荔枝LcBES1的克隆、表达及功能分析

[目的]基于荔枝花穗RNA-seq数据,克隆荔枝BR信号途径的LcBES1,并对其序列特征、表达特点、基因功能及亚细胞定位进行分析.[方法]利用qRT-PCR对LcBES1在不同组织中的表达进行研究,利用MEGA 6软件进行系统进化树分析.通过转基因分析其在拟南芥中的基因功能,利用烟草瞬时转化系统分析其亚细胞定位.[结...     

苹果PGIP基因部分家族成员启动子的克隆与功能分析

根据已发表的苹果基因组序列设计特异引物,克隆了苹果（Malus × domestica Borkh.）品种‘秦冠’和‘太平洋玫瑰’中PGIP家族基因PGIP1和PGIP2的启动子片段,序列分析结果表明PGIP1与PGIP2启动子序列相似度为71%,‘秦冠’和‘太平洋玫瑰’的PGIP1、PGIP2启动子相似度都达到了99%。两个品种PGIP1启动子中TATA-box和CAAT-box的数量明显多于PGIP2,PGIP1和PGIP2启动子分别存在茉莉酸甲酯和水杨酸响应元件,暗示PGIP1和PGIP2存在不同的抗病响应途径。构建了启动子︰︰GUS融合表达载体,通过对农杆菌介导法转化烟草叶片中的GUS活性分析,比较了不同启动子的活性。结果表明：PGIP1启动子活性在品种间差异不显著;PGIP2启动子活性在品种间差异显著,‘秦冠’是‘太平洋玫瑰’的2.37倍,可能与品种抗病性差异有关;同一品种中PGIP1启动子活性显著高于PGIP2。     

荔枝水孔蛋白基因LcPIP的克隆与组织特异性表达研究

 从荔枝组织中克隆了9个质膜水孔蛋白基因（LcPIP）的cDNA全长序列,并研究了其组织特异性表达。结果表明：9个LcPIP分为PIP1和PIP2两类。定量PCR结果表明9个LcPIPs在荔枝的不同组织中均表达,其中LcPIP1-2、LcPIP1-4、LcPIP2-1、LcPIP2-2在花中的表达量相对较高,LcPIP1-1在茎中相对较高,LcPIP1-2在果肉中较高,LcPIP2-5在种子中的表达量仅次于果皮,而LcPIP1-3在叶中最高,LcPIP2-4在根中最高。LcPIP1-1、LcPIP2-4、LcPIP2-5在果皮中表达量较高,而LcPIP2-3在果皮中特异表达,且在所有成员中表达量最高,可能与果皮组织水分运输有关。     

‘嘎啦’苹果MYB12基因启动子的克隆与功能分析

以‘嘎啦’苹果基因组为模板,用PCR方法扩增得到苹果MYB12基因启动子(ProMYB12)。利用在线分析网站Plant CARE和PLACE对启动子上存在的顺式作用元件进行了预测。构建pCAMBI0390-ProMYB12-GUS植物瞬时表达载体并转化农杆菌,瞬时转染野生型番茄Micro-Tom(Lycopersion esculentumcv.Micro-Tom)用以研究启动子的启动活性。结果表明:克隆获得的启动子长度为1 290bp。启动子序列中存在大量的顺式作用元件,既有转录必备的TATA-box和CAAT-box,也存在非生物胁迫和植物激素响应元件以及大量的光调控元件,此外还存在一些组织特异性表达元件。‘嘎啦’苹果MYB12启动子驱动的GUS基因在番茄的花和种子处有较高的表达量,表现出一定的组织特异性。     

海南杜鹃热诱导基因RhRCA1启动子的克隆与功能分析

为了阐述RhRCA1基因的调控机制,以海南杜鹃(Rhododendron hainanense)3年生扦插苗为材料,分析RhRCA1高温响应和组织表达特性;并克隆获得RhRCA1的启动子序列,将该启动子分别驱动荧光素酶报告基因在烟草中瞬时表达、GUS报告基因在拟南芥中稳定表达,分析该启动子活性、组织特异性和热诱导性。结果显示:(1)25℃条件下,RhRCA1表达量极低,37℃处理后其表达量显著升高,呈现典型的热诱导特性,并且RhRCA1在绿色组织中的表达量显著高于非绿色组织,呈现组织特异性;(2)获得的启动子长1 624 bp,其含有多个非生物胁迫响应、光响应、组织特异性等相关元件;(3)构建RhRCA1启动子和萤光素酶融合的植物表达载体,在烟草叶片中瞬时表达,荧光成像结果表明RhRCA1启动子能强烈响应高温胁迫;(4)构建RhRCA1启动子和GUS融合的植物表达载体,转化拟南芥植株筛选至T3代,GUS染色结果显示,高温能显著诱导RhRCA1启动子在拟南芥子叶、成熟叶、茎、萼片、果荚等绿色组织中的表达。研究结果表明RhRCA1启动子是1个兼具高温诱导型和组织特异性的启动子,可应用于植物...     

甘菊BADH基因启动子的克隆与瞬时表达分析

为了给菊花 [Dendronthema ×grandiflora (Ramat.) Kitam.] 的转基因育种提供诱导型启动子,借鉴5′RACE策略,利用锚定PCR步移的方法克隆了甘菊 [Dendranthema lavandulifolium (Fisch. ex Trautv.) Makino] 甜菜碱醛脱氢酶（betaine aldehyde dehydrogenase,BADH）基因的4个启动子序列,分别命名为DBP11、DBP12、DBP21和DBP22（GenBank登录号：DQ497620~DQ497623）,序列长度分别为1 230、1 249 、1 273 和574 bp。4个启动子序列对应区域的同源性在89%以上。其中DBP12和DBP21分別是甘菊BADH基因DlBADH1和DlBADH2（GenBank登录号：DQ011151和DQ011152）的启动子序列,DBP11和DBP22为甘菊BADH基因家族中其它成员的启动子序列。序列分析表明,上述启动子序列中均含有多处与水分胁迫和脱落酸诱导相关的顺式作用元件。在表达载体pCAMBIA1305.2的基础上,用所克隆的4个甘菊BADH基因启动子序列分别置换驱动其报告基因表达的35S启动子,建立了新的植物表达载体。用其转化农杆菌,并用叶盘法侵染甘菊,瞬时表达的结果表明,这些启动子序列均具备驱动报告基因表达的功能。     

荔枝漆酶基因LcLac 的克隆与表达分析

 为了探明荔枝漆酶基因LcLac的表达与果皮褐变之间的关系,通过筛选‘妃子笑’荔枝果皮cDNA文库和3′-RACE技术,克隆获得了荔枝LcLac全长cDNA序列（EU 527187.1）。该序列全长1 779 bp,5′-UTR长26 bp,3′-UTR长52 bp,含一个1 701 bp的完整开放阅读框,编码含566个氨基酸残基的多肽。该氨基酸残基序列与欧亚槭树等物种的漆酶蛋白相似性较高。荧光定量PCR结果表明,LcLac在荔枝花中表达量最高,果肉中表达量最低。在采后贮藏前期,随LcLac表达量上升果皮褐变指数升高,且褐变指数较高的‘妃子笑’果皮中LcLac表达量高于褐变指数低的‘紫娘喜’果皮。贮藏中后期严重褐变果皮中LcLac表达量比贮藏前期低。采后贮藏前期果皮中LcLac的上调表达可能对其褐变起促进作用。     

荔枝ANS基因的克隆及其序列分析

以3个不同发育时期的‘糯米糍’荔枝果皮和幼嫩叶片为试材,对其ANS基因进行了克隆及分析。结果表明:花青素合成酶(Anthocyanidin synthase,ANS)是花色素苷生化合成途径中的一个关键酶。利用同源克隆方法从荔枝果皮中克隆得到了1个ANS基因,该基因开放阅读框的长度为1 074bp,编码357个氨基酸。以基因组为模板,扩增得到了1 279bp的核苷酸序列与cDNA序列比对发现,基因组序列中还有1个内含子,位置在504～708bp之间。通过系统发育分析发现,该基因编码的蛋白与葡萄、可可豆、柑橘等聚为一类。     

桃PpPGIP1 启动子的分离与功能分析

 通过染色体步移法分离桃[Prunus persica（L.）Batch]PpPGIP1 上游启动子序列,利用生物信息学方法对其功能进行初步预测;构建该启动子植物表达载体并通过农杆菌介导法转化烟草,研究不同激素诱导条件下GUS 蛋白瞬时表达情况;并利用实时荧光定量RT-PCR 技术分析了PpPGIP1 在水杨酸（SA）、茉莉酸甲酯（MeJA）、脱落酸（ABA）和1–氨基环丙烷–1–羧酸（ACC）诱导下的表达特性,获得了长度为1 018 bp 的桃PpPGIP1 启动子序列。该序列与梅和中国李PGIP 启动子序列的同源性分别为90%和89%;该启动子序列含有SA、MeJA、ABA 和ETH 诱导调控相关的抗病与胁迫顺式作用元件;ABA 和ACC 可以诱导PpPGIP1 启动子调控GUS 基因的表达;实时荧光定量RT-PCR 分析结果表明：SA、MeJA、ABA 和ACC 都可以诱导桃叶片中PpPGIP1 的表达。PpPGIP1 可能参与SA、MeJA、ABA 和ETH的信号转导,在桃抵抗病原菌和逆境胁迫方面起着非常重要的作用。     

鸭梨多酚氧化酶基因CDS区的克隆及表达

以鸭梨(Pyrus bretschneideri Rehd.)果皮基因组DNA为模板,根据已经发表的多酚氧化酶(polyphenol oxidase,PPO)基因保守序列设计引物,利用PCR技术,克隆得到鸭梨多酚氧化酶编码区序列,将此核酸序列克隆到载体pGEM-T,酶切鉴定后测序,结果表明该段序列含有1782个核苷酸,编码593个氨基酸。对鸭梨多酚氧化酶基因片段的一致性分析和进化树分析表明,该片段与沙梨(Pyrus pyrifolia,AB056680)、苹果(Malus domestica,L29450)、李(Prunuss alicina,AY866432)以及杏(Prunus armeniaca,AF020786)的一致性分别为99%、96.3%、82%和51.4%,绘制的进化树和形态学分类地位相一致。将该片段连接到表达载体pET39b上,获得的重组子命名为pET39b-PPO,热激法转化表达受体大肠杆菌BL21(DE3)菌株,用IPTG进行诱导。SDS-PAGE分析表明,PPO基因在大肠杆菌中被诱导表达蛋白质相对分子质量约66ku,检测表明具有多酚氧化酶的活性。     

Effect of temperature on growth and flowering of litchi (Litchi chinensis Sonn.) cultivars

Summary

Moderate day/night temperatures (20/15° v. 15/10°C) increased vegetative growth and reduced flowering in the seven litchi cvs Tai So, Bengal, Souey Tung, Kwai May Pink, Kwai May Red, Salathiel and Wai Chee. At higher temperatures (25/20° and 30/25°C), vegetative growth was promoted further and flowering eliminated. Temperature also influenced the type of inflorescence formed. More leaves were formed on the panicles of trees growing at 20/15° than at 15/10°C. All terminal shoots on all cultivars produced panicles at 15/10°C. The relative order for the amount of flowering at 20/15°C was: ‘Wai Chee’>‘Salathiel’>‘Kwai May Pink’>‘Tai So’>‘Bengal’>‘Souey Tung’>‘Kwai May Red’. Cultivars which were vigorous at high temperatures produced fewer panicles at 20/15°C and fewer leafless panicles at 15/10°C. Only small differences were observed in the leaf water potential and the nutrient status of the shoots at different temperatures. Vigour and flowering of the cultivars in the glasshouse generally reflected field performance in subtropical Australia (Lat. 27°S). Low vigour could be useful for selecting litchi cultivars for good fruiting in environments with warm autumns and winters.     

The effect of fruiting status on nutrient composition of litchi (Litchi chinensis Sonn.) during the flowering and fruiting season

Summary

The pattern of leaf composition was investigated in three litchi orchards (cvs Bengal and Tai So) in subtropical Australia (Lat 27°S). Leaves were sampled from fruiting and non-fruiting branches from flowering to fruit harvest. The response to fruiting varied with the nutrient and the orchard. The major effect of fruiting was to reduce leaf K in two of the orchards. Leaf N, P, Zn and Na were lower in fruiting branches in a third of the orchards, while leaf Ca, Mg, Mn and B levels were higher. The levels of these nutrients in the other two orchards, and those of Cu and Fe, were not significantly affected by fruiting status. Strong seasonal effects from flowering to harvest on the leaf contents of most nutrients followed the typical pattern as leaves aged. Most nutrients were more stable during flowering to just after fruit set. The levels of most nutrients were similar in both leaf and fruit. The major exceptions were K which was about twice as high in the fruit and B which was five times higher in the leaves. The uptake of all nutrients continued throughout fruit growth in proportion to the accumulation of fruit dry weight and reached a maximum rate during the final period of aril (flesh) development. The relative order for final content in the fruit was: K>N>P>Mg>Ca>Na>Fe>Zn>Cu>Mn>B. The choice of branch type and time of sampling would influence the diagnosis for N, K, Ca, Mg and Fe at these sites. It is recommended that leaves for diagnostic purposes in litchi be collected from fruiting branches two to six weeks after fruit set.     

The roles of cytokinins and abscisic acid in the pericarp of litchi (Litchi chinensis Sonn.) in determining fruit size

Summary

Variations in the concentrations of cytokinins (CTKs) and abscisic acid (ABA) were studied in the pericarp of litchi (Litchi chinensis Sonn.) fruit using the large-fruited cv. ‘Erdanli’ (55.3 g per fruit) and the small-fruited cv. ‘Huaizhi’ (20.9 g per fruit), as well as large fruit (32.4 g) from early blooms and small (20.8 g) fruit from late blooms on the same inflorescences of cv. ‘Feizixiao’ from the same commercial orchard in Guangdong, China in 2000. ‘Erdanli’ had higher concentrations of CTKs than cv. ‘Huaizhi’ on three out of six sampling dates during fruit development, and lower concentrations of abscisic acid (ABA) from 40 d after anthesis. In cv. ‘Feizixiao’, fruit from early blooms had higher concentration of CTKs than fruit from late blooms at all sampling dates, and lower concentrations of ABA on three out of five sampling dates. Therefore, the former had a higher CTK:ABA ratio. These data suggest that a high CTK:ABA ratio favours fruit growth in litchi.     

Growth responses and endogenous IAA and iPAs changes of litchi (Litchi chinensis Sonn.) seedlings induced by arbuscular mycorrhizal fungal inoculation

Seedlings of litchi (Litchi chinensis Sonn.) were inoculated with two arbuscular mycorrhizal (AM) fungal species, Glomus intraradices and Gigaspora margarita. Plant growth response and morphological changes induced by AM inoculation were investigated. Plant endogenous indoleacetic acid (IAA) and isopentenyl adenosines (iPAs) concentrations were determined. With mycorrhizal infection rate of 9.0–18.8%, plant biomasses increased by 13.5–30.1%. Leaf number, leaflet number, total leaf area and first order lateral root number were significantly increased by AM inoculation. Although G. margarita significantly increased plant P content and uptake, no significant difference in N nutrition was observed between mycorrhizal and non-mycorrhizal plants. Enzyme-linked immunosorbent assay (ELISA) indicated the changes in IAA and iPAs induced by AM inoculation. IAA concentrations in shoots and in roots were 5–7 times and 2–5 times higher in mycorrhizal than in non-mycorrhizal plants, respectively. The iPAs concentrations increased by 1.7 times in shoots and by 1.9–2.5 times in roots, due to mycorrhizal inoculation. We suggest that the changes in endogenous phytohormone level may be responsible for morphological alteration induced by AM inoculation.     

The relationship between yield and assimilate supply in lychee (Litchi chinensis Sonn.)

Summary

Experiments were conducted on lychee (Litchi chinensis Sonn.) in subtropical Australia (lat. 27° –29°S) to evaluate the role of assimilates on fruit retention. All the leaves of the last flush, all the leaves of the previous flush (about eight leaves per terminal shoot), or all the old leaves were removed from trees. Medium (3–5.cm diameter) or large branches (5–10.cm diameter) were girdled and defoliated after fruit set, and fruit retention compared with ungirdled and undefoliated branches. Other branches were girdled and defoliated between anthesis and fruit harvest. Finally, 20, 50 or 80% of the flowering panicles were defruited on large trees. Defoliated trees had 35 to 45% lower yields than the controls. This was despite the treatment with all the old leaves removed having a much lower leaf area index than the other defoliation treatments (1.7 vs. 2.3 and 2.8). Leaves next to the inflorescences are more important for yield than the older leaves. Fruit retention was very low on girdled branches that had been defoliated, especially when the leaves were removed in the first 20.d after anthesis. This suggests that the yields of girdled branches were determined by the availability of assimilates soon after fruit set. In contrast, the number of fruit retained on ungirdled branches was unrelated to the number of leaves, with defoliation having no effect on yield. Fruit on these branches were supported by resources from elsewhere in the tree. Thinned trees had similar yields to those of unthinned plots (65–82.kg tree^–1). Thinning apparently increased fruit retention in the remaining clusters, under a higher leaf:fruit ratio. There were large differences in the concentrations of starch in the tree, and seasonal changes, with starch declining from flowering to fruit harvest. In contrast, there were only small responses to the treatments, suggesting that the fruit were mainly dependent on current photosynthesis. Photosynthesis in the leaves behind the fruit clusters was more important than photosynthesis in the older shaded leaves.     

荔枝FLOWERING LOCUST(FT)同源基因cDNA全长克隆及其表达

应用RT-PCR方法在首次克隆获得荔枝AP1同源基因cDNA全长基础上,又得到2个荔枝FT同源基因cDNA全长,分别命名为LcFT1和LcFT2(基因登录号分别为:JN214350、JN214351)。LcFT1基因开放阅读框522 bp,编码174个氨基酸,推测蛋白质分子质量为19.65 ku,等电点为8.68。LcFT2基因开放阅读框522 bp,编码174个氨基酸,推测蛋白质分子质量为19.56 ku,等电点为7.34。蛋白质二级结构预测表明,LcFT1和LcFT2蛋白都具有4个α螺旋,10个β折叠区。同源分析表明,LcFT1和LcFT2基因在不同植物中的一致性为72%～82%。半定量RT-PCR分析表明,三月红荔枝花芽分化期LcFT1和LcFT2基因只在叶中表达,并且在成熟叶中表达量最多。研究将有助于进一步了解荔枝开花的分子机理及其成花的生物学发育过程。     

荔枝钻蛀性害虫种类调查及防治

对我国荔枝产区的钻蛀性害虫种类进行调查,并对主要害虫的发生规律进行描述,针对不同害虫提出了科学的防治措施。结果表明,发现的荔枝钻蛀性害虫共19种,隶属于3目12科,其中蛀干害虫9种,蛀果害虫10种;主要钻蛀性害虫为荔枝蒂蛀虫Conopomorpha sinensis Bradley、龟背天牛Aristobia testudo(Voet)和荔枝尖细蛾Conopomorpha litchiella Bradley。     

热水结合酸浸处理对荔枝果皮色素含量与酶活性的影响

研究了９８℃热水３ｓ结合酸浸处理的荔枝果皮的色素含量与酶活性变化。结果表明：在（２±０．５）℃贮藏条件下,９８℃热水３ｓ结合酸浸处理降低果皮叶绿素、花色素苷与类胡萝卜素的含量,也降低类黄酮、总酚的含量与多酚氧化酶、过氧化物酶的活性,改变上述指标在贮藏期间的动态变化曲线。讨论了热水处理技术的应用前景。     

胚珠发育与荔枝花型的关系

通过荔枝胚珠发育观察发现,胚珠发育特点同花蕾外部开矿及不同花类型密切相关,提出了荔 枝不同类型花歧化发育的假设模式：胚珠在孢子母细胞时败育,严重萎缩,导致雄花的形成;若胚珠在功能大孢子出现前后败育,则在胚珠形成椭圆的、内含皱折的空腔,决定雄能花的形成;若胚珠能通过２核胚囊期,则建成雌蕊结构完整的雌能花。     

Interaction of temperature and vegetative flush maturity influences shoot structure and development of lychee (Litchi chinensis Sonn.)

Potted lychee trees (cv. Tai so) of varying vegetative flush maturity were grown under a range of temperature regimes and monitored for subsequent shoot structure and development. A combination of low temperature (15/17 or 18/13 °C day/night) and high vegetative flush maturity was necessary for floral initiation to occur. Exposure to high temperatures (28/23 °C) invariably resulted in the production of vegetative shoots, irrespective of flush maturity. Strong floral initiation was marked by the emergence of terminal panicles and accompanying axillary panicles. A decrease in vegetative flush maturity or increase in temperature (e.g. 23/18 °C) resulted in a decrease in axillary shoot formation and the production of several intermediate shoot structures. These included leafy panicles, stunted panicles, partially emerged buds and non-emergent swollen buds, often produced on the same tree. At 23/18 °C, closer synchronisation of initial flush maturity was required for the production of a consistent shoot-type. Trees with synchronised mature flushes (I-2) at 23/18 °C resulted in the production of swollen terminal buds. Healthy trees were maintained in this state for at least 11 months. These results indicate that both temperature and flush maturity can influence subsequent shoot structure of lychee. In the absence of either a strong floral temperature (18/13 °C) or strong vegetative temperature (28/23 °C), slight differences in initial flush maturity have greater impact on the type of emerging shoot formed.