Isolation and characterization of a new d-limonene synthase gene with a different expression pattern in Citrus unshiu Marc

We isolated a new d-limonene synthase gene (CitMTSE2) from Satsuma mandarin (Citrus unshiu Marc.) and compared its mRNA expression pattern with that of the previously isolated CitMTSE1. The predicted protein of CitMTSE2 showed 84.2% identity to that of CitMTSE1 at the amino acid level and possessed the typical structure and chemical features of general monoterpene synthases, such as transit peptides in the N-terminus, sequence motifs of RR and DDXXD, and cation-dependent reactions. A functional test demonstrated that CitMTSE2 is a d-limonene synthase gene.Under different expression regulations in response to the fruit developmental stage, the mRNA expression of CitMTSE2 indicated a specificity to fruit tissue and was different from that of CitMTSE1. Occurring mainly in peel at an early stage of fruit development, the mRNA expression profile of CitMTSE2 was similar to that of d-limonene biosynthesis in C. unshiu.     

温州蜜柑线粒体基因的克隆与表达分析

 根据其它植物atp6、cob和coxⅡ基因保守区设计特异引物, 利用RT - PCR技术从‘国庆1号’温州蜜柑cDNA中分别扩增出特异性片段, 将其克隆至pBluescrip t ( sk + ) 载体上进行测序。同源性分析表明, 所克隆的atp6、cob和coxⅡ与其他植物的对应氨基酸区域同源性分别达到85%、98%和96%以上。RT - PCR分析表明它们在叶片、花瓣以及不同时期的花蕾中都有表达, 在幼果中没有表达。Southern分析表明它们在温州蜜柑基因组DNA中为多拷贝。     

温州蜜柑叶片光合作用的光抑制

中午或模拟中午强光处理后,温州蜜柑叶片表观量子效率（ＡＱＹ）、最大荧光（Ｆｍ）、初始荧光（Ｆｏ）、可变荧光（Ｆｖ）、光化学效率（Ｆｖ／Ｆｍ）、光化学猝灭系数（ｑｐ）和电子传递速率（ＥＴＲ）下降。经强光处理后,虽然叶片的净光合速率（Ｐｎ）和光呼吸（Ｐｒ）均下降,但由于Ｐｒ降低幅度小于Ｐｎ,使Ｐｒ／Ｐｎ比值升高。     

低温胁迫对温州蜜柑光合作用的影响

低温处理使温州蜜柑叶片的气孔导度、净光合速率、光合表观量子效率及光合作用的光饱和光强和ＣＯ２饱和浓度降低,细胞间隙ＣＯ２浓度、光补偿点和ＣＯ２补偿点提高。虽然１０℃处理植株数小时后即出现光合速率下降,但叶绿素荧光测定结果显示,只有在３℃低温处理后,反映光系统Ⅱ光化学效率的Ｆｖ／Ｆｍ比值和Ｔ１／２才有较明显下降,而Ｆ０则明显升高。低温在３０～４０μｍｏｌ·ｍ－２·ｓ－１弱光下对光合的影响明显大于黑暗下。     

NaCl胁迫对转GFP国庆1号温州蜜柑愈伤组织生理效应的影响

以转GFP国庆1号温州蜜柑愈伤组织为试验材料,研究了不同浓度NaCl处理对转GFP国庆1号温州蜜柑愈伤组织增殖量及愈伤组织可溶性蛋白含量、可溶性糖含量、脯氨酸含量、SOD活性、CAT活性的影响。结果表明,随着NaCl浓度的增大,愈伤组织的增殖量、SOD活性、CAT治洼下降;可溶性蛋白含量和脯氨酸含量增加。     

柑桔配方施肥对产量品质及抗寒性的效应

本试验以10-11年生枳砧温州蜜柑龟井为试材，以含氮、磷、钾不同比例的五种复合肥和尿素（CK）为处理，进行配方施肥试验，结果表明，合理的配方施肥能够提高座果率和产量，改善果实的品质，增强树体的抗寒性。     

柑橘完熟采收增糖效应及其机理

研究了完熟采收对宫川温州蜜柑果实精积累影响和光合产物在果实内的分配特性及运输机制。结果表明:完熟采收可使柑橘汁囊中葡萄糖、果糖、蔗糖含量明显提高。光合产物主要通过背维管束输入汁囊,完熟采收期间分配到汁囊的光合产物占整个果实18%,而分配到可食组织(维管束+囊瓣皮+汁囊)的光合产物却超过60%。从着色期到完熟采收期,维管束到囊瓣皮再到汁囊存在一个由高到低的~(14)C光合产物的放射性比活度梯度,其中从囊瓣皮到汁囊的梯度都随汁囊糖积累而稳步上升。上述结果表明完熟采收有利于提高果实含糖量,光合产物通过存在于运输路径中由高到低的光合产物梯度推动而持续输入汁囊是完熟采收增糖的主要原因。     

亚硫酸氢钠处理减轻低温对温州蜜柑光合作用的影响

 低温胁迫使温州蜜柑叶片的净光合速率(Pn)、光系统Ⅱ的光化学效率(Fv/Fm)及光合电子传递速率(ETR)下降，反映跨膜质子动力势的叶绿素毫秒延迟发光(ms-DIE)减弱，叶片中的ATP含量降低。低温胁迫前，用NaHSO₃ 5 mmol/L涂于叶片表面，可使处理植株叶片的Pn和Fv/Fm分别少下降了11.5%和11.6%，ETR和ATP含量几乎没有下降，ms-DLE的下降幅度减少。可见，在柑橘上施用NaHSO₃能够减轻短期低温对光合机构及光合作用的影响。     

高温处理对温州蜜柑幼果膜脂过氧化作用和乙烯生成的影响

以3年生枳砧兴津温州蜜柑盆栽苗为试材,研究高温处理对柑桔幼果细胞膜脂过氧化作用和内原乙烯水平的影响,及其与幼果脱落的关系,以探讨柑桔高温热害的生理机制.结果表明:高温处理使SOD活性下降,MDA含量增加;膜脂脂肪酸组分中亚麻酸比例减少,硬脂城比例上升;致使股脂不饱和脂肪酸指数下降;加速了内原乙烯合成;加剧了幼果脱落.细胞膜脂脂质过氧化产物MDA含量与不饱和脂肪酸指数呈极显著负相关(r=-0.8407),而与幼果累积落果率呈极显著正相关(r=0.8707).细胞膜脂过氧化作用加剧可能是高温热害加速幼果脱落的初始反应.     

灌溉方法与地面覆盖对柑桔果实品质、产量与冠幕光水平的影响

灌溉方法与果园地面覆盖对'Mihowase'温州蜜柑果实品质、产量、果实大小分布与冠幕光水平有一定的影响。试验在南非西开普地区连续进行了两个季节（1994／1995和1995／1996）,设微喷、滴灌及滴灌配合TyvekTM（一种白色防渗纤维纸）地面覆盖等3个处理。结果表明：处理间果实颜色、可溶性固形物含量与果汁百分率均无明显差别。然而,滴灌及滴灌结合地面覆盖处理降低果实含酸量,因而趋于提高果实固酸比。处理不显著影响果实选择性采收百分率、产量与果实大小分布。TyvekTM地面覆盖显著（P＜0．05）提高进入冠幕下部的地面反射光水平。     

柑橘体细胞胚发生基因CsHB1特异肽段多克隆抗体的制备及其蛋白动态检测

【目的】构建柑橘HD-ZIP II转录因子CsHB1基因的原核表达系统,制备多克隆抗体,并检测抗体在‘伏令夏橙’胚性愈伤体胚诱导阶段中的特异性,为研究CsHB1基因在柑橘体细胞胚发生过程中的功能奠定基础。【方法】构建柑橘HD-ZIP II转录因子CsHB1基因的原核表达载体p GEX4T-CsHB1-N,转化大肠杆菌诱导目的融合蛋白的表达,并制备获得多克隆抗体anti-CsHB1-N。通过Western blot检测抗体在原核表达系统和柑橘体细胞胚诱导阶段的特异性,并分析体细胞胚诱导阶段CsHB1蛋白水平表达的动态变化。【结果】重组原核表达载体p GEX4T-CsHB1-N在Escherichia coli中高效表达出了分子质量约为49 ku的GST-CsHB1-N融合蛋白,并纯化获得多克隆抗体anti-CsHB1-N;经过Western blot分析表明,多克隆抗体可与‘伏令夏橙’愈伤体胚诱导阶段表达的目的蛋白特异结合;蛋白表达结果分析表明,在胚性愈伤组织中CsHB1蛋白呈现高的表达量,随着诱导培养的进行,呈现了先下降后上升的波动变化。【结论】多克隆抗体特异性好,可与‘伏令夏橙’胚性愈伤组织中目的蛋白特异性结合,可用于CsHB1基因功能分析。     

Somatic embryogenesis and plant regeneration in black pepper (Piper nigrum L.): I. Direct somatic embryogenesis from tissues of germinating seeds and ontogeny of somatic embryos

Summary

A protocol was developed for induction, maturation and germination of somatic embryos from the tissues of germinating seeds of black pepper (Piper nigrum L.). Explants were cultured on growth regulator – free solid SH medium maintained in the dark. The first somatic embryos developing directly from the explant tissue were noticed after 60 d of culture. Somatic embryos originated from a ring-like tissue on the micropylar region of the seeds. Sucrose concentration of the medium was found to be crucial for the induction of somatic embryos, and 30 g l^–1 was found to be the optimum. Maturation and germination of somatic embryos were achieved on the same medium. Suspension culture enhanced the process of maturation and germination. Regenerated plants were established in soil. Histology confirmed the ontogeny and each stage of development. Growth regulators were found to inhibit the induction of somatic embryogenesis. Cytological analysis of the regenerated plants revealed the normal chromosome number of 2n=52.     

Morphohistological examination on somatic embryogenesis of Musa acuminata cv. Mas (AA)

Somatic embryogenesis is a routine method in obtaining mass-production of plantlets especially in Musa spp. Somatic embryogenesis is a process characterized by a series of morphological changes leading to plant regeneration. Therefore it is important to identify stages in somatic embryogenesis through morphological and histological characteristics. In this study, stages of somatic embryogenesis through morphohistological study could differentiate between embryogenic callus consisting of somatic embryos and non-embryogenic callus. In Musa spp., globular to torpedo shaped somatic embryos resulted in good suspension cultures followed by recovery of somatic embryos during developmental stage. The morphological observations were supported by histological sections of structures at all particular stages. Histological examination also revealed the vascular connection in germinated somatic embryos which later turned into a complete plantlet.     

Efficient plant regeneration through somatic embryogenesis from anthers and ovaries of six autochthonous grapevine cultivars from Galicia (Spain)

An efficient protocol for plant regeneration through somatic embryogenesis was developed for the first time in five autochthonous grapevine cultivars (Treixadura, Torrontés, Mencía, Merenzao and Brancellao) from Galicia (north-western Spain). Improvements of the induction protocol for the cv. Albariño in respect to previously reported data were also made. Media containing NN salts and MS vitamins supplemented with combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and benzyladenine (BA) were effective in inducing somatic embryogenesis. The addition of casein hydrolysate produced the best results for up to four cultivars (Albariño, Treixadura, Merenzao and Brancellao). Somatic embryogenesis was also induced in explants collected during the binucleate pollen microsporogenesis stage (R3 flower stage) of all cultivars with the exception of Treixadura, suggesting that under appropriate conditions explants can display longer windows of competence. Transfer of embryogenic callus to differentiation medium produced callus proliferation and somatic embryo development proliferation by secondary embryogenesis. However, an extensive process of precocious embryo germination was observed reducing the efficiency of secondary embryo proliferation. This situation was overcame by the use of differentiation medium lacking growth regulators (DM1 medium), which allowed reducing precocious germination by half on average and improving embryo proliferation by secondary embryogenesis. Transfer of normally developed, ungerminated isolated embryos to germination medium allowed obtaining very high percentages of embryo germination (up to 97% in Mencía, more than 87% averaging all cultivars). A comparison of plant conversion between precociously and normally germinated embryos showed that precocious germination in differentiation medium reduced plant conversion, even at high rates depending on the cultivar (from 93% to 39% in Brancellao, from 86% to 61% averaging all cultivars).     

香蕉延长因子1A（MaEF1A）基因的分离及特征分析

采用筛选香蕉果实采后cDNA文库的方法,首次获得了一条长为1 341 bp的cDNA序列,命名为MaEF1A。生物信息学分析结果表明,它与麝香百合延长因子1A 3的mRNA有84%的同源性,与烟草延长因子1A的mRNA有83%的同源性,推测它可能为编码香蕉延长因子1A（elongation factor-1 alpha...     

Direct somatic embryogenesis and plant regeneration from leaf explants of Limoniumsinensis (Girard) Kuntze

The potentialities of direct somatic embryogenesis and plant regeneration from leaf explants of Limoniumsinensis var. Golden Diamond invitro were investigated. Young whole leaf and cut leaf explants when cultured on MS basal medium supplemented with each of the growth regulators N⁶-benzyladenine (BA) (0.44–2.2 μM) or thidiazuron (TDZ) (4.54 μM) alone or in combination with a fixed concentration of α-naphthalene acetic acid (NAA) (1.07 μM) produced somatic embryos directly. More than 90% of the leaf explants produced white, globular somatic embryos on BA (2.2 μM) and NAA (1.07 μM) supplemented MS basal medium within 1 week of inoculation. Most of the embryos matured further and converted after 8 weeks of culture on the same medium. Histological observation showed that the somatic embryos originated from single cells of epidermal layer of leaf. Histological evidence of formation of shoot and root poles during conversion of the embryos confirmed that these structures were true somatic embryos. After conversion the plantlets were further placed on MS medium containing 0.44 μM BA and 4.5 μM IBA for better shoot and root growth. About 90% of the plantlets transferred to the mixture of soil:perlite:vermiculite (1:1:1) in small plastic pots acclimatized successfully. Of these 85.5% plants survived after transferring into earthen pots containing a mixture of soil, coarse sand and cattle manure (1:1:1) under greenhouse or shady open condition.     

番木瓜丝氨酸/苏氨酸蛋白激酶类抗病基因同源序列的克隆与特征分析

根据丝氨酸/苏氨酸蛋白激酶类抗病基因产物催化结构域Ⅰ和Ⅸ的氨基酸保守序列设计简并引物,PCR扩增番木瓜叶片基因组DNA和cDNA。在NCBI中用BlastX比对发现,5个来自基因组DNA的克隆和4个来自叶片cDNA的克隆可能编码的氨基酸残基序列属于STK蛋白激酶类抗病基因同源序列片段和受体样蛋白激酶基因的同源片段,命名为gPK1-gPK5和cPK1-cPK4,在GenBank注册得到序列号分别为AY693385-AY693389,AY738762-AY738765。除克隆gPK5含有内含子之外,其它都发现了连续阅读框ORF。通过分析除Gpk5之外的8个克隆可能编码氨基酸序列中丝氨酸/苏氨酸蛋白激酶类蛋白激酶的9个催化保守结构域Ⅰ-Ⅸ,并利用ClustalW软件对8个克隆和已报道的抗病蛋白水稻Xa21和西红柿Pto氨基酸残基序列进行分子系统进化树分析发现,8个克隆不仅是受体样蛋白激酶基因的同源片段而且是抗病候选基因片段。     

Direct somatic embryogenesis from explants obtained from in vitro germinated embryonic axes of Camellia sinensis (L.) O. Kuntze

Summary

Studies on direct somatic embryogenesis in several types of explant from in vitro plantlets of tea cultivar TRI 2025 were undertaken to select those most suitable to induce cotyledonary-type somatic embryos. Mature zygotic embryonic axes were surface-sterilised and cultured on MS medium without growth regulators containing 0.6% (w/v) agar. Results showed that 65% of embryonic axes that converted into plantlets at the fifth week of culture had succulent leaves. Several types of explant (normal leaves, large and small succulent leaves, hypocotyl segments and root tips) were isolated from in vitro plantlets at the fifth week and cultured on half-strength MS medium containing 2 mg l^–1 6-benzylaminopurine (BAP) and 0.2 mg l^–1 naphthalene acetic acid (NAA). Morphological and histological observations on somatic embryogenesis were made. The results indicated that somatic embryos were produced at high frequency (25 – 50%) directly from the surface of hypocotyl segments (HS) and large succulent leaves (LSL) after 6 weeks of culture. Efficient somatic embryogenesis was induced in small succulent leaves (SSL) after 16 weeks. Most somatic embryos originated directly from the cortical tissues of HS or the upper epidermal layers of SSL or LSL. HS and SSL from in vitro plantlets gave the highest production of typical, firm somatic embryos for use in tea improvement programmes and for in vitro conservation of tea germplasm.