荔枝果皮过氧化物酶的纯化及部分酶学性质研究

经硫酸铵分级盐析、DEAE-Sepharose和Sephadex G-75柱层析分离,从荔枝果皮中分离提纯了过氧化物酶(POD),该酶被纯化了12．5倍,产率为1．9％。经SDS-PAGE确定为单一条带。该酶最适反应温度为35℃,对热具有较强的稳定性,经75℃处理30min,酶活性只损失50％。最适pH约为6．5,但在pH4．0—8．0范围内活力仍比较稳定。该酶在25℃和0．05mol／L磷酸缓冲液(pH7．0)条件下对愈创木酚、邻苯二酚和没食子酸的Km分别是2．75、12．4和12．8mmol／L。二硫苏糖醇和抗坏血酸能完全抑制POD活性,L-半胱氨酸、柠檬酸、FeS04、GSH、SDS和ZnS04对POD活性有一定的抑制作用,而FeCl,和CuSOt对POD则有较好的激活作用。     

Intergeneric Hybridisation between Litchi (Litchi chinensis Sonn.) and Longan (Dimocarpus longan Lour.)

The breeding barriers between commercial litchi (Litchi chinensisSonn.) and longan (Dimocarpus longan Lour.) cultivars were investigatedby conducting reciprocal pollinations. This work has shown thatit is possible to generate intergeneric hybrids using litchias the female parent. Investigation of comparative in vivo pollentube growth demonstrated that there is discrimination againstcross- compared to self-pollen at all sites in the pistil. Pollentubes were frequently observed in the ovary after cross-pollinationin litchi but rarely in longan. Fruit production was reducedafter crossing in both longan and litchi. Isozyme analysis usingphosphoglucose isomerase revealed that hybrid progeny only developedwhen litchi was the maternal parent. Morphologically the hybridplants were similar to the maternal parent but leaves were smaller.Three types of seeds developed in litchi following pollinationwith longan pollen. These were (1) normal seeds with a developedtesta and embryo, (2) seeds with aborted embryos but normaltesta development, and (3) seedless fruit where the ovule remainedthe same size as at anthesis without further development ofembryo or testa. The potential germplasm available to improvethese crops within the Sapindaceae is discussed.Copyright 1994,1999 Academic Press Litchi, Litchi Longan, Dimocarpus, hybridisation, isozyme     

Identifying Litchi (Litchi chinensis Sonn.) Cultivars and Their Genetic Relationships Using Single Nucleotide Polymorphism (SNP) Markers

Litchi is an important fruit tree in tropical and subtropical areas of the world. However, there is widespread confusion regarding litchi cultivar nomenclature and detailed information of genetic relationships among litchi germplasm is unclear. In the present study, the potential of single nucleotide polymorphism (SNP) for the identification of 96 representative litchi accessions and their genetic relationships in China was evaluated using 155 SNPs that were evenly spaced across litchi genome. Ninety SNPs with minor allele frequencies above 0.05 and a good genotyping success rate were used for further analysis. A relatively high level of genetic variation was observed among litchi accessions, as quantified by the expected heterozygosity (He = 0.305). The SNP based multilocus matching identified two synonymous groups, ‘Heiye’ and ‘Wuye’, and ‘Chengtuo’ and ‘Baitangli 1’. A subset of 14 SNPs was sufficient to distinguish all the non-redundant litchi genotypes, and these SNPs were proven to be highly stable by repeated analyses of a selected group of cultivars. Unweighted pair-group method of arithmetic averages (UPGMA) cluster analysis divided the litchi accessions analyzed into four main groups, which corresponded to the traits of extremely early-maturing, early-maturing, middle-maturing, and late-maturing, indicating that the fruit maturation period should be considered as the primary criterion for litchi taxonomy. Two subpopulations were detected among litchi accessions by STRUCTURE analysis, and accessions with extremely early- and late-maturing traits showed membership coefficients above 0.99 for Cluster 1 and Cluster 2, respectively. Accessions with early- and middle-maturing traits were identified as admixture forms with varying levels of membership shared between the two clusters, indicating their hybrid origin during litchi domestication. The results of this study will benefit litchi germplasm conservation programs and facilitate maximum genetic gains in litchi breeding programs.     

Aldose reductase inhibitors from Litchi chinensis Sonn

Diabetes is one of the major risk factors for cataract. Aldose reductase has been reported to play an important role in sugar-induced cataract. In this study, we conducted pharmacological investigations upon experimental rat lenses using extracts of the fruits of Litchi chinensis (Sapindaceae). Of the extracts and organic fractions of L. chinensis tested, a MeOH extract and an EtOAc fraction were found to be potent inhibitors of rat lens aldose reductase (RLAR) in vitro — their IC₅₀ values being 3.6 and 0.3 μg/mL, respectively. From the active EtOAc fraction, four minor compounds with diverse structural moieties were isolated and identified as d-mannitol (1), 2,5-dihydroxybenzoic acid (2), delphinidin 3-O-β-galactopyranoside-39,59-di-O-β-glucopyranoside (3), and delphinidin 3-O-β- galactopyranoside-39-O-β-glucopyranoside (4). Among these, 4 was found to be the most potent RLAR inhibitor (IC₅₀ = 0.23 μg/mL), and may be useful in the prevention and/or treatment of diabetic complications.     

荔枝若干重要果实品质性状的QTL分析

以‘马贵荔＇ב焦核三月红＇76株F_1代群体为试材,检测了酒石酸、苹果酸、蔗糖含量和单果重4个果实性状分离的情况。结果表明,4个性状表现为连续分布,具有数量性状的典型特征,与4个果实性状连锁的QTL位点23个,其中控制酒石酸的QTL为2个,控制苹果酸的QTL为4个,控制蔗糖的QTL为12个和控制单果重的QTL为5个。各QTL的LOD值在3.15～5.61之间,可解释13.85%～88.3%的表型变异。     

Multiple shoot induction and plant regeneration in litchi (Litchi chinensis Sonn.)

Multiple shoot induction in Litchi chinensis Sonn. (litchi) has been achieved by two methods: (1) direct germination of litchi seeds in 6-benzylaminopurine (20 mg l^–1)-supplemented MS liquid medium and supported on a filter-paper bridge and (2) in planta treatment with 6-benzylaminopurine (100 μg on alternate days) of the axillary bud regions of plants germinated and maintained under sterile conditions. While the former method resulted in as many as 27.5±8.6 shoot buds from the cotyledonary node within 4 weeks, the latter yielded on average approximately 8 shoot buds from each treated node in 8 weeks. The cytokinin treatment in planta consisted of placing sterile filter paper moistened with sterile distilled water over the node and adding different concentrations of 6-benzylaminopurine. Both methods of multiple shoot induction were found to be effective for the five genotypes of litchi that were tested. The shoots elongated and rooted directly in vermiculite after a pulse treatment with IBA (25 mg/ml) for 15 min. Fungus growth which is a serious problem in litchi tissue culture, was controlled using a fungicide, Bavistin, and by eliminating organic nutrients from the growth medium. Received: 27 July 1998 / Revision received: 30 September 1998 / Accepted: 27 October 1998     

Identification of polysaccharides from pericarp tissues of litchi (Litchi chinensis Sonn.) fruit in relation to their antioxidant activities

A large number of polysaccharides are present in the pericarp tissues of harvested litchi fruits. A DEAE Sepharose fast-flow anion-exchange column and a Sephadex G-50 gel-permeation column were used to isolate and purify the major polysaccharides from litchi fruit pericarp tissues. Antioxidant activities of these major polysaccharide components were also evaluated. An aqueous extract of the polysaccharides from litchi fruit pericarp tissues was chromatographed on a DEAE anion-exchange column to yield two fractions. The largest amount of the polysaccharide fraction was subjected to further purification by gel filtration on Sephadex G-50. The purified product was a neutral polysaccharide, with a molecular weight of 14 kDa, comprised mainly of 65.6% mannose, 33.0% galactose and 1.4% arabinose. Analysis by Smith degradation indicated that there were 8.7% of (1-->2)-glycosidic linkages, 83.3% of (1-->3)-glycosidic linkages and 8.0% of (1-->6)-glycosidic linkages in the polysaccharide. Furthermore, different polysaccharide fractions extracted and purified from litchi fruit pericarp tissues exhibited strong antioxidant activities. Among these fractions, the purified polysaccharide had the highest antioxidant activity and should be explored as a novel potential antioxidant.     

水分胁迫对荔枝叶片氮和核酸代谢的影响及其与抗旱性的关系

水分胁迫下,荔枝叶片蛋白酶活性和Ｐｒｏ 含量增加,ＰＤＨ 活性和可溶性蛋白质含量下降;抗旱性强的品种蛋白酶活性增加的幅度和蛋白质含量下降的幅度小于抗旱性弱的品种, 而ＰＤＨ 活性下降的幅度和Ｐｒｏ 含量上升的幅度均大于抗旱性弱的品种。水分胁迫引起荔枝叶片核酸、ＤＮＡ 和ＲＮＡ 含量下降,ＤＮＡ 含量下降的幅度小于ＲＮＡ 含量下降的幅度;ＤＮａｓｅ 和ＲＮａｓｅ 活性上升,ＤＮａｓｅ 活性上升的幅度小于ＲＮａｓｅ 活性上升的幅度。抗旱性强的品种核酸、ＤＮＡ 和ＲＮＡ 含量下降的幅度以及ＤＮａｓｅ 和ＲＮａｓｅ 活性上升的幅度均小于抗旱性弱的品种。     

中国96个荔枝种质资源的EST-SSR遗传多样性分析

根据本实验室已获得的荔枝果皮cDNA文库EST序列,通过SSRIT在线检索,从3391条EST序列中,发现305条含有SSR,占整个文库EST的8.99%。利用SSR-ESTs序列共设计100对EST-SSR引物,其中62对在荔枝上有扩增产物,50对有扩增多态性,即具有一定的通用性。接着从96份荔枝种质中选取12个品种的基因组DNA,开展核心引物筛选,共筛选出多态性较好的EST-SSR分子标记30个;这30个EST-SSR分子标记在96份资源共扩出284条带,不同引物的扩增条带在3～18条之间,平均9.47条,其中有282条为多态性带,多态率高达99.30%,每对引物的Nei's基因多样度范围为0.186～0.396,香农信息指数范围为0.318～0.558;此外,系统聚类分析结果表明,在相似系数0.5525处,可将96份荔枝种质资源分成了8大类群,该8大类群基本与其生态类型和植物学性状特征相符。在此基础上,还对荔枝的主栽品种和特殊种质进行鉴别,结果表明,该30个EST-SSR分子标记在不同品种间可产生较清晰可辨的多态性差异,为荔枝品种以及种质资源鉴别和鉴定的分子指纹的构建奠定了良好基础。     

蕈树叶芳香精油成分分析及其抗氧化活性研究

为了解蕈树叶芳香精油化学组分及抗氧化活性,采用气相色谱-质谱联用技术(GC-MS)结合Kovats保留指数(K I)比较的方法对其进行了成分分析,并运用二种体外方法对其抗氧化活性进行了测定。结果表明蕈树叶精油以倍半萜烯类为主(占62.39%),主要特征成分为双环大根香叶烯(10.71%)、(E)-丁香烯(9.96%)和α-依兰油烯(8.92%)。该精油具有中等程度的自由基清除活性和抗脂质过氧化活性。其抗氧化活性可能与精油中的酚类物质5-羟基白菖莆烯(2.97%)以及醇类物质1-表橙椒醇(3.12%)和(Z)-白檀油烯醇(2.12%)等化合物有关。     

Somatic embryogenesis and plant regeneration of litchi (Litchi chinensis Sonn.) from leaves of mature phase trees

Embryogenic cultures were induced from leaflets from new vegetative flushes of mature ‘Brewster’ litchi trees on B5 medium containing 400 mg l⁻¹ glutamine, 200 mg l⁻¹ casein hydrolysate, 30 g l⁻¹ sucrose, 4.52 μM 2,4-D, 9.30 μM kinetin and 3 g l⁻¹ gellan gum in darkness. Embryogenic cultures consisting of proembryonic cells and masses were maintained either on semi-solid MS medium supplemented with 4.52 μM 2,4-D and 0.91 μM zeatin or as embryogenic suspension cultures in liquid medium of the same composition. Maturation of somatic embryos occurred on semi-solid MS medium with 5–20% (v/v) filter-sterilized coconut water in darkness. Recovery of plants from somatic embryos was improved with 14.4 μM GA₃ on half-strength MS medium with 0.2 g l⁻¹ activated charcoal under a 16 h photoperiod provided by cool white fluorescent lights (60–80 μmol s⁻¹ m⁻²). Plants have been successfully acclimatized in the greenhouse.     

In Vitro Embryo Culture and Induction of Multiple Shoots in Lychee (Litchi chinensis Sonn.)

Immature embryos of different sizes and ages from commercialvarieties of lychee (Litchi chinensis Sonn.) were cultured ina range of different media. Embryos as small as 3 mm could becultured using in vitro techniques and subsequently grown intoplants. MS solid medium with 2% sucrose supplemented with 150ml l^–1 coconut water was most effective in stimulatingthe germination of immature lychee embryos. Embryos of lycheewere treated to induce adventitious buds from embryonic shootsas a means of achieving multiplication. The different varietiesexhibited differences in response, with Bengal embryonic shootsproducing 15 adventitious buds after pretreatment with 100 mgl^–1 BAP for 3 h. Root formation was achieved in 65% ofadventitious shoots using MS medium supplemented with 0.5 mgl^–1 NAA and activated charcoal. These plants were successfullydeflasked and grown on in the glasshouse. This technique providesof means of producing some multiple shoots from lychee embryosand has value for multiplication in a breeding program wherea method of micropropagation is unavailable. Litchi chinensis Sonn., lychee, embryo culture, multiple shoots, in vitro     

应用RAPD标记研究野生荔枝种质资源

应用RAPD方法对海南60份野生荔枝进行基因组多态性分析,20个随机引物共产生165条RAPD带,DNA片段大小在200～1500bp之间,其中121条为多态性带,占总带数的73．3％,并利用遗传距离进行聚类分析。结果表明,60份野生荔枝可归为6类,表明种群存在一定的遗传变异,也为分析野生荔枝群体间及群体内的遗传多样性探索了有效方法。     

荔枝雌蕊发育过程中钙分布变化与细胞程序性死亡

应用焦锑酸钾沉淀法研究了荔枝雌花和雄花雌蕊发育过程中钙的分布变化。在大孢子母细胞阶段,雌花近珠孔内珠被细胞和花柱细胞的钙沉淀颗粒主要分布在细胞壁和细胞间隙,少部分在液泡;雌花花柱维管细胞中含有很多的钙沉淀颗粒;在雄花的近珠孔内珠被细胞钙沉淀颗粒大多在液泡中;雄花花柱细胞和维管细胞中钙沉淀颗粒很少。大孢子母细胞减数分裂后,雌花雌蕊继续发育,雄花雌蕊败育。雌花维管中的钙沉淀颗粒数量减少,可能被转运到将要发生花粉萌发和受精的部位。雌花近珠孔内珠被细胞壁的钙沉淀颗粒分布增加,花柱细胞从上（近柱头）到下（近子房）钙沉淀颗粒量递增。雄花近珠孔内珠被细胞发生程序性死亡：液泡中的钙进入细胞核启动细胞程序性死亡,核周隙与质膜腔形成连续的通道,钙在核与细胞质之间的流动不受限制;在特定的时间段,钙沉淀颗粒出现在线粒体、过氧化物体和线型内质网的外膜上。钙在细胞中重新分布可能触发和调节细胞程序性死亡的进程。缺乏钙沉淀颗粒的雄花花柱细胞迅速解体。     

Changes in Cytokinins before and during Early Flower Bud Differentiation in Lychee (Litchi chinensis Sonn.)

Lychee (Litchi chinensis) has been analyzed for cytokinins in buds before and after flower bud differentiation, using reversephase high performance liquid chromatography in combination with Amaranthus bioassay and gas chromatography-mass spectrometry-selected ion monitoring. Four cytokinins, zeatin, zeatin riboside, N⁶-(δ²-isopentenyl)adenine, and N⁶-(δ⁶-isopentenyl) adenine riboside, were detected in buds. There was an increase of cytokinin activity in the buds during flower bud differentiation. In dormant buds, the endogenous cytokinin content was low, and the buds did not respond to exogenous cytokinin application. Application of kinetin promotes flower bud differentiation significantly after bud dormancy. These results are interpreted as an indication that the increase in endogenous cytokinin levels during flower bud differentiation may be correlative rather than the cause of flower bud initiation.     

Fingerprinting and analysis of genetic diversity of litchi (Litchi chinensis Sonn.) accessions from different germplasm collections using microsatellite markers

Litchi (Litchi chinensis Sonn.) is a fruit crop with a clear niche for expansion in countries with subtropical climates. One of the main limitations for breeding purposes and optimum germplasm management in this species is the confusion in cultivar denomination among different producing countries and germplasm collections worldwide. Litchi cultivar identification is still mainly based on morphological characters, and homonymies and synonymies are very frequent. To address this gap, a molecular study was conducted to characterize litchi accessions from two of the main litchi-producing regions for the export market, Mauritius and Réunion, and to compare them to those obtained from litchi cultivars from different origins conserved in a germplasm collection in Spain. Eleven simple sequence repeat (SSR) loci were used to characterize molecular polymorphisms among 88 litchi accessions conserved in Mauritius, Réunion, and Spain. A total of 67 amplification fragments were detected with those 11 SSRs, with an average of 6.1 bands/SSR. Three primer pairs seemed to amplify more than one locus. The mean expected and observed heterozygosities over the eight single locus SSRs averaged 0.53 and 0.61, respectively. The total value for the probability of identity was 9.78?×?10^?4. Molecular characterization revealed the existence of 42 different genetic profiles. Several synonymies and homonymies in litchi cultivar nomenclature both within and across geographical regions were found. This comparative study provides the basis for the standardization of litchi cultivar nomenclature over the studied regions and in other litchi-producing countries.     

Floral organogenesis and development of Litchi chinensis Sonn.cv.Guiwei

利用SV11立体显微镜和JSM-6360LV型扫描电镜观察‘桂味'荔枝花器官的发生和发育过程.结果表明:花序原基最先发生,然后形成数个大小不等的单花原基;4个萼片原基的发生不同步,其中一侧对位先发生;6～10枚雄蕊原基以轮状方式几乎同时发生;心皮原基最后发生,2～3枚(稀4枚)心皮原基同时出现,随后进行侧向生长,逐渐合拢形成子房.雌花中,花柱、柱头分化明显,雄蕊退化.雄花中,花丝细长,花药饱满,雌蕊退化或发育不完全.两性花中,雌雄蕊发育完全.花粉粒近球形,具3孔沟,表面为条纹状纹饰.