冬小麦春化作用相关cDNA克隆Vrc79的分子克隆

在低温需求型植物的发育过程中,春化作用是诱导植物成花的必要因子。在冬小麦春化进程中存在着一个核酸代谢的关键期,为了探讨春化作用的分子机理,以不春化及脱春化的冬小麦京冬1号（Ｔｒｉｔｉｃｕｍ　ａｅｓｔｉｖｕｍＬ．ｃｖ．Ｊｉｎｄｏｎｇ　Ｎｏ．１）胚芽的ｍＲＮＡ为对照,通过减法杂交构建了富集冬小麦春化相关基因的ｃＤＮＡ文库,经过差异筛选分离到了一个仅在春化２１ｄ这一关键期表达,而在不春化、春化４ｄ、脱春化不表达的春化相关。ｃＤＮＡ克隆Ｖｒｃ７９。Ｎｏｒｔｈｅｍ杂交及同源性分析表明它是一个在植物中新发现的不同于低温胁迫诱导基因的春化特异克隆,其对春化需求型植物的成花诱导可能起重要的调控作用。     

用差异显示PCR技术克隆冬小麦春化设定cDNA克隆实验系统的建立

差异显示PCR技术是一种新近发展起来的在真核细胞中检测与克隆特异表达基因的手段。本文建立了用此技术克隆春化相关基因的研究系统,并提供了诸如总RNA的提取、污染DNA的去除、sscDNA的逆转录、PCR参数、扩增cDNA的电泳、特异cDNA的收集与重扩增等在方法上的细节。用这种技术分离了一个仅在春化20天这一特异时期表达的春化设定cDNA克隆VPC28。这些结果也适用于更加广泛的类似研究。     

Existence of homologous sequences corresponding to cDNA of the ver gene in diverse higher plant species

INTRODUCT1ONIn the vast majority of higher plalls, a transitionfrom vegetative growth to reproductive developmentis strongly influenced by a set of environmental fac-tors, such as photoperiod, temperatu-re etc. Bothwinter trait and biennia1 plants require a period of1ow temperature fOr switching from vegetative to re-productive growth, and this process is known as ver-na1ization. Several physiological and genetic inves-tigations showed that the vrngenes control the ver-nalization traits of…     

Changes of Soluble Protein Components in Winter Wheat (Triticum aestivum L.) Shoots During Vernalization and its Relation to Morphogenesis

Winter wheat “Nong Da 139” and spring wheat “Zhong 8022” were used for this experiment. The effect of low temperature treatment at different periods on protein content and composition of the seedling shoots as well as subsequent development was studied. The main results are as follows: 1. More than 40 days of vernalizing treatment were required for a rapid and uniform earing by winter wheat “Nong Da 139”. After 14–21 days vernalization, the winter wheat is possible to ear in summer, but in an irregular manner. The results indicate that during the whole vernalization process, the effect of low temperature on earing development varies with the duration of treatment. At the earlier stage, it is likely that the Iow temperature induces a change in physiological process, and in the later stage, it only accelerates the development. It is there- fore, suggested that there are two distinct processes existing during vernalization. The transformation from the former to the latter state appears to occur at the middle period of vernalization process. 2. The protein metabolic inhibitors, such as ethionine and p-fluorophenylalanine, interfere with the vernalizing process of winter wheat also at its middle period. 3. With low temperature treatment for different periods, the soluble protein content and composition are found to be changed in winter wheat shoots. At the middle-stage of vernalization (after 14 days low temperature treatment), not only is the content of protein increased twice as compared with the control, but new proteins (on electrophorestic gel) are also produced. On the contrary, there was no difference in protein bands for spring wheat. Spring wheat not to be treated by low temperature, has already possessed the proteins that appeared in vernalized winter wheat shoots. These results demonstrated that the days of 14–21 are the critical time for the vernalization of the winter wheat. The new proteins synthesized at this stage might be the factor of the determination whether or not the plants will transform from vegetative stage into reproductive phase.     

小麦春化相关基因的分子克隆与功能分析

小麦春化相关基因的分子克隆与功能分析@种康$中国科学院植物研究所!北京100093@许智宏$中国科学院植物研究所!北京100093@谭克辉$中国科学院植物研究所!北京100093小麦;;基因;;分子克隆     

Relationships among vernalization, shoot apex development and frost tolerance in wheat

BACKGROUND AND AIMS: Frost tolerance of wheat depends primarily upon a strong vernalization requirement, delaying the transition to the reproductive phase. The aim of the present study was to learn how saturation of the vernalization requirement and apical development stage are related to frost tolerance in wheat. METHODS: 'Mironovskaya 808', a winter variety with a long vernalization requirement, and 'Leguan', a spring variety without a vernalization requirement, were acclimated at 2 degrees C at different stages of development. Plant development (morphological stage of the shoot apex), vernalization requirement (days to heading) and frost tolerance (survival of the plants exposed to freezing conditions) were evaluated. KEY RESULTS: 'Mironovskaya 808' increased its frost tolerance more rapidly; it reached a higher level of tolerance and after a longer duration of acclimation at 2 degrees C than was found in 'Leguan'. The frost tolerance of 'Mironovskaya 808' decreased and its ability to re-acclimate a high tolerance was lost after saturation of its vernalization requirement, but before its shoot apex had reached the double-ridge stage. The frost tolerance of 'Leguan' decreased after the plants had reached the floret initiation stage. CONCLUSIONS: The results support the hypothesis that genes for vernalization requirement act as a master switch regulating the duration of low temperature induced frost tolerance. In winter wheat, due to a longer vegetative phase, frost tolerance is maintained for a longer time and at a higher level than in spring wheat. After the saturation of vernalization requirement, winter wheat (as in spring wheat) established only a low level of frost tolerance.     

Vernalization-induced flowering in wheat is mediated by a lectin-like gene VER2

A vernalization-related gene VER2 was isolated from winter wheat ( Triticum aestivum L.) using a differential screening approach. The deduced VER2 is a lectin-like protein of 300 amino acids, which contains the presence of a jacalin-like GWG domain. RNA in situ hybridization results demonstrated that VER2 gene expression is restricted to the marginal meristems of immature leaves in vernalized wheat seedlings. No hybridization signal was detected in the epidermal tissue and vascular bundles. However, "devernalization" resulted in the silencing of VER2 gene activity. The gene expression pattern of VER2 induced by jasmonate was similar to that induced by vernalization. Antisense inhibition of VER2 in transgenic wheat showed that heading and maturation time were delayed up to 6 weeks compared with non-transformed wheat and the pBI121empty-vector-transformed wheat. Tissue degeneration at the top of the spike was also noticed in the antisense inhibited transgenic wheat. These results suggest that VER2 plays an important role in vernalization signaling and spike development in winter wheat.     

植物春化作用的分子机理

春化作用在控制高等植物开花中起着重要的作用。本文综述了近年来以拟南芥（Arabidopsis thaliana）和冬小麦（Triticum aestivum）为主要研究对象进行的有关春化作用分子机制的研究;概括和分析了已经分离得到的与春化有关的基因的功能及其调控方式以及各基因间的相互作用。     

利用反义RNA技术分析春化相关基因VER17在冬小麦花发育过程中的功能

为了研究小麦春化相关基因VER17的功能,应用反义RNA技术,将VER17基因的反义片段构建到载体pBI121上,通过花粉管通道法获取转基因小麦.对T0代转基因植株GUS染色以及PCR等分子鉴定,得到14株含反义VERJ7基因片段的阳性转基因植株.对T0代和T1代的表型观察结果显示,VER17反义转基因植株开花时间延迟,并且穗的顶部和基部小花出现明显的退化.表明春化相关基因VER17在小麦发育过程中可能起到促进植物开花以及穗顶端和基部花发育的作用,减少小花退化,同时对雄蕊的发育也有影响.     

Developmental traits affecting low-temperature tolerance response in near-isogenic lines for the Vernalization locus Vrn-A1 in wheat (Triticum aestivum L. em Thell)

Investigation of low-temperature (LT) tolerance in cereals has commonly led to the region of the vyn-A1 vernalization gene or its homologue in related genomes. Two cultivars, one a non-hardy spring wheat and one a very cold-hardy winter wheat, whose growth habits are determined by the Vrn-A1 (spring habit) and vrn-A1 (winter habit) alleles, were chosen to produce reciprocal near-isogenic lines (NILs). These lines were then used to determine the relationship between rate of phenological development and the degree and duration of LT tolerance gene expression. Each allele was isolated in the genetic backgrounds of the non-hardy spring wheat 'Manitou' and the very cold-hardy winter wheat 'Norstar'. The effects of each allele on phenological development and low-temperature tolerance (LT50) were determined at regular intervals over a 4 degrees C acclimation period of 0-98 d. The vegetative/reproductive transition, as determined by final leaf number (FLN), was found to be a major developmental factor influencing LT tolerance. Possession of a vernalization requirement increased both the length of the vegetative growth phase and LT tolerance. Similarly, increased FLN in spring Norstar and winter Manitou NILs delayed their vegetative/reproductive transition and increased their LT tolerance relative to Manitou. Although the winter Manitou NILs had a lower FLN than the spring Norstar NILs, they were able to extend their vegetative stage to a similar length by increasing the phyllochron (interval between the appearance of successive leaves). Cereal plants have four ways of increasing the length of the vegetative phase, all of which extend the time that low-temperature tolerance genes are more highly expressed: (1) vernalization; (2) photoperiod responses; (3) increased leaf number; and (4) increased length of the phyllochron.     

植物春化用的分子机理

春化作用在控制高等植物开花中起着重要的作用。本文综述了近年来以拟南芥(Arabidopsis thaliana)和冬小麦(Triticum aestivum)为主要研究对象进行的有关春化作用分子机制的研究; 概括和分析了已经分离得到的与春化有关的基因的功能及其调控方式以及各基因间的相互作用。     

Vernalization-induced changes of the DNA methylation pattern in winter wheat.

Vernalization is a cold treatment that induces or accelerates flowering and insures that temperate-zone plants will not flower until after winter. There is evidence that vernalization results in DNA demethylation that induces flowering. Differences in DNA methylation can be determined using methylation-sensitive amplified fragment length polymorphisms (AFLPs). Methylation-sensitive AFLPs utilize restriction enzyme isoschizomers that are differentially sensitive to methylation, producing polymorphisms related to methylation differences as opposed to sequence differences. Near-isogenic lines (NILs) have been developed for spring vs. winter habit in wheat (Triticum aestivum) and allow for the study of a single vernalization locus. In this study, differences in the methylation pattern were determined for spring and winter NILs, as well as for unvernalized and vernalized individuals. Winter wheat was more highly methylated than spring wheat and methylation-related AFLPs were produced between winter and spring wheat. Changes in the methylation pattern were observed at the end of vernalization, one week after the end of vernalization, and four weeks after the end of vernalization of winter wheat. However, the most methylation differences were observed one week after removal of winter wheat from cold treatment. Our data suggest that there is not only a vernalization-induced demethylation related to flower induction, but there is also a more general and non-specific demethylation of sequences unrelated to flowering. Two methylation-related AFLPs induced by vernalization were shared among all of the winter NILs.     

Vernalization,a Switch in Initiation of Flowering,Promotes Differentiation and Development of Spikelet in Winter Wheat

Vernalization is a decisive physiological process for heading, flowering and graining of biennial plants. Variable duration of low-temperature treatment has effects on lateral morphogenesis, such as spike initiation, floral development and graining rate in winter wheat ( Triticum aestivum L.). The investigation data showed that the duration of vernalization treatment was a decisive factor for the initiation of spike relevant to the time of initiation; the longer the duration at low temperature, the earlier the spike initiation in winter wheat. In the process of the spike differentiation, relatively lower temperature and longer differential time benefited for spike differentiation. Under laboratory condition, a low-temperature treatment for 45 d was optial for flower differentiation and graining in winter wheat. It is novelly recognized that vernalization treatment is essential for development of both spikes and spikelets, besides for promoting initiation of differentiation in winter wheat.     

An early meiosis cDNA clone from wheat

Meiosis occupies only a very short period of the life cycle of higher plants but it is a crucial process ensuring the correct passage and maintenance of genetic information from parent to offspring. A clone (designated pAWJL3) has been isolated from a cDNA library generated from RNA prepared from young wheat florets at early meiosis. The clone was identified through cross-hybridisation to a cDNA clone from maize that, in turn, had been isolated by hybridisation to a Lilium meiosis-specific cDNA clone. The genes encoding the sequence represented in the wheat cDNA clone have been assigned to chromosomes in wheat. The clone, pAWJL3, represents a small family of genes with about 20 members located on the short arms of group 3 and 5 chromosomes. The chromosomal regions harbour genes known to control chromosomal pairing in wheat. DNA prepared from a deletion mutation affecting one of the major genes controlling pairing, Ph2 located on the short arm of 3DS, lacks the 3DS-specific members of the pAWJL3 family bands. The genes are shown to be expressed only after leptotene and predominantly at zygotene and pachytene of meiosis I. The deduced amino acid sequence encoded by the cDNA clone shows two domains, one with three leucine-rich, 24-amino acid repeats and the other with four leucine heptad repeats that resemble those found in basic leucine zipper proteins.     

Developmental Regulation of Low-temperature Tolerance in Winter Wheat

Vernalization and photoperiod genes have wide-ranging effectson the timing of gene expression in plants. The objectives ofthis study were to (1) determine if expression of low-temperature(LT) tolerance genes is developmentally regulated and (2) establishthe interrelationships among the developmental stages and LTtolerance gene expression. LT response curves were determinedfor three photoperiod-sensitive LT tolerant winter wheat (Triticumaestivum L. em Thell) genotypes acclimated at 4 °C under8 h short-day (SD) and 20 h long-day (LD) photoperiods from0 to 112 d. Also, three de-acclimation and re-acclimation cycleswere used that bridged the vegetative/reproductive transitionpoint for each LD and SD photoperiod treatment. A vernalizationperiod of 49 d at 4 °C was sufficient for all genotypesto reach vernalization saturation as measured by minimum finalleaf number (FLN) and confirmed by examination of shoot apicesdissected from crowns that had been de-acclimated at 20 °CLD. Before the vegetative/reproductive transition, both theLD- and SD-treated plants were able to re-acclimate to similarLT₅₀(temperature at which 50% of the plants are killed by LTstress) levels following de-acclimation at 20 °C. De-acclimationof LD plants after vernalization saturation resulted in rapidprogression to the reproductive phase and limited ability tore-acclimate. The comparative development of the SD (non-flowering-inductivephotoperiod) de-acclimated plants was greatly delayed relativeto LD plants, and this delay in development was reflected inthe ability of SD plants to re-acclimate to a lower temperature.These observations confirm the hypothesis that the point oftransition to the reproductive stage is pivotal in the expressionof LT tolerance genes, and the level and duration of LT acclimationare related to the stage of phenological development as regulatedby vernalization and photoperiod requirements. Copyright 2001Annals of Botany Company Triticum aestivum L., wheat, low-temperature tolerance, vernalization, photoperiod, phenological development     

Effect of Trypsin on the Vernalization Process in Winter Wheat

This paper deals with the experimental results of the effect of trypsin on the vernalization process in the winter wheat. The variations of both the trypsin-like enzyme activity and the soluble protein content during vernalization in winter wheat seedlings were assayed. The results are as follows: 1. When the vernalization was progressing to the middle stage (around 25 days), the seedlings of the winter wheat were moved into the room temperature for continuous culture. These seedlings possessed the ability to earing after this kind of treatment, but earing development was rather late. Whereas the development of the earing was much earlier by treating them with 100 ppm trypsin just after moving into the room temperature condition. 2. Earing could not be induced by treatment with trypsin (100 ppm) in nonvernalized winter wheat. 3. Vernalization process was promoted in the initial period by trypsin under the low temperature, but it was inhibited in the middle and there was no remarkably effect on the developmental process in the later period. 4. There was no effect by the treatment of trypsin on the spring wheat under the conditions with or without vernalization. 5. The trypsinlike enzyme activity during the cold codication was increased in the initial period, then decreased remarkably later, but at the same time the soluble protein content increased rapidly. These results indicate that at the middle-later period of the vernalization the synthesis of some specific proteins is very important for the proceeding of varnalization.     

Temperature Response of Vernalization in Wheat: Modelling the Effect on the Final Number of Mainstem Leaves

This paper outlines a modelling approach which predicts theeffect of both continuous and intermittent low temperature regimeson the final number of leaves in winter wheat. The model takesaccount of the balance between the concurrent processes of leafprimordium initiation and rate of saturation of vernalization,and their response to temperature. The inverse of the time tosaturation of vernalization, at which stage final leaf numberis set, is modelled as a linear function of vernalizing temperature,between 0 and 17 °C. The rate of leaf primordium initiationis modelled using the established linear relationship betweenrate and temperature above 0 °C. Final leaf number is hencethe product of the number of leaf primordia initiated once vernalizationis saturated. In the model, genotypes are characterized by (1)the slope and intercept of the linear response of the rate ofsaturation of vernalization to temperature in the vernalizingrange, and (2) by a development rate towards floral transitionat on-vernalizing temperatures (above 17 °C). The modelis tested against data from experiments where six cultivarsof winter wheat plants of different ages were exposed to a rangeof low temperature regimes, including continuous and intermittentvernalizing temperatures. Overall, the model predicted, withr ²values of 70–90%, the final leaf number across a rangeof six to 21 leaves. Prediction of final leaf number for somecultivars was better in continuous than in intermittent vernalizingregimes. This modelling approach can explain the often-conflictingreports of the effectiveness of different temperatures for vernalization,and the interaction of plant age and vernalization effectiveness. Triticum aestivum L.; wheat; vernalization; rate; temperature; leaf number; modelling; phenology; flowering     

Wheat SOC1 functions independently of WAP1/VRN1, an integrator of vernalization and photoperiod flowering promotion pathways

Heading time in bread wheat ( Triticum aestivum L.) is determined by three characters – vernalization requirement, photoperiodic sensitivity and narrow-sense earliness (earliness per se) – which are involved in the phase transition from vegetative to reproductive growth. The wheat APETALA1 ( AP1 )-like MADS-box gene, wheat AP1 ( WAP1 , identical with VRN1 ), has been identified as an integrator of vernalization and photoperiod flowering promotion pathways. A MADS-box gene, SUPPRESSOR OF OVEREXPRESSION OF CO 1 ( SOC1 ) is an integrator of flowering pathways in Arabidopsis . In this study, we isolated a wheat ortholog of SOC1 , wheat SOC1 ( WSOC1 ), and investigated its relationship to WAP1 in the flowering pathway. WSOC1 is expressed in young spikes but preferentially expressed in leaves. Expression starts before the phase transition and is maintained during the reproductive growth phase. Overexpression of WSOC1 in transgenic Arabidopsis plants caused early flowering under short-day conditions, suggesting that WSOC1 functions as a flowering activator in Arabidopsis . WSOC1 expression is affected neither by vernalization nor photoperiod, whereas it is induced by gibberellin at the seedling stage. Furthermore, WSOC1 is expressed in transgenic wheat plants in which WAP1 expression is cosuppressed. These findings indicate that WSOC1 acts in a pathway different from the WAP1 -related vernalization and photoperiod pathways.