Characterisation of microsatellites from Actinidia chinensis

We have identified a set of informative microsatellite markers for genome analysis in kiwifruit and related Actinidia species. A small-insert genomic library was constructed from Actinidia chinensis DNA, and screened for microsatellites. About 1.2% of the total colonies hybridised to a (GA)₈ probe, 0.4% to (GT)₈, and 0.1% to a mixture of three different trinucleotide repeat probes, (CAA)₅, (GAA)₅ and (CTA)₅. From the DNA sequences of 35 hybridising clones, 18 primer pairs were designed, and used to amplify genomic DNA from 38 individual plants, representing 30 different accessions of ten Actinidia species. The banding patterns for most of the dinucleotide repeats showed a high degree of polymorphism in the diploid and tetraploid A. chinensis, and in the hexaploid A. deliciosa (kiwifruit). Heterozygosity levels of up to 100% were found among eight diploid accessions of A. chinensis examined, and the number of different-sized bands among all the species varied from 3 to 36 for each microsatellite. One simple CT microsatellite gave 21 bands with sizes suggesting that the number of repeats ranged from 9 to 37. The highest number of bands (36) and the largest size variation (>100 bp) were observed with a complex microsatellite harbouring four different repeat motifs. The majority of primer pairs amplified bands from most of the ten Actinidia species tested. The most polymorphic primer pairs were used successfully to fingerprint a range of closely related varieties of kiwifruit (A. deliciosa).Abbreviations PCR polymerase chain reaction - RFLP restriction fragment length polymorphism - VNTR variable number of tandem repeats     

Hydraulic resistance of developing Actinidia fruit

Background and Aims

Xylem flows into most fruits decline as the fruit develop, with important effects on mineral and carbohydrate accumulation. It has been hypothesized that an increase in xylem hydraulic resistance (R_T) contributes to this process. This study examined changes in R_T that occur during development of the berry of kiwifruit (Actinidia deliciosa), identified the region within the fruit where changes were occurring, and tested whether a decrease in irradiance during fruit development caused an increase in R_T, potentially contributing to decreased mineral accumulation in shaded fruit.

Methods

R_T was measured using pressure chamber and flow meter methods, the two methods were compared, and the flow meter was also used to partition R_T between the pedicel, receptacle and proximal and distal portions of the berry. Dye was used as a tracer for xylem function. Artificial shading was used to test the effect of light on R_T, dye entry and mineral accumulation.

Key Results

R_T decreased during the early phase of rapid fruit growth, but increased again as the fruit transitioned to a final period of slower growth. The most significant changes in resistance occurred in the receptacle, which initially contributed 20 % to R_T, increasing to 90 % later in development. Dye also ceased moving beyond the receptacle from 70 d after anthesis. The two methods for measuring R_T agreed in terms of the direction and timing of developmental changes in R_T, but pressure chamber measurements were consistently higher than flow meter estimates of R_T, prompting questions regarding which method is most appropriate for measuring fruit R_T. Shading had no effect on berry growth but increased R_T and decreased dye movement and calcium concentration.

Conclusions

Increased R_T in the receptacle zone coincides with slowing fresh weight growth, reduced transpiration and rapid starch accumulation by the fruit. Developmental changes in R_T may be connected to changes in phloem functioning and the maintenance of water potential gradients between the stem and the fruit. The effect of shade on R_T extends earlier reports that shading can affect fruit vascular differentiation, xylem flows and mineral accumulation independently of effects on transpiration.     

Endogenous cytokinin in developing kiwifruit is implicated in maintaining fruit flesh chlorophyll levels

Background and Aims

Green kiwifruit (Actinidia deliciosa) retain high concentrations of chlorophyll in the fruit flesh, whereas in gold-fleshed kiwifruit (A. chinensis) chlorophyll is degraded to colourless catabolites during fruit development, leaving yellow carotenoids visible. The plant hormone group the cytokinins has been implicated in the delay of senescence, and so the aim of this work was to investigate the link between cytokinin levels in ripening fruit and chlorophyll de-greening.

Methods

The expression of genes related to cytokinin metabolism and signal transduction and the concentration of cytokinin metabolites were measured. The regulation of gene expression was assayed using transient activation of the promoter of STAY-GREEN2 (SGR2) by cytokinin response regulators.

Key Results

While the total amount of cytokinin increased in fruit of both species during maturation and ripening, a high level of expression of two cytokinin biosynthetic gene family members, adenylate isopentenyltransferases, was only detected in green kiwifruit fruit during ripening. Additionally, high levels of O-glucosylated cytokinins were detected only in green kiwifruit, as was the expression of the gene for zeatin O-glucosyltransferase, the enzyme responsible for glucosylating cytokinin into a storage form. Season to season variation in gene expression was seen, and some de-greening of the green kiwifruit fruit occurred in the second season, suggesting environmental effects on the chlorophyll degradation pathway. Two cytokinin-related response regulators, RRA17 and RRB120, showed activity against the promoter of kiwifruit SGR2.

Conclusions

The results show that in kiwifruit, levels of cytokinin increase markedly during fruit ripening, and that cytokinin metabolism is differentially regulated in the fruit of the green and gold species. However, the causal factor(s) associated with the maintenance or loss of chlorophyll in kiwifruit during ripening remains obscure.     

One-Step Lipophilization of Proteins Using Fatty Acyl Anhydride

Nucleotide sequences of mRNAs were compared between major calcium-sensitive caseins of cow (α_s1-casein) and rat (α-casein). A best fit alignment of the two sequences showed homology of 81% and 69% for the 5′- and 3′-untranslated regions, respectively. Homology in the comparable coding region of the mature asl-casein (76% of total codons) was remarkably lower at amino acid level (46%) than at nucleotide level (69%). The low conservation at amino acid level is explained by the unusual nucleotide substitution pattern (random at all three positions of codons) in contrast to synonymous substitutions at the third position revealed on comparison of other related proteins. The evolutionary distances among the number of the casein family were estimated by comparing known nucleotide sequences of the signal peptides which were the most conserved coding regions in the family. The divergence time for most distantly related caseins (both rat α-casein/rat β-casein and rat α-casein/mouse ε-casein) was estimated to be about 170 million years.     

猕猴桃L-艾杜糖脱氢酶基因的克隆及转化

以猕猴桃果实为材料,利用电子克隆技术获得了L-艾杜糖脱氢酶(L-Idonate dehydrogenase,IDH)基因,然后将其转入番茄中,为了解猕猴桃IDH基因如何调控AsA的降解奠定基础。结果表明,获得的猕猴桃IDH基因cDNA全长为1 239bp,含有一个1 080bp的开放阅读框,编码359个氨基酸;成功构建了IDH基因植物表达载体pWR-IDH及工程农杆菌,以番茄叶片及茎段为受体,通过农杆菌介导法进行转化,获得了4株经PCR和RT-PCR检测呈阳性的转基因植株。     

猕猴桃园节肢动物群落重建及主要类群的生态位

为研究猕猴桃节肢动物群落重建过程和主要类群生态位移,本试验系统调查野生猕猴桃和人工栽植不同树龄(3年、9年、14年)猕猴桃园节肢动物群落,测定群落特征指标和主要类群的时空生态位。结果表明,群落包含3纲15目74科,约90种。不同桃园节肢动物群落的主要类群不同,各类群占群落总数的比例差异显著,野生猕猴桃园群落中各类群分布较均匀,栽植桃园群落中害虫比例较大。野生猕猴桃园和栽植猕猴桃园之间,群落的多样性指数、均匀度和优势度差异均极显著,群落稳定性表现为野生>9年>14年>3年。各类群的时空二维生态位也会随着栽植时间增长发生变化：蜘蛛和蝇类的生态位在不同树龄的猕猴桃园中都保持在较高水平;人工栽植初期,山楂叶螨和叶蝉占据的时空生态位较宽,成为优势类群,小薪甲与山楂叶螨生态位重叠较大,竞争激烈;随后,小薪甲和瘿蚊的时空二维生态位逐渐增大,到14年桃园中已经取代了山楂叶螨和叶蝉,成为主要害虫。总之,人工干扰的节肢动物群落重建是一个次级演替过程,与自然生态系统群落明显不同。     

Towards aspect-oriented functional--structural plant modelling

Background and Aims

Functional–structural plant models (FSPMs) are used to integrate knowledge and test hypotheses of plant behaviour, and to aid in the development of decision support systems. A significant amount of effort is being put into providing a sound methodology for building them. Standard techniques, such as procedural or object-oriented programming, are not suited for clearly separating aspects of plant function that criss-cross between different components of plant structure, which makes it difficult to reuse and share their implementations. The aim of this paper is to present an aspect-oriented programming approach that helps to overcome this difficulty.

Methods

The L-system-based plant modelling language L+C was used to develop an aspect-oriented approach to plant modelling based on multi-modules. Each element of the plant structure was represented by a sequence of L-system modules (rather than a single module), with each module representing an aspect of the element''s function. Separate sets of productions were used for modelling each aspect, with context-sensitive rules facilitated by local lists of modules to consider/ignore. Aspect weaving or communication between aspects was made possible through the use of pseudo-L-systems, where the strict-predecessor of a production rule was specified as a multi-module.

Key Results

The new approach was used to integrate previously modelled aspects of carbon dynamics, apical dominance and biomechanics with a model of a developing kiwifruit shoot. These aspects were specified independently and their implementation was based on source code provided by the original authors without major changes.

Conclusions

This new aspect-oriented approach to plant modelling is well suited for studying complex phenomena in plant science, because it can be used to integrate separate models of individual aspects of plant development and function, both previously constructed and new, into clearly organized, comprehensive FSPMs. In a future work, this approach could be further extended into an aspect-oriented programming language for FSPMs.     

五个猕猴桃种的皮孔、气孔器和叶片下表皮微观特征

为了探讨不同种类猕猴桃皮孔、气孔器和叶片下表皮特征的差异及其分类学意义,该文利用光学显微镜观察五个猕猴桃种共计9份样品材料的一年生枝条韧皮部上皮孔的形态,结果发现供试猕猴桃皮孔呈长椭圆形或长梭形,不同种皮孔的长×宽、皮孔密度、皮孔面积和皮孔面积的百分比值存在差异但与种类划分无明显规律,皮孔的宽可明显区分所选4个中华猕猴桃品种。利用扫描电镜观察气孔器和叶片下表皮特征,结果发现猕猴桃气孔器呈宽椭圆形或椭圆形,仅分布在叶背的叶肉区域,气孔器类型有辐射型、环列型、不规则型和不等型4种。中华猕猴桃的气孔器是辐射型,美味猕猴桃的气孔器是环列型,阔叶猕猴桃和毛花猕猴桃的气孔器是不规则型,长果猕猴桃的气孔器是不等型。保卫细胞围绕气孔器排列,与表皮水平或突起。叶片下表皮细胞一般为不规则形或乳状突起,垂周壁浅波状或深波状;表面覆着颗粒状纹饰和分叉单细胞非腺毛,其中阔叶猕猴桃为不分叉单细胞非腺毛,长果猕猴桃多为双分叉单细胞非腺毛,其余为多分叉单细胞非腺毛。部分具有鳞片状蜡质层。气孔器外拱盖内缘浅波状,纹饰光滑或有颗粒状物。测量猕猴桃气孔器长×宽、气孔长轴、气孔器密度,结果发现不同种之间存在差异但与猕猴桃种类划分无明显规律,其气孔器类型与种类划分一致,4个中华猕猴桃品种可通过气孔器的长、气孔长轴和密度大小来区分。此外,新种长果猕猴桃的微表观形态与其他种类存在明显差异,为其识别提供基本的微观依据。