Characterization of two cDNA clones for mRNAs expressed during ripening of melon (Cucumis melo L.) fruits

In vitro translation of mRNAs and polyacrylamide gel electrophoresis of proteins from melons revealed that several mRNAs increased in amount during ripening, indicating the existence of other ripening genes in addition to those cloned previously. To identify ripening-related genes we have screened a ripe melon cDNA library and isolated two novel cDNA clones (MEL2 and MEL7) encoding unidentified proteins. Southern analysis revealed that MEL2 and MEL7 are encoded by low-copy-number genes. The MEL2 cDNA clone is near full-length, corresponds to a 1600 nucleotide mRNA that accumulates during ripening and encodes a predicted protein rich in hydrophobic amino acids. The MEL7 cDNA clone is full-length, corresponds to a mRNA of 0.7 kb which accumulates during early ripening stages and is also present at low levels in other organs of the melon plant. The MEL7 predicted polypeptide is 17 kDa and shows significant homology with the major latex protein from opium-poppy. Wounding and ethylene treatment of unripe melon fruits 20 days after anthesis showed that MEL2 and MEL7 mRNAs are only induced by ethylene.     

Organisation and expression of a wound/ripening-related small multigene family from tomato

cDNA clones derived from a ripe tomato fruit cDNA library were used to investigate changes in the abundance of specific mRNAs in ripening fruit and wounded leaves. mRNAs related to one cDNA clone (pTOM 13) were expressed in both situations. This clone was used to identify homologous sequences in a tomato genomic library. Three groups of related clones that hybridised to the pTOM 13 cDNA insert were identified and subcloned into plasmid vectors. Genomic Southern analysis of tomato DNA using gene-specific DNA fragments isolated from the subcloned DNAs indicated that all pTOM 13 closely related genes had been isolated. RNA dot blot analysis with these DNA fragments as probes indicated differential expression of this small multigene family in leaves and fruit.     

Structure and expression of an ethylene-related mRNA from tomato.

Messenger RNAs homologous to a cDNA clone (pTOM 13) derived from a ripe-tomato-specific cDNA library are expressed during tomato fruit ripening and after the wounding of leaf and green fruit material. Both responses involve the synthesis of the hormone ethylene. Accumulation of the pTOM 13--homologous RNA during ripening is rapid and sustained, and reaches its maximum level in orange fruit. Following mechanical wounding of tomato leaves a pTOM 13--homologous RNA shows rapid induction within 30 minutes, which occurs before maximal ethylene evolution (2-3 h). This RNA also accumulates following the wounding of green tomato fruit. Northern blot analysis of poly(A)+ RNA indicates that the length of the mRNA is about 1400 nucleotides. Nucleotide sequence analysis showed the cDNA insert to contain the complete coding region of the pTOM 13 protein (33.5 kD) and an unusual 5' structure of ten dT-nucleotides. Hybridisation of the pTOM 13 cDNA insert to Southern blots of tomato DNA indicates the presence of only a small number of homologous sequences in the tomato genome.     

兜兰DEFICIENS(DEF)-和GLOBOSA(GLO)-like基因的克隆及表达分析

以‘同色兜兰’品种为材料,采用RT-PCR和RACE技术获得了DEFICIENS(DEF)-和GLOBOSA(GLO)-like基因的cDNA全长,命名为PcDEF和PcGLO,并用半定量RT-PCR和实时PCR研究了PcDEF和PcGLO在花芽发育过程和不同组织部位的表达特性。结果表明,PcDEF和PcGLO的全长cDNA分别为1 039bp和934bp,分别编码224和210个氨基酸;蛋白比对表明,PcDEF和PcGLO蛋白都具有典型MADS-box蛋白的MADS和K结构域;蛋白同源性分析显示,PcDEF和PcGLO与已登录的其它兰科植物的DEF/AP3和GLO/PI蛋白的相似性分别在75%～96%和87%～98%;系统进化树分析表明,PcDEF和PcGLO分别属于B类MADS-box蛋白家族的AP3和PI亚家族。表达分析显示,PcDEF和PcGLO在花芽发育中均有表达,PcDEF在成熟花、唇瓣和花瓣中的表达量高,在蕊柱、萼片、苞叶和根中次之,在花茎和叶中较低,在子房中几乎不表达;PcGLO在各组织中均有不同丰度的表达。     

Differential expression of expansin gene family members during growth and ripening of tomato fruit

cDNA clones encoding homologues of expansins, a class of cell wall proteins involved in cell wall modification, were isolated from various stages of growing and ripening fruit of tomato (Lycopersicon esculentum). cDNAs derived from five unique expansin genes were obtained, termed tomato Exp3 to Exp7, in addition to the previously described ripening-specific tomato Exp1 (Rose et al. (1997) Proc Natl Acad Sci USA 94: 5955–5960). Deduced amino acid sequences of tomato Exp1, Exp4 and Exp6 were highly related, whereas Exp3, Exp5 and Exp7 were more divergent. Each of the five expansin genes showed a different and characteristic pattern of mRNA expression. mRNA of Exp3 was present throughout fruit growth and ripening, with highest accumulation in green expanding and maturing fruit, and lower, declining levels during ripening. Exp4 mRNA was present only in green expanding fruit, whereas Exp5 mRNA was present in expanding fruit but had highest levels in full-size maturing green fruit and declined during the early stages of ripening. mRNAs from each of these genes were also detected in leaves, stems and flowers but not in roots. Exp6 and Exp7 mRNAs were present at much lower levels than mRNAs of the other expansin genes, and were detected only in expanding or mature green fruit. The results indicate the presence of a large and complex expansin gene family in tomato, and suggest that while the expression of several expansin genes may contribute to green fruit development, only Exp1 mRNA is present at high levels during fruit ripening.     

Isolation and characterization of a fruit-specific cDNA and the corresponding genomic clone from tomato

Differential screening of a cDNA bank constructed from ripe tomato fruit mRNA allowed the isolation of cDNA clone 2A11 which is entirely fruit-specific, is expressed at steadily increasing levels from anthesis to breaker, and accounts for approximately 1% of the messenger RNA in mature tomato fruit. A genomic clone corresponding to the 2A11 cDNA was isolated from a tomato genomic library. Sequence comparison of the cDNA clone with the genomic clone shows they are identical over the shared region with the genomic clone possessing a single large intron near the 5 end of the message.The open reading frame of 2A11 would encode a sulfur-rich polypeptide 96 amino acids in length. The identity of the putative protein is unknown. In situ hybridization shows that the 2A11 message is found throughout the pericarp cells in a tomato fruit. In contrast, in situ hybridization of early ripening stages with a polygalacturonase probe shows higher mRNA levels in cells of the outer pericarp and cells surrounding the vascular regions of the pericarp.     

Cloning and molecular characterization of a strawberry fruit ripening-related cDNA corresponding a mRNA for a low-molecular-weight heat-shock protein

We have isolated and characterized a cDNA from a strawberry fruit subtractive library that shows homology to class-I low-molecular-weight (LMW) heat-shock protein genes from other higher plants. The strawberry cDNA (clone njjs4) was a 779 bp full-length cDNA with a single open reading frame of 468 bp that is expected to encode a protein of ca. 17.4 kDa with a pI of 6.57. Southern analysis with genomic DNA showed several high-molecular-weight hybridization bands, indicating that the corresponding njjs4 gene is not present as a single copy in the genome. This strawberry gene was not expressed in roots, leaves, flowers and stolons but in fruits at specific stages of elongation and ripening. However, a differential pattern of mRNA expression was detected in the fruit tissues achenes and receptacle. The njjs4 gene expression increased in achenes accompanying the process of seed maturation whereas in the receptacle, a high mRNA expression was detected in the W2 stage, during which most of the metabolic changes leading to the fruit ripening are occurring. Our results clearly show a specific relationship of this njjs4 strawberry gene with the processes of seed maturation and fruit ripening, and strongly support that at least some of the class-I LMW heat-shock protein-like genes have a heat-stress-independent role in plant development, including fruit ripening.     

cDNA cloning and characterisation of novel ripening-related mRNAs with altered patterns of accumulation in the ripening inhibitor (rin) tomato ripening mutant

A cDNA library produced from mRNA isolated from the pericarp of wild-type tomato fruit (Lycopersicon esculentum Mill. cv Ailsa Craig) at the first visible sign of fruit ripening was differentially screened to identify clones whose homologous mRNAs were present at reduced levels in fruit of the tomato ripening mutant, ripening inhibitor,rin. Five clones were isolated (pERT 1, 10, 13, 14, 15). Accumulation of mRNA homologous to each of these clones increased during the ripening of wild-type fruit and showed reduced accumulation in ripening rin fruit. The levels of three of them (homologous to ERT 1, 13 and 14) were increased by ethylene treatment of the mutant fruit. A further clone, ERT 16 was identified for a mRNA present at a high level in both normal and mutant fruit at early stages of ripening. Database searches revealed no significant homology to the DNA sequence of ERT 14 and 15; however, DNA and derived amino acid sequence of ERT 1 both contain regions of homology with several reported UDP-glucosyl and glucuronosyl transferases (UDPGT) and with a conserved UDPGT motif. A derived amino acid sequence from the ERT 10 cDNA contains a perfect match to a consensus sequence present in a number of dehydrogenases. The ERT 13 DNA sequence has homology with an mRNA present during potato tuberisation. The presence of these mRNAs in tomato fruit is unreported and their role in ripening is unknown. The ERT 16 DNA sequence has homology with a ripening/stress-related cDNA isolated from tomato fruit pericarp.     

Expression of six expansin genes in relation to extension activity in developing strawberry fruit.

Expansins are proteins which have been demonstrated to induce cell wall extension in vitro. The identification and characterization of six expansin cDNAs from strawberry fruit, termed FaExp3 to FaExp7, as well as the previously identified FaExp2 is reported here. Analysis of expansin mRNAs during fruit development and in leaves, roots and stolons revealed a unique pattern of expression for each cDNA. FaExp3 mRNA was present at much lower levels than the other expansin mRNAs and was expressed in small green fruit and in ripe fruit. FaExp4 mRNA was present throughout fruit development, but was more strongly expressed during ripening. FaExp5 was the only clone to show fruit specific expression which was up-regulated at the onset of ripening. FaExp6 and FaExp7 mRNAs were present at low levels in the fruit with highest expression in stolon tissue. During fruit development FaExp6 had the highest expression at the white, turning and orange stages whereas expression of FaExp7 was highest in white fruit. The expression profiles of FaExp2 and FaExp5 in developing fruit were similar except that FaExp2 was induced at an earlier stage. Analysis of expansin protein by Western blotting using an antibody raised against CsExp1 from cucumber hypocotyls identified two bands of 29 and 31 kDa from developing fruit. Protein extracts from developing fruit were assayed for extension activity. Considerable rates of extension were observed with extracts from ripening fruit, but no extension was observed with protein from unripe green fruit. These results demonstrate the presence of at least six expansin genes in strawberry fruit and that during ripening the fruit acquires the ability to cause extension in vitro, characteristic of expansin action.     

Tomato alcohol dehydrogenase. Expression during fruit ripening and under hypoxic conditions.

We have isolated and sequenced a partial tomato alcohol dehydrogenase (Adh) cDNA clone. Expression of tomato Adh was studied at the messenger RNA level in seedlings, roots, and fruit. High induction was observed under hypoxic conditions, both in tomato seedlings and in roots. In addition, the Adh mRNA was present at the mature green and pink stage of the tomato fruit, and was highly induced in late ripening. Moreover, an artificial ripening treatment resulted in at least 50-fold induction compared to the mature green mRNA level. Genomic DNA gel blotting suggested the presence of a multigene family for Adh in tomato.