达量较高,在果肉中的表达量次之,而在茎和根中表达量较低;不同浓度的蔗糖、葡萄糖和果糖处理龙眼叶片后,DlSWEET1在叶片中的表达量均有显著升高;低温、干旱及MeJA(茉莉酸甲酯)处理可显著提高DlSWEET1的表达。农杆菌侵染本氏烟草发现DlSWEET1蛋白定位在细胞膜和细胞核。糖转运活性分析证明DlSWEET1蛋白可以转运葡萄糖、果糖、蔗糖和甘露糖。草莓中瞬时转化DlSWEET1可以显著提升果实中的可溶性糖含量。【结论】瞬时过表达DlSWEET1导致转基因草莓果实的可溶性糖含量增加,为进一步解析DlSWEET1在龙眼果实糖积累中的作用提供理论依据。

基于HPLC指纹图谱和多组分含量测定结合化学模式识别研究的龙眼核质量评价

目的 建立龙眼Dimocarpus longan Lour.核HPLC指纹图谱及含量测定方法。方法 建立13批龙眼核指纹图谱,结合指纹图谱相似度评价及化学模式识别研究对龙眼核质量进行分析,并测定其中没食子酸、柯里拉京、鞣花酸的含量。结果 13批龙眼核样品相似度在0.919~0.999,标定共有峰11个,并指认出3种成分;聚类分析(HCA)将13批龙眼核聚为3类,主成分分析(PCA)提取到3个主成分,正交偏最小二乘判别分析(OPLS-DA)筛选出5个差异性成分;定量分析得到13批龙眼核中没食子酸、柯里拉京金、鞣花酸的含量范围依次为0.65~1.8 mg·g^(-1)、3.02~5.46 mg·g^(-1)、3.18~4.49 mg·g^(-1)。结论 建立的HPLC指纹图谱及含量测定方法重复性、稳定性良好,能够为龙眼核质量评价提供参考。

Optimization of Extraction Process for Longan(Dimocarpus longan Lour.)Leaves Using Multi-indicator Comprehensive Evaluation Method

[Objectives]This study aimed to optimize the extraction process of longan(Dimocarpus longan Lour.)leaves by multi-indicator comprehensive evaluation method.[Methods]The contents of gallic acid ethyl ester,astragalin,quercetin,luteolin and kaempferol in longan leaves were determined simultaneously by HPLC.Using the multi-indicator comprehensive evaluation method,the methanol concentration,solid to liquid ratio and extraction time for extraction of longan leaves were optimized by orthogonal test.[Results]The optimal extraction process of longan leaves are as follows:methanol concentration of 100%,solid to liquid ratio of 1∶5,and extraction time of 50 min.[Conclusions]The optimized process is simple and feasible,and it can be used to determine the contents of different ingredients in longan leaves.

龙眼拟茎点霉(Phomopsis longanae Chi)的生物学特性研究

龙眼拟茎点霉（Phomopsis longanae Chi）是导致采后龙眼果实腐烂的主要病原菌,研究了温度、湿度、pH值、光照、营养、越冬越夏等条件对龙眼拟茎点霉菌丝生长、产孢和孢子萌发的影响。结果表明：龙眼拟茎点霉生长适温为25℃,最适pH6~7;产生子座的最适温度为22~25℃,最适pH6,还需要一定的光照;以蔗糖、葡萄糖、可溶性淀粉和D-果糖为碳源和以蛋白胨、硫酸铵和硝酸铵为氮源的培养基最有利于龙眼拟茎点霉的生长发育;分生孢子的大量萌发要求高湿度和营养刺激,最适萌发温度25~28℃,最适pH6~7,在分生孢子器中,分生孢子的存活时间长于1年;该菌菌丝的致死温度为59℃（10 min）,分生孢子为50℃（10 min）。

龙眼DlAGO4和DlAGO6启动子的克隆及活性分析

【目的】Argonaute(AGO)蛋白是RNA介导的沉默复合物RISC的核心成分,参与植物生长发育、组织形成、细胞增殖凋亡、病毒防御、逆境响应等多种生物过程。探究龙眼DlAGO4和DlAGO6基因启动子区域的顺式作用元件,以及重组表达载体在不同激素处理下的表达模式,可为进一步研究DlAGO4和DlAGO6基因的功能提供参考。【方法】使用常规PCR技术克隆龙眼DlAGO4和DlAGO6基因启动子序列,采用BPDG、PLACE和PlantCARE等生物信息学工具进行DlAGO4和DlAGO6基因启动子序列的生物信息学分析,并构建全长启动子与GUS基因融合表达载体,转化烟草叶片,进行瞬时表达,通过GUS组织化学染色分析启动子的活性。【结果】DlAGO4基因启动子片段长度1514 bp,DlAGO6基因启动子片段长度1784 bp。两个启动子序列均含有TATA-box和CAAT-box核心元件、茉莉酸甲酯、脱落酸和光响应元件;DlAGO4启动子序列还含有水杨酸、赤霉素响应元件和厌氧应答调控元件;DlAGO6启动子序列还含有生长素及昼夜节律调控元件。2个启动子片段均可驱动GUS基因表达,表达强度弱于CaMV35S。MeJA和ABA处理可显著提高转DlAGO6启动子烟草叶片中GUS基因的相对表达水平,SA和MeJA处理可提高转DlAGO4启动子烟草叶片GUS基因的相对表达水平。【结论】成功克隆了龙眼DlAGO4和DlAGO6基因启动子,二者为激素诱导型启动子,具有驱动下游GUS表达的活性,可能参与了植物体胚发育以及对激素的响应。

龙眼蛋白对C57BL/6小鼠及RAW264.7巨噬细胞的炎症因子的影响研究

目的探讨龙眼蛋白对C57BL/6小鼠及RAW264.7巨噬细胞的炎症因子影响及作用机制。方法采用反向色谱-质谱法(reverse-phase liquid chromatography-mass spectrometry,RPLC-MS)鉴定龙眼蛋白的组成。采用100 mg/(kg·d)和200 mg/(kg·d)龙眼蛋白腹腔注射C57BL/6小鼠,酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)测定小鼠血液炎症因子的表达,苏木精-伊红(hematoxylin-eosin,HE)染色法染色分析肺部炎症病理反应;用0.2、0.4、0.6 mg/mL龙眼蛋白处理小鼠RAW264.7巨噬细胞,噻唑蓝[3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide,MTT]法测定细胞活力,实时荧光定量聚合酶链式反应法(real-time quantitative polymerase chain reaction,Q-PCR)和ELISA检测炎症因子表达,蛋白质免疫印迹法(western blot,WB)分析腺苷酸活化蛋白激酶(AMP-activatedprotein kinase,AMPK)/核因子κB(nuclear factor kappa-B,NF-κB)信号通路相关蛋白表达情况。结果通过RPLC-MS结合数据库检索,共鉴定到肽段覆盖率在30%以上的蛋白质25种。腹腔注射100 mg/(kg·d)龙眼蛋白使小鼠肺部产生炎症病理反应,小鼠血清中的炎症因子[血清淀粉样蛋白A(serum amyloid A,SSA)、超敏C反应蛋白(hypersensitive C-reactive protein,hs-CRP)、降钙素原(procalcitonin,PCT)、白细胞介素-6(interleukin-6,IL-6)]均显著升高。0.2 mg/mL龙眼蛋白处理使RAW264.7细胞IL-6、白细胞介素-8(interleukin-8,IL-8)、肿瘤坏死因子-α(tumor necrosis factorα,TNF-α)的m RNA和蛋白表达均呈剂量依赖式上升,AMPK磷酸化水平表达下调,p65磷酸化水平表达上调。结论龙眼蛋白能够促进小鼠和RAW264.7巨噬细胞的炎症反应,其作用机制可能是龙眼蛋白促进了AMPK/NF-κB信号通路相关炎症因子的表达。

Indicator Analysis of Off-season Longan Flower Reversal

[Objectives] This study was conducted to analyze the internal causes of flower reversal in Longan （ Dimocarpus longan Lour.） trees. [Methods] With flowering trees and flower reversal trees as experimental materials, the variations in sugar and starch in mature leaves, tender leaves, mature branches, twigs and terminal buds after flower forcing were analyzed. [Results] During flowering process, sugar content showed the greatest difference between flowering and flower reversal trees, and the difference was the greatest in mature leaves. Trees with mature leaves having a sugar content above 44.71 mg/g were found to be more prone to flowering, while those with leaf sugar content lower than 27.80 mg/g were susceptible to flower reversal. In addition, longan trees with a higher sugar content in tender leaves were not prone to flower reversal. [Conclusions] In future, whether off-season flower forcing can be performed on longan trees could be judged through the detection of tree leaves, which is of great significance to prevention of flower reversal in off-season longan production.

基于UHPLC-Q-Orbitrap MS技术的龙眼叶乙酸乙酯部位体内代谢研究

目的 采用超高效液相色谱-四级杆/静电场轨道阱高分辨质谱(UHPLC-Q-Orbitrap MS)技术研究龙眼叶乙酸乙酯在大鼠体内的代谢产物。方法 大鼠灌胃给予龙眼叶乙酸乙酯部位后,收集其尿液、血浆以及粪便样品。样品前处理后,采用Waters HSS T3色谱柱(100 mm×2.1 mm, 1.8μm);流动相为0.1%-甲酸水溶液(A)和含0.1%甲酸的100%甲醇(B),梯度洗脱。在正、负离子模式下采集样品数据分析。结果 从大鼠尿液、血清及粪便样品中共鉴定出包括原型成分在内的44个代谢产物,其中分别从尿液、血清及粪便样品中检测到10、20、26个。主要代谢途径有硫酸化、氧化、葡萄糖醛酸化等。结论 通过液质联用技术分析了龙眼叶乙酸乙酯部位在大鼠体内的代谢产物,为龙眼叶药效物质基础的阐明提供了一定的参考依据。

龙眼DlSWI3、DlARP、DlSWC和DlBSH亚基的分子特性及其在体胚发生早期的基因表达模式

[目的]探究龙眼Switching 3(SWI3)、Actin-related protein(ARP)、SWR1 complex(SWC)和Bushy(BSH)亚基的潜在功能。[方法]利用生物信息学分析其分子进化特性,用转录组和实时荧光定量PCR(real-time quantitative PCR,q RT-PCR)分析基因在体胚发生早期胚性愈伤组织(embryogenic callus,EC)、不完全胚性紧实结构(incomplete embryogenic compact structures,ICpEC)和球形胚(globular embryos,GE)阶段及在EC中响应激素的表达模式。[结果]龙眼含4个DlS WI3、10个DlA RP、4个DlS WC和1个DlB SH蛋白,它们大多不稳定且主要定位在细胞核、细胞质和叶绿体上。DlS WI3和DlA RP家族扩张受基因复制驱动,而4个DlSWC和1个DlBSH基因由单拷贝产生。DlS WI3、DlA RP、DlS WC和DlB SH结构域各异,进化上与拟南芥、水稻和甜橙一致。启动子预测表明,DlSWI3、DlARP、DlSWC和DlBSH亚基的基因含多种激素、胁迫、光和生长发育相关元件。q RT-PCR分析显示,从EC分化到GE,DlARP4、DlARP5-3、DlARP9和DlBSH逐渐下调,而DlSWI3C、DlARP8和DlSWC2逐渐上调,且DlSWI3D在GE特异高表达;DlARP5-3、DlSWC2、DlSWI3D和DlBSH在脱落酸(abscisic acid,ABA)处理12 h时被显著上调,DlARP4、DlARP5-3、DlSWC2、DlSWI3D和DlBSH在茉莉酸甲酯(methyl jasmonate,MeJ A)处理4 h时被显著上调,5个基因被赤霉素(gibberellin A3,GA3)或水杨酸(salicylic acid,SA)下调,DlARP4被ABA下调。[结论]DlS WI3、DlA RP、DlS WC、DlB SH亚基在进化上高度保守,它们可能通过响应激素诱导影响染色质重塑活性,调控龙眼早期体胚发育。

HPLC法测定龙眼肉中的几种核苷类物质

目的:建立HPLC法测定龙眼肉中腺苷、尿苷和腺嘌呤的含量。方法:采用NucleosilC18反相柱(250mm×4mm),以0.01mol/L的KH2PO4:乙腈=92:8为流动相,流速为1ml/min,检测波长为254nm,柱温40℃,用外标法,测定了龙眼肉中腺苷、尿苷和腺嘌呤的含量。结果与结论:分析中选取腺苷的线性范围0.1～20.0μg/ml,相关系数r=0.9999,回收率94.59%,RSD3.10%;尿苷的线性范围0.05～10.0μg/ml,相关系数r=1,回收率97.49%,RSD4.50%;腺嘌呤线性范围0.05～10.0μg/ml,相关系数r=1,回收率103.11%,RSD1.90%。该方法简便快速,线性关系良好。

18个龙眼品种果肉中核苷物质的HPLC定量测定

采用HPLC法同时分离测定了龙眼肉中的为尿嘧啶、胞苷、尿苷、胸腺嘧啶、肌苷、鸟苷、胸苷、腺嘌呤、腺苷等多种核苷成分,并用外标法计算各种核苷的含量。结果发现,鸟苷、尿苷、腺苷含量较高,多数龙眼品种为30～70μg/g;胸腺嘧啶、胞苷含量次之,普遍在10～30μg/g之间;胸苷、尿嘧啶含量较低,普遍在3～10μg/g之间;龙眼肉中还含有少量的腺嘌呤和次黄嘌呤核苷。如果以鸟苷、次黄嘌呤核苷、腺苷等核苷含量的多少来衡量龙眼品种的优劣,则石硖、双孖木、古山二号、储良、大乌圆、东壁、福眼等为较优的龙眼品种。

拟茎点霉侵染对采后龙眼果皮LOX活性和膜脂脂肪酸组分的影响

用拟茎点霉(Phomopsis longanae Chi)侵染福眼龙眼(Dimocarpus longan Lour.‘Fuyan’)果实,研究龙眼果皮脂氧合酶(LOX)活性、膜脂脂肪酸组分和细胞膜透性的变化及其与果皮褐变的关系。结果表明:拟茎点霉侵染导致龙眼果皮褐变指数、LOX活性和细胞膜透性增加,不饱和脂肪酸组分[亚油酸(C18:2)、亚麻酸(C18:3)和花生一烯酸(C20:1)]下降而饱和脂肪酸组分[棕榈酸(C16:0)和硬脂酸(C18:0)]增加,脂肪酸不饱和指数(IUFA)和脂肪酸不饱和度下降。拟茎点霉侵染提高龙眼果皮LOX活性和加速膜脂不饱和脂肪酸的降解,从而破坏细胞膜系统的完整性,导致膜系统区室化功能丧失,使多酚氧化酶与酚类物质接触,引起酚类物质的酶促氧化和黑褐色高聚物形成,导致龙眼果皮褐变。

石硖龙眼HPLC指纹图谱的研究

采用高效液相色谱(HPLC)法,建立了石硖龙眼的HPLC指纹图谱。色谱条件为:Nucleosil C18反相柱(250mm×4mm,5μm),流动相为0.01mol/L的KH2PO4和甲醇,二元梯度洗脱,流速0.8ml/min,检测波长260nm,柱温4℃。结果表明:14批次的石硖龙眼水溶性提取物色谱图共有峰13个。经"中药指纹图谱鉴别分析系统"统计分析,共有峰相对保留时间的相对标准偏差在1.2%以下,相似度均在0.967以上。符合指纹图谱的要求,可建立共有模式,作为科学评价石硖龙眼产品质量的检测方法。

龙眼咖啡酰辅酶A-O-甲基转移酶(DLCCoAOMT)基因的克隆和表达分析

【目的】克隆龙眼咖啡酰辅酶A-O-甲基转移酶(DLCCoAOMT)基因,分析其序列特征和在低温胁迫下不同组织中的表达情况并进行原核表达研究。【方法】采用RT-PCR和RACE技术从龙眼叶片中克隆DLCCoAOMT基因,用生物信息学方法对获得的氨基酸序列进行分析,利用荧光定量PCR研究DLCCoAOMT基因在不同组织中的表达。【结果】克隆得到DLCCoAOMT基因,GenBank登录号为JN093023。该cDNA全长993 bp,具有1个744 bp的完整开放阅读框(ORF),编码247个氨基酸。序列分析表明,DLCCoAOMT编码的氨基酸序列与其它植物的CCoAOMT蛋白有很高的相似性。系统进化树分析显示,龙眼DLCCoAOMT与桦木属的CCoAOMT蛋白亲缘关系较近。利用荧光定量技术进行组织表达模式分析发现,DLCCoAOMT基因在根、茎、叶中均有表达。总体上表达量在根和茎中较多,叶中较少,随着低温胁迫时间延长,DLCCoAOMT基因在各组织的表达量也发生变化。原核表达结果表明,DLCCoAOMT基因在大肠杆菌中获得表达。【结论】从龙眼中克隆到咖啡酰辅酶A-O-甲基转移酶基因,该基因可能参与调控低温胁迫。

龙眼DCL家族基因的启动子分析及时空表达

为了解龙眼DCL基因的功能,该试验对龙眼基因组数据提取的DlDCL1、DlDCL2、DlDCL3和DlDCL4基因序列进行启动子顺式作用元件及其受miRNA调控的分析;并以龙眼胚性愈伤组织为材料,研究了DlDCLs不同基因成员在非生物胁迫和外源激素处理下的表达情况。结果显示:(1)龙眼DCL基因启动子中除了TATA和CAAT外,还具有大量的光反应元件、激素应答元件、胁迫响应元件、组织特异性调控元件及植物生长发育相关的顺式调控元件,提示龙眼DCL基因启动子转录活性可能受到光、激素信号及逆境胁迫因素的诱导。(2)对调控龙眼DCL基因的miRNA进行筛选,结果显示DlDCL1受miR162和miR1024调控,DlDCL4受miR390和miR396调控。(3)实时荧光定量PCR显示,在一定浓度范围内,外源激素GA3、ABA和ETH均能下调DlDCLs基因的表达,而高浓度ETH处理则显著上调DlDCLs的表达。(4)高浓度蔗糖(6%)处理时DlDCL2、DlDCL3和DlDCL4显著上调表达,而低浓度(0.1%)处理时DlDCL1显著上调表达;不同温度处理下,DlDCL1在34℃时显著上升,DlDCL3随着温度的提高相对表达量逐渐减低;而DlDCL2和DlDCL4表达量差异不明显;NaCl胁迫处理下,DlDCLs在1h处理时表达量下调,但在其他不同时间点则上调表达。研究表明,龙眼DCL基因在外源激素及非生物胁迫处理下,并非是简单的一对一响应,而是存在较为复杂的响应机制。