Construction of a Plant Transformation-ready Expression cDNA Library for Thellungiella halophila Using Recombination Cloning

Salt cress （Thellungiella halophila）, a close relative of the model plant Arabidopsis thaliana L., is an extremophile that is adapted to harsh saline environments. To mine salt-tolerance genes from this species, we constructed an entry cDNA library from the salt cress plant treated with salt-stress by using a modified cDNA synthesis and an improved recombinationassisted cDNA library construction method that is completely free of manipulations involving restriction enzymes and DNA ligase. This cDNA library construction procedure is significantly simplified and the quality of the cDNA library is improved. This entry cDNA library was subsequently shuttled into the destination binary vector pCB406 designed for plant transformation and expression via recombination-assisted cloning. The library is plant transformation ready and is used to transform Arabidopsis on a large scale in order to create a large collection of transgenic lines for functional gene mining.     

Construction of a Plant Transformation-ready Expression cDNA Library for Thellungiella halophila Using Recombination Cloning

Salt cress(Thellungiella halophila),a close relative of the model plant Arabidopsis thaliana L.,is an extremophile that isadapted to harsh saline environments.To mine salt-tolerance genes from this species,we constructed an entry cDNA libraryfrom the salt cress plant treated with salt-stress by using a modified cDNA synthesis and an improved recombination-assisted cDNA library construction method that is completely free of manipulations involving restriction enzymes andDNA ligase.This cDNA library construction procedure is significantly simplified and the quality of the cDNA library isimproved.This entry cDNA library was subsequently shuttled into the destination binary vector pCB406 designed for planttransformation and expression via recombination-assisted cloning.The library is plant transformation ready and is used totransform Arabidopsis on a large scale in order to create a large collection of transgenic lines for functional gene mining.     

Construction of a cDNA library of Aphis gossypii Glover for use in RNAi

Aphis gossypii Glover is an important insect pest that functions as a viral vector and mediates approximately 45 different viral diseases. As part of a strategy for control of A. gossypii, we investigated the functions of genes using RNAi. To this end, a cDNA library was constructed for various genes and for selecting appropriate targets for RNAi mediated silencing. The cDNA library was constructed using the Gateway cloning system with site‐specific recombination of bacteriophage λ. It was used to carry out single step cloning of A. gossypii cDNAs. As a result, a cDNA library with a titer of 8.4 × 10⁶ was constructed. Since the sequences in this library carry att sites, they can be cloned into various binary vectors. This library will be of value for various studies. For later screening of selected genes, it is planned to clone the library into virus‐induced gene silencing (VIGS) vectors, which makes it possible to analyze gene function and allow subsequent transfection of plants. Such transfection experiments will allow testing of RNAi‐induced insecticidal activity or repellent activity to A. gossypii, and result in the identification of target genes. It is also expected that the constructed cDNA library will be useful for analysis of gene functions in A. gossypii.     

Comparative genomic analysis of 1047 completely sequenced cDNAs from an Arabidopsis-related model halophyte, <Emphasis Type="Italic">Thellungiella halophila</Emphasis>

Background  

Thellungiella halophila (also known as T. salsuginea) is a model halophyte with a small size, short life cycle, and small genome. Thellungiella genes exhibit a high degree of sequence identity with Arabidopsis genes (90% at the cDNA level). We previously generated a full-length enriched cDNA library of T. halophila from various tissues and from whole plants treated with salinity, chilling, freezing stress, or ABA. We determined the DNA sequences of 20 000 cDNAs at both the 5'- and 3' ends, and identified 9569 distinct genes.     

紫外线强烈诱导的谷胱甘肽转移酶基因的功能鉴定

植物谷胱甘肽转移酶（glutathione S-transferases,GSTs)基因家族在逆境反应和植物生长发育过程中都起着非常重要的作用。为了阐明GST在紫外辐射下是否对植物有保护作用,以紫外强烈诱导表达的GST、cDNA为探针,筛选拟南芥cDNA文库,获得了这种GST的全长cDNA;利用此cDNA构建植物表达载体,并通过农杆菌介导法转化拟南芬,使其在拟南芥中得到大量表达;通过对转基因植株的紫外辐射耐性分析,证实了该GST的过量表达可明显增强拟南芥对紫外辐射损伤作用的耐受性。     

FOX-superroots of Lotus corniculatus, overexpressing Arabidopsis full-length cDNA, show stable variations in morphological traits

Using the full-length cDNA overexpressor (FOX) gene-hunting system, we have generated 130 Arabidopsis FOX-superroot lines in bird's-foot trefoil (Lotus corniculatus) for the systematic functional analysis of genes expressed in roots and for the selection of induced mutants with interesting root growth characteristics. We used the Arabidopsis-FOX Agrobacterium library (constructed by ligating pBIG2113SF) for the Agrobacterium-mediated transformation of superroots (SR) and the subsequent selection of gain-of-function mutants with ectopically expressed Arabidopsis genes. The original superroot culture of L. corniculatus is a unique host system displaying fast root growth in vitro, allowing continuous root cloning, direct somatic embryogenesis and mass regeneration of plants under entirely hormone-free culture conditions. Several of the Arabidopsis FOX-superroot lines show interesting deviations from normal growth and morphology of roots from SR-plants, such as differences in pigmentation, growth rate, length or diameter. Some of these mutations are of potential agricultural interest. Genomic PCR analysis revealed that 100 (76.9%) out of the 130 transgenic lines showed the amplification of single fragments. Sequence analysis of the PCR fragments from these 100 lines identified full-length cDNA in 74 of them. Forty-three out of 74 full-length cDNA carried known genes. The Arabidopsis FOX-superroot lines of L. corniculatus, produced in this study, expand the FOX hunting system and provide a new tool for the genetic analysis and control of root growth in a leguminous forage plant.     

Enhanced resistance against bacterial wilt in transgenic tomato (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis>) lines expressing the <Emphasis Type="Italic">Xa21</Emphasis> gene

To enhance bacterial wilt resistance in tomato plants and simplify the protocol of Agrobacterium tumefaciens mediated gene transfer, parameters affecting transformation efficiency in tomato have been optimized. A. tumefaciens strain EHA101, harboring a recombinant binary expression vector pTCL5 containing the Xa21 gene under the control of the CaMV 35S promoter was used for transformation. Five cultivars of tomato (Rio Grande, Roma, Pusa Ruby Pant Bahr and Avinash) were tested for transformation. Transformation efficiency was highly dependent on preculture of the explants with acetosyringone, acetosyringone in co-cultivation media, shoot regeneration medium and pre-selection after co-cultivation without selective agent. One week of pre-selection following selection along with 400 μM acetosyringone resulted in 92.3% transient GUS expression efficiency in Rio Grande followed by 90.3% in Avinash. The presence and integration of the Xa21 gene in putative transgenic plants was confirmed by polymerase chain reaction (PCR) and Southern blot analyses with 4.5–42.12% PCR-positive shoots were obtained for Xa21 and hygromycin genes, respectively. Transgenic plants of the all lines showed resistance to bacterial wilt. T₁ plants (resulting from self-pollination of transgenic plants) tested against Pseudomonas solanacearum inoculation in glasshouse, showed Mendelian segregation.     

Towards Arabidopsis genome analysis: monitoring expression profiles of 1400 genes using cDNA microarrays

cDNA microarrays containing 1443 Arabidopsis thaliana genes were analyzed for expression profiles in major organs of Arabidopsis plants. Novel expression profiles were identified for many coding sequences with putative gene identifications. Expression patterns of novel sequences provided clues to their possible functions. The results demonstrate how microarrays containing a large number of Arabidopsis genes can provide a powerful tool for plant gene discovery, functional analysis and elucidation of genetic regulatory networks.     

Functional analysis of endo‐1,4‐β‐glucanases in response to Botrytis cinerea and Pseudomonas syringae reveals their involvement in plant–pathogen interactions

Plant cell wall modification is a critical component in stress responses. Endo‐1,4‐β‐glucanases (EGs) take part in cell wall editing processes, e.g. elongation, ripening and abscission. Here we studied the infection response of Solanum lycopersicum and Arabidopsis thaliana with impaired EGs. Transgenic TomCel1 and TomCel2 tomato antisense plants challenged with Pseudomonas syringae showed higher susceptibility, callose priming and increased jasmonic acid pathway marker gene expression. These two EGs could be resistance factors and may act as negative regulators of callose deposition, probably by interfering with the defence‐signalling network. A study of a set of Arabidopsis EG T‐DNA insertion mutants challenged with P. syringae and Botrytis cinerea revealed that the lack of other EGs interferes with infection phenotype, callose deposition, expression of signalling pathway marker genes and hormonal balance. We conclude that a lack of EGs could alter plant response to pathogens by modifying the properties of the cell wall and/or interfering with signalling pathways, contributing to generate the appropriate signalling outcomes. Analysis of microarray data demonstrates that EGs are differentially expressed upon many different plant–pathogen challenges, hormone treatments and many abiotic stresses. We found some Arabidopsis EG mutants with increased tolerance to osmotic and salt stress. Our results show that impairing EGs can alter plant–pathogen interactions and may contribute to appropriate signalling outcomes in many different biotic and abiotic plant stress responses.     

An Arabidopsis gene isolated by a novel method for detecting genetic interaction in yeast encodes the GDP dissociation inhibitor of Ara4 GTPase.

The Arabidopsis Ara proteins belong to the Rab/Ypt family of small GTPases, which are implicated in intracellular vesicular traffic. To understand their specific roles in the cell, it is imperative to identify molecules that regulate the GTPase cycle. Such molecules have been found and characterized in animals and yeasts but not in plants. Using a yeast system, we developed a novel method of functional screening to detect interactions between foreign genes and identified this Rab regulator in plants. We found that the expression of the ARA4 gene in yeast ypt mutants causes exaggeration of the mutant phenotype. By introducing an Arabidopsis cDNA library into the ypt1 mutant, we isolated a clone whose coexpression overcame the deleterious effect of ARA4. This gene encodes an Arabidopsis homolog of the Rab GDP dissociation inhibitor (GDI) and was named AtGDI1. The expression of AtGDI1 complemented the yeast sec19-1 (gdi1) mutation. AtGDI1 is expressed almost ubiquitously in Arabidopsis tissues. The method described here indicates the physiological interaction of two plant molecules, Ara4 and GDI, in yeast and should be applicable to other foreign genes.     

Preferential expression and immunogenicity of HIV-1 Tat fusion protein expressed in tomato plant

HIV-1 Tat plays a major role in viral replication and is essential for AIDS development making it an ideal vaccine target providing that both humoral and cellular immune responses are induced. Plant-based antigen production, due to its cheaper cost, appears ideal for vaccine production. In this study, we created a plant-optimized tat and mutant (Cys30Ala/Lys41Ala) tat (mtat) gene and ligated each into a pBI121 expression vector with a stop codon and a gusA gene positioned immediately downstream. The vector construct was bombarded into tomato leaf calli and allowed to develop. We thus generated recombinant tomato plants preferentially expressing a Tat-GUS fusion protein over a Tat-only protein. In addition, plants bombarded with either tat or mtat genes showed no phenotypic difference and produced 2–4 μg Tat-GUS fusion protein per milligram soluble plant protein. Furthermore, tomato extracts intradermally inoculated into mice were found to induce a humoral and, most importantly, cellular immunity.     

Overexpression of thaumatin-like protein in transgenic tomato plants confers enhanced resistance to Alternaria solani

Abstract

A cDNA encoding thaumatin-like protein (TLP) from rice was cloned into the binary vector pMON410 under the control of the CaMV 35S promoter for Agrobacterium-mediated transformation of tomato. All putative transformants were tested for the integration and expression of the chimeric gene by polymerase chain reaction (PCR) for hygromycin resistance gene (hph) and enzyme-linked immunosorbent assay (ELISA) for TLP respectively. Constitutive, high-level expression of TLP was observed in transgenic plants. The transgenic lines exhibited increased resistance to Alternaria solani, the early blight pathogen compared to non-transgenic tomato plants.     

Molecular cloning of a tomato leaf cDNA encoding an aspartic protease, a systemic wound response protein

A full-length cDNA encoding an aspartic protease (LeAspP) has been cloned from a tomato leaf cDNA library. Using LeAspP cDNA as a probe in gel blots, LeAspP mRNA was shown to be systemically induced in tomato leaves by wounding. Application of methyl jasmonate to leaves of intact tomato plants, or supplying systemin to young tomato plants through their cut stems, induces synthesis of LeAspP mRNA. LeAspP message is regulated in tomato similar to several systemic wound response proteins (swrps) that are part of the defense response in tomato plants directed against herbivore attacks.