Cloning,molecular analysis,and developmental expression of 3 oleosin cDNA isoforms in coconut (Cocos nucifera L.)

Coconut stores its lipid reserves in the endosperm, specifically in oil bodies that contain proteins called oleosins which stabilise their structure. This study reports the complete cDNA sequences of 3 genes for 3 isoforms of oleosin termed CnOLE500a, CnOLE500c, and CnOLE300a with 396, 375, and 381 nucleotides, respectively. Their predicted amino acid sequences were 131, 124, and 126 residues in length, respectively, with molecular weights of 13,787, 12,982, and 12,988 Da, respectively. The complete CnOLE500a cDNA sequence had 83.1% similarity with that of CnOLE500c, while CnOLE300a cDNA had only 50.7% and 46.5% similarity with CnOLE500a and CnOLE500c, respectively. None of the 3 coconut oleosins had the 18 amino acid insertion characteristics of H-class oleosins, thus they belonged to the L-class of oleosins. However, phylogenetic analysis showed that CnOLE300a was more related to H-class oleosins from dicots. All 3 isoforms were highly expressed at all stages of coconut endosperm development. CnOLE500c exhibited 6% higher expression than CnOLE500a and 15% higher than CnOLE300a at all stages of coconut endosperm development. However, oleosin proteins were barely detectable in the solid endosperm of coconut at 5–6 months, but this increased 20-fold at 6–7 months, and increased by a further 2- and 12-fold, at 7–8 and 8–9 months, respectively.     

荔枝类甜蛋白基因的克隆与表达分析

以‘妃子笑’荔枝（Litchi chinenesis）为试材,通过PCR方法克隆了荔枝类甜蛋白基因LcTLP（登录号JF682821）的cDNA全长和完整开放阅读框（ORF）对应的gDNA序列。序列分析表明：该基因无内含子,cDNA序列含有一个672 bp的ORF,编码223个氨基酸残基序列。荧光定量PCR结果表明：LcTLP在花中表达量最高,其次是果皮,在果肉中表达量最低;在果实发育过程中,果皮中LcTLP表达量先上升后下降,采后果皮中的表达量高于采前,炭疽菌侵染诱导LcTLP的表达,失水和低温能抑制LcTLP的表达。     

欧李番茄红素β–环化酶基因ChLCYb的分离与功能鉴定

利用RACE技术和RT-PCR相结合,从欧李[Cerasus humilis(Bge)Sok]果实中克隆获得长度为1798 bp的番茄红素β–环化酶(lycopeneβ-cyclase)基因ChLCYb的cDNA全长序列,开放读码框为1515 bp,编码503个氨基酸。序列分析发现,ChLCYb具有植物LCYb保守区(Plant LCYb conserved region)、FAD/NAD(P)结合区(Dinucleotide-binding signature)、"Cyclase motif 1"、"Cyclase motif 2"和"lycopeneβ-cyclase motif"等典型结构特征,在N–端存在1~84个氨基酸残基组成的转运肽信号序列,在85~106、209~227、373~391和460~480氨基酸区域包含4个跨膜结构域。荧光定量PCR结果表明,ChLCYb在叶中表达量最高,其次是幼果,在根中最低;在欧李果实发育过程中,果皮中ChLCYb表达量高于果肉,果皮中ChLCYb表达量先升高,在花后90 d左右表达量最高,然后呈缓慢下降趋势,而果肉中ChLCYb表达量相对平稳。ChLCYb表达模式与果皮和果肉中β–胡萝卜素含量的积累呈显著正相关(r=0.824和r=0.712,P0.05)。利用大肠杆菌工程菌体系诱导表达了ChLCYb蛋白,并证实其可催化工程菌株中番茄红素向β–胡萝卜素转化,其转化效率达71.22%。在番茄中超量表达ChLCYb促进果实中番茄红素向β–胡萝卜素的转化和积累,转基因株系L-11号果实中的β–胡萝卜素含量高达692.18μg·g~(-1) DW,是非转基因对照的4.42倍。     

Assessment of volatile and non-volatile organic compounds in the liquid endosperm of young ‘Nam Hom’ coconut (Cocos nucifera L.) at two stages of maturity

SUMMARY

Young coconut (Cocos nucifera L.) fruit collected 6–8 months after anthesis (MAA) contain a high volume of water [20% (w/w)] and can provide a refreshing drink. The present study investigated volatile organic compounds (VOCs), as aroma components, and physico-chemical changes in the liquid endosperm of young ‘Nam Hom’ coconut at two stages of maturity: the one-layer stage (6 MAA) and the two-layer stage (7.0–7.5 MAA) of the flesh. The liquid endosperm of coconut fruit becomes sweeter and more turbid late during maturation. Lauric acid (C_12:0) and myristic acid (C_14:0) were the main free fatty acids in the liquid endosperm of coconut. Short-chain fatty acid (C₁₀–C₁₂) concentrations increased slightly, and longer-chain fatty acid (C₁₄–C₁₈) concentrations declined when fruit matured from the one- to the two-layer stage of flesh. Solvent extractions showed a 1.4-fold increase in the total concentration of VOCs in the liquid endosperm during fruit development. This was related to a major increase in squalene and sterols, to 21.7% (w/w) of the total VOC and non-volatile organic compounds, whereas the concentrations of terpenes and esters remained stable. Although a series of alkanes existed in the liquid endosperm of coconut, the main aroma components were 2-methyl-1-butyl acetate and terpenes such as D-limonene, α-pinene, and 3-carene. We conclude that, when coconut fruit developed from the one- to the two-layered flesh stage, the liquid endosperm not only became sweeter and contained higher concentrations of aroma volatile and non-volatile components, but also became oily and less translucent.     

Sequence and developmental expression of a seed storage protein gene in pecan

Summary

A cDNA clone, designated pepl, that encodes a putative seed storage protein in pecan (Carya illinoinensis) was isolated and characterized. Northern blot analysis indicated that this gene is embryo-specific, and is temporally expressed during the reserve accumulation stage of pecan seed development. The nucleotide sequence covers the partial coding region (135 base pairs) with a 3’ non-coding region of 219 base pairs. The deduced amino acid sequence consists of 45 amino acids that bears close identity to seed storage proteins from other plants.     

Virus-induced PpCHLH gene silencing in peach leaves (Prunus persica)

Summary

Compared with other model plants or crops, studies on the molecular biology of fruit trees have lagged behind due to technical difficulties in gene transformation and manipulation. Therefore, developing an efficient system for gene manipulation is of particular significance in fruit trees. Here, we report on a method for virus-induced gene silencing (VIGS) by syringe-infiltrating a tobacco rattle virus (TRV) vector containing a specific target gene sequence into peach (Prunus persica) leaves to analyse gene function. The target gene (PpCHLH) was a 4,445 bp sequence encoding the H subunit of magnesium chelatase and was first cloned as a cDNA. This gene (PpCHLH) is reported to be related to chlorophyll biosynthesis, and any loss of function leads to a decrease in chlorophyll content, with concomitant yellow or white colour changes in the leaves. To silence the PpCHLH gene, a 1:1 mixture of Agrobacterium tumefaciens strain GV3101 cultures containing pTRV1 or a pTRV2 vector construct with a 650 bp cDNA fragment of the PpCHLH gene was infiltrated into leaves of 4 – 5 week-old peach seedlings. After 15 d, the inoculated areas of the green leaves faded and finally turned yellow or white. Loss of PpCHLH gene function was confirmed by semi-quantitative RT-PCR, real-time qRT-PCR, and siRNA northern blot analysis. The virus-induced gene silencing (VIGS) technique developed here could be used for further molecular studies on fruit trees.     

Cloning,silencing, and prokaryotic expression of strawberry sucrose synthase gene FaSus1

The sucrose synthase (Sus) gene is regarded to be involved in strawberry fruit ripening; however, direct molecular and biochemical evidence is lacking. Here, the coding cDNA sequence of FaSus1 was cloned by RT-PCR and both a hairpin-mediated RNA interference pBI121 vector and a recombinant E. coli expression PET28a vector were constructed. These vectors were then used to transform strawberry fruit and for prokaryotic expression in strain BL21, respectively. The results showed that hairpin-mediated RNA silencing of FaSus1 led to a decrease in sucrose content and inhibited fruit ripening. Enzymatic activity analysis of FaSus1 showed that the reaction system contains 0.309 mg of recombinant FaSus1 protein, reaching 0.617 mg/mL. The cleavage activity of the enzyme was 0.25 U with a specific activity of 0.812 U/mg, whilst the synthetic activity of the enzyme was 0.057 U with a specific activity of 0.185 U/mg. This study provides physiological, molecular, and biochemical evidence to demonstrate that the FaSus1 has high sucrolysis activity and plays an important role in the regulation of strawberry fruit ripening.     

MaCDPK7, a calcium-dependent protein kinase gene from banana is involved in fruit ripening and temperature stress responses

Calcium-dependent protein kinase (CDPK) is an important Ca²⁺ sensor in plant development and responses to stress stimuli. Banana fruit is a typical climacteric- and chilling-sensitive fruit. The roles of CDPK genes in the ripening and chilling response of banana fruit are unclear. We isolated a cDNA fragment with full-length coding MaCDPK7 (HM061075) from fruit peel tissue. Induction of MaCDPK7 expression in fruit peel was observed 0.5 h after phytohormone ethylene treatment, earlier than the up-regulation of MaACO1 and MaACS1, coding a 1-aminocyclopropane-1-carboxylate oxidase and 1-aminocyclopropane-1-carboxylate synthase, respectively. Penetration of calcium signaling blockers, EGTA, or LaCl₃ inhibited the ripening and gene expression of MaCDPK7, MaACO1, and MaACS1 in the in vitro cultured peel pieces, but Ca²⁺ application removed the inhibitory effect of EGTA and LaCl₃. This suggested that MaCDPK7 might be a positive regulator involved in the calcium signaling in banana fruit ripening. Under temperature stresses, we found that MaCDPK7 gene expression increased 3 h after hot water dipping (HWD). The HWD-treated fruits exhibited markedly less chilling injury (CI) than control fruits in cold storage. Stored at 7°C (CI temperature) dramatically increased MaCDPK7 gene expression, while pre-treatment of HWD repressed the induction in cold storage. These results show that the MaCDPK7 gene is involved in regulating banana fruit ripening and chilling resistance induced by heat treatment.     

DNA markers for variety identification in date palm (Phoenix dactylifera L.)

Summary

Simple sequence repeats (SSRs; microsatellites) are currently the favoured type of molecular marker for identifying plant germplasm. However, identifying polymorphic SSRs and then using them to distinguish closely-related varieties can be time-consuming. Polymorphic markers originating from particularly labile regions of the genome are likely to be easier to develop and also have the potential to identify markers that have higher polymorphic information contents. Genomic regions that vary in somaclonal “off-types” are a possible source of such labile regions of the genome. Thirty-seven primer pairs, developed from sequences that differed between normal and mantled somaclonal mutant oil palm (Elaeis guineensis Jacq.) plants, were used in polymerase chain reactions to screen DNA from 18 varieties of date palm (Phoenix dactylifera L.). From the resulting polymorphisms, three primer pairs were selected which, when used in combination, could identify each of the date palm varieties, unambiguously. The polymorphic bands were isolated, sequenced, and new internal primers were designed. However, all of the amplifications using these new primers yielded only monomorphic bands, indicating that the variation among these date palm varieties lay mainly at or near the original primer sites, and that the internal sequences were conserved.     

Identification and structure of six members of the hexokinase gene family in Vitis vinifera: Cloning,expression, and functional analysis of a putative chloroplast stromal-type hexokinase

Summary

Hexokinase catalyses the first step in the metabolism of glucose, but has also been proposed to be involved in sugar sensing and signalling in both yeasts and plants. The substrates of hexokinase such as glucose (Glc) and fructose (Fruc) are also the most important sugars during grape berry ripening. The grapevine proteome database was analysed to investigate the roles of hexokinases in grape berry growth and development. Six hexokinase genes displaying high nucleotide sequence identity (72 – 87%) with hexokinase genes in other species were identified. Most of the Vitis vinifera hexokinase (VvHXK) genes had a highly conserved genomic structure consisting of nine exons and eight introns. A search for cis–regulatory elements in the promoter regions of all six hexokinase genes revealed that most were probably regulated by light, sugar, phytohormones, or abiotic stress. The isolation and expression of a hexokinase cDNA from V. vinifera ‘Sultanine’, named VvHXK3, was also reported. Analysis of the predicted amino acid sequence of VvHXK3 suggested that the protein could be a chloroplast-stromal hexokinase with a possible transit peptide cleavage site after amino acid residue 26. The VvHXK3 gene was differentially expressed in a variety of organs including berries, leaves, roots, and pollen, but its expression was highest in berries during their early development and at the start of ripening. To determine its function, VvHXK3 cDNA was expressed in a triple mutant yeast strain that lacked the ability to phosphorylate Glc and Fru and, therefore, was unable to grow on these sugars as sole carbon source. Mutant yeast cells that expressed VvHXK3 grew on both Glc and Fru, indicating that VvHXK3 could complement this mutant and had hexokinase activity.     

Effect of low temperatures on pulp browning and endogenous abscisic acid and ethylene concentrations in peach (Prunus persica L.) fruit during post-harvest storage

Summary

Changes in the severity of pulp browning, endogenous abscisic acid (ABA) and ethylene concentrations, and related gene expression in peach (Prunus persica L. ‘Bayuecui’) fruit stored at the biological freezing-point temperature (BFT; 1ºC), at 4ºC, or at room temperature (20ºC; as a control) were investigated.The results showed that fruit stored at room temperature showed climacteric changes in ethylene and ABA concentrations and in levels of expression of the corresponding ethylene synthetase genes (PpACS1, PpACO1) and the gene for a key enzyme of ABA synthesis (PpNCED), concomitant with a significant decrease in fruit firmness and an increase in juice yield. Meanwhile, two sucrose synthase genes (PpSUS1 and PpSUS2) were expressed at relatively low levels. However, fruit firmness decreased slightly and chilling injury (CI) increased significantly 40 d after storage (DAS) at 4ºC, with a significant accumulation of proline [from 8.33 µg g^–1 (at t = 0 d) to 9.125 µg g^–1 (at t = 40 d)]. In contrast, fruit firmness decreased slowly and pulp browning increased slightly 80 DAS at the BFT ( 1ºC), with a lower accumulation of proline than in fruit stored at 4ºC. The concentrations of soluble sugars and titratable acidity decreased during storage at 1ºC, 4ºC, or 20ºC, but there was a peak of soluble sugars at a late stage of storage at the BFT ( 1ºC). The fruit browning index was significantly and positively correlated (R ² = 0.967) with ABA concentration. The decrease in ABA concentration in cold-stored fruit (at both 1ºC and 4ºC) inhibited ethylene production. Moreover, there was no significant correlation between CI in fruit and ethylene production. In conclusion, the decrease in ABA concentration reduced ethylene concentrations and inhibited the development of pulp browning, which resulted in an improved taste, but crisper fruit, when fruit were stored at the BFT (–1ºC).     

苹果酰基辅酶A结合蛋白2 编码基因MdACBP2的克隆和表达分析

 酰基辅酶A结合蛋白（ACBP）是一个保守的蛋白家族,在酰基酯辅酶A的转运过程中发挥重要作用。在水稻和拟南芥中鉴定的ACBP除了参与长链酰基辅酶A的转运以外,还参与了植物对生物和非生物胁迫的反应。为了探讨在苹果树中是否也存在参与植物对逆境反应的ACBP,并可用于改良苹果品种,以提高抗逆能力,从苹果品种‘鸡冠’中克隆、鉴定了一个ACBP基因,命名为MdACBP2。MdACBP2包含一个378 bp的开放阅读框,编码包含125个氨基酸残基的蛋白质;推定的氨基酸序列中包含有一个ACB结构域,无信号肽序列。序列和结构特征表明该基因是ACBP家族ClassⅠ亚族的成员。利用荧光定量PCR技术检测MdACBP2的表达,发现在苹果的根、叶、花、幼果、枝皮、芽等组织中均有表达,在叶中的表达量最高,在幼果中的表达量最低,这一表达模式暗示MdACBP2可能参与多种生理途径。不同胁迫处理的时序表达分析表明,MdACBP2的表达能被轮纹病病原菌Botryosphaeria dothidea侵染和10% PEG渗透胁迫所抑制,低温胁迫则能诱导MdACBP2的表达,1 mmol·L^-1 Pb(NO₃)₂处理对MdACBP2的表达没有显著影响。推测MdACBP2可能参与了苹果对低温胁迫的防御反应。     

甜瓜果实蔗糖磷酸合成酶基因cDNA片段的克隆及表达分析

根据在GenBank中登录的番茄、马铃薯等蔗糖磷酸合成酶基因的保守序列设计引物, 采用RT-PCR方法从甜瓜花后25 d的果实总RNA中扩增出目标cDNA片段, 克隆到pMD18-T载体中。序列分析表明, 该片段与番茄蔗糖磷酸合成酶氨基酸序列同源性为98.9% , GenBank中登记号为DQ355797。通过Northern blot检测其在甜瓜果实不同发育时期的表达变化, 结果表明该基因在甜瓜果实花后25 d开始表达,随着果实的成熟, 表达量升高。     

杧果Rab蛋白基因MiRab11的克隆及表达分析

根据前期对杧果（Mangifera indica L.）胁迫差异分析中获得的Rab11 片段,采用RT-PCR结合RACE 技术,从‘四季杧’混合组织材料（叶、茎、花和果实）中克隆了一个Rab11 完整编码区序列,命名为MiRab11。其cDNA 全长902 bp,包含完整开放阅读框657 bp,编码218 个氨基酸。同源性分析表明MiRab11 与葡萄和柑橘的同源性最高（相似度均为97%）。实时荧光定量检测结果表明：MiRab11在杧果叶、茎、花和果实中均有表达,在嫩茎和花后70 d 的成熟果实中的表达较高;在果实形成及发育初期表达量略有下调,但在果实成熟过程中表达量明显上调;逆境胁迫（低温、盐、干旱）处理可引起在叶或茎中上调表达;不同信号物质（脱落酸、水杨酸、双氧水）刺激均可引起叶中的表达上调。结果表明,MiRab11 可能在杧果果实成熟和胁迫反应中发挥重要作用。     

草莓9–顺式–环氧类胡萝卜素双加氧酶基因FaNCED的克隆及表达分析

 采用RT-PCR和RACE技术从草莓果实中克隆ABA合成途径中关键基因FaNCED,该cDNA全长2 228 bp,具有完整的开放阅读框（ORF）,共1 827个碱基,编码609个氨基酸。序列分析表明,FaNCED编码的氨基酸序列与其他植物的NCED蛋白有很高的同源性。系统进化树分析显示,草莓NCED与同为蔷薇科的湖北海棠聚为一类,与橙、柠檬、温州蜜柚、葡萄等非跃变型果实NCED蛋白亲缘关系较近。实时荧光定量PCR分析发现,FaNCED基因在草莓根、茎、叶、花萼和果实中都有表达;在果实成熟过程中FaNCED的表达出现两次高峰,分别在大白果期和红果期,且在红果期表达量最高,并与ABA积累相吻合;FaNCED在果实采后1 d表达略有下降,之后急剧上升,ABA含量变化与FaNCED基因的表达基本一致。FaNCED可能参与调控ABA的合成并在草莓成熟中起一定作用。     

Cloning and expression analysis of three genes encoding ubiquitins in papaya (Carica papaya L.)

Papaya (Carica papaya L.) fruit have a short shelf-life due to rapid ripening induced by ethylene, which usually results in a high percentage of product loss. However, little is known about the genetic mechanism of ripening and the attributes of fruit quality. Ubiquitin (UBQ) proteins have received increasing attention because they play important roles in response to ripening and in regulating certain developmental processes in plants. In the present study, three genes encoding UBQ proteins, CpUBI1, CpUBI2, and CpUBI3, were isolated from papaya fruit. The lengths of the cDNAs of CpUBI1, CpUBI2, and CpUBI3 were 1,485 bp, 1,642 bp, and 529 bp, encoding 306, 308, and 156 predicted amino acids, respectively. Sequence analysis demonstrated that the CpUBI1 and CpUBI2 proteins contained four consecutive structural domains of the UBQ superfamily, while CpUBI3 contained a ribosomal domain structure of the S27a superfamily. RT-qPCR analysis demonstrated that ethephon treatment increased CpUBI gene expression significantly, compared to 1-methylcyclopropene (1-MCP) treatment and the untreated controls. Levels of CpUBI1 and CpUBI2 gene expression were significantly higher than CpUBI3. These results suggested that CpUBI1, CpUBI2, and CpUBI3 might have different roles during papaya fruit ripening and softening.     

藤稔’葡萄VvGAI基因的克隆、亚细胞定位及时空表达分析

 以‘藤稔’葡萄（Vitis vinifera × V. labrusca‘Fujiminori’）的茎、叶、花和果为试材,采用RT-PCR结合RACE技术,克隆获得1个推测为葡萄赤霉素响应因子VvGAI基因的cDNA序列,全长2 295 bp,其编码590个氨基酸。该基因在GenBank基因数据库的登录号为HQ834311。序列分析表明：VvGAI与杨树、拟南芥、水稻的同源基因的氨基酸序列相似性分别为68.56%、62.79%和54.16%。半定量与定量PCR结果都表明,VvGAI在葡萄茎尖、叶、花和果等营养与生殖器官中均有表达,但在茎尖中的表达最高,是一个与快速生长和分裂关系密切的基因。50 mg · L^-1赤霉素处理后,各阶段果实中VvGAI表达量趋势与对照基本一致,但水平低于对照,其中幼果中表达量最高。洋葱表皮细胞的瞬时表达显示,VvGAI蛋白定位于细胞核。     

Pre-harvest growth and development,measured as accumulated degree days,determine the post-harvest green life of banana fruit

Summary

We aimed to define a more robust indicator for banana harvest date that ensures an optimal fruit green life (GL). Our hypothesis was that development rather than growth would account for GL more accurately. To this end, five indicators were compared: one related to fruit size (i.e., growth, expressed as the diameter of fruit); two related to fruit age [i.e., development, expressed as the age of fruit measured in the number of days or in degree days (°Cd) from inflorescence emergence]; and two related to metabolism during maturation (i.e., the concentrations of malate and citrate in the pulp). Different treatments (e.g., fruit removal, leaf shading, bunch bagging, defoliation, water deficit, and flooding) were applied to modify the fruit growth rate. On different dates between the emergence of the inflorescence and harvest, fruit GL and the five indicators were measured. The results showed that there was a decreasing exponential relationship between GL and accumulated °Cd from inflorescence emergence (r² = 0.77). This was more reliable than the relationships between GL and fruit diameter (r² = 0.39), or between GL and fruit age, expressed in days (r² = 0.39). Relationships were also established between GL and malate or citrate concentrations, but they were not sufficiently reliable to estimate GL. The results illustrate that GL is related to fruit development, and that °Cd is a more reliable criterion for harvest date than the number of days, or fruit diameter, because it is less sensitive to different fruit growth rates. Banana growers in the French West Indies usually use fruit diameter and age in days to determine harvest date. However, they face problems of fruit ripening during transportation. The use of °Cd as an indicator may help to determine the optimum harvest date more accurately.     

Comparative studies on the postharvest physiology of fruit from different species of Annona (custard apple)

Summary

Fruit of three Annona species, viz. cherimoya (A. cherimola Mill), sugar apple (A. squamosa L.) and custard apple (atemoya, Annona X) were ripened in ethylene-free air and under propylene. Differences were found in patterns of respiration, ethylene production and fruit firmness changes during ripening. Cherimoya and custard apple fruits showed two successive rises in respiration rate whereas sugar apple fruits showed only one. Ethylene production showed one main peak but the onset of rapid ethylene production occurred after the beginning of the respiration climacteric in all three species. Custard apple fruit had acceptable eating quality (as judged by sensory assessments and chemical analyses of pulp) for up to four days after first detectable softening, when ripened at 20°C in ethylene-free air or under propylene. Fruit ripened in ethylene-free air had better ripe fruit quality than fruit ripened under propylene.