Cloning and characterization of a SnRK1-encoding gene from <Emphasis Type="Italic">Malus hupehensis</Emphasis> Rehd. and heterologous expression in tomato

Sucrose non-fermenting-1-related protein kinase-1 (SnRK1) plays an important role in metabolic regulation in plant. To understand the molecular mechanism of amino acids and carbohydrate metabolism in Malus hupehensis Rehd. var. pinyiensis Jiang (Pingyi Tiancha, PYTC), a full-length cDNA clone encoding homologue of SnRK1 was isolated from PYTC by Rapid Amplification of cDNA Ends (RACE). The clone, designated as MhSnRK1, contains 2063 nucleotides with an open reading frame of 1548 nucleotides. The deduced 515 amino acids showed high identities with other plant SnRK1 genes. Quantitative real-time PCR analysis revealed this gene was expressed in roots, stems and leaves. Exposing seedlings to nitrate caused and initial decrease in expression of the MhSnRK1 gene in roots, leaves and stems in short term. Ectopic expression of MhSnRK1 in tomato mainly resulted in higher starch content in leaf and red-ripening fruit than wild-type plants. This result supports the hypothesis that overexpression of SnRK1 causes the accumulation of starch in plant cells. All the results suggest that MhSnRK1 may play important roles in carbohydrate and amino acid metabolisms.     

Gene expression and genetic mapping analyses of a perennial ryegrass glycine-rich RNA-binding protein gene suggest a role in cold adaptation

A perennial ryegrass cDNA clone encoding a putative glycine-rich RNA binding protein (LpGRP1) was isolated from a cDNA library constructed from crown tissues of cold-treated plants. The deduced polypeptide sequence consists of 107 amino acids with a single N-terminal RNA recognition motif (RRM) and a single C-terminal glycine-rich domain. The sequence showed extensive homology to glycine-rich RNA binding proteins previously identified in other plant species. LpGRP1-specific genomic DNA sequence was isolated by an inverse PCR amplification. A single intron which shows conserved locations in plant genes was detected between the sequence motifs encoding RNP-1 and RNP-2 consensus protein domains. A significant increase in the mRNA level of LpGRP1 was detected in root, crown and leaf tissues during the treatment of plants at 4°C, through which freezing tolerance is attained. The increase in the mRNA level was prominent at least 2 h after the commencement of the cold treatment, and persisted for at least 1 week. Changes in mRNA level induced by cold treatment were more obvious than those due to treatments with abscisic acid (ABA) and drought. The LpGRP1 protein was found to localise in the nucleus in onion epidermal cells, suggesting that it may be involved in pre-mRNA processing. The LpGRP1 gene locus was mapped to linkage group 2. Possible roles for the LpGRP1 protein in adaptation to cold environments are discussed.     

Ectopic Expression of Plant RNA Chaperone Offering Multiple Stress Tolerance in <Emphasis Type="Italic">E. coli</Emphasis>

Members of the plant glycine-rich RNA-binding proteins (GR-RBPs) family have been reported in flowering, development, circadian rhythms, biotic and abiotic stresses. Particularly, GR-RBPs are reported to function as RNA chaperones, promoting growth and acclimation during cold shock. It is indispensable to further question the efficacy and mechanism of GR-RBPs under various environmental strains. Monitoring the expression of stress-regulated proteins under stress conditions has been a beneficial strategy to study their functional roles. In an effort to elucidate the NtGR-RBP1 function, stress markers such as salinity, drought, low temperature and heat stresses were studied. The NtGR-RBP1 gene was expressed in E. coli followed by the exposure to stress conditions. Recombinant E. coli expressing NtGR-RBP1 were more tolerant to stresses, e.g., salinity, drought, cold and heat shock. Recombinants exhibited higher growth rates compared to control in spot assays. The tolerance was further confirmed by monitoring the growth in liquid culture assays. Cells expressing NtGR-RBP1 under salt (500 mM NaCl), drought (20% PEG), cold (4 and 20 °C) and heat stresses (50 °C) had enhanced growing ability and better endurance. Our study supports the notion that the protective role of NtGR-RBP1 may contribute to growth and survival during diverse environmental stresses.     

Gossypium arboreum GHSP26 enhances drought tolerance in Gossypium hirsutum

Heat‐shock proteins (HSP) are molecular chaperones for protein molecules. These proteins play an important role in protein–protein interactions such as, folding and assisting in the establishment of proper protein conformation and prevention of unwanted protein aggregation. A small HSP gene GHSP26 present in Gossypium arboreum responds to dehydration. In the present study, an attempt was made to overcome the problem of drought stress in cotton. A cDNA of GHSP26 was isolated from G. arboreum, cloned in plant expression vector, pCAMBIA‐1301 driven by the cauliflower mosaic virus 35S promoter and introduced into Gossypium hirsutum. The integration and expression studies of putative transgenic plants were performed through GUS assay; PCR from genomic DNA, and quantitative real‐time PCR analysis. Transgenic cotton plants showed an enhanced drought tolerance, suggesting that GHSP26 may play a role in plant responsiveness to drought. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

<Emphasis Type="Italic">IiSDD1</Emphasis>, a gene responsive to autopolyploidy and environmental factors in <Emphasis Type="Italic">Isatis indigotica</Emphasis>

In plants, stomata play a pivotal role in the regulation of gas exchange and are distributed throughout the aerial epidermis. SDD1, a gene isolated from Arabidopsis thaliana has been demonstrated to specialize in stomatal density and distribution. In our present study, a comprehensive survey of global gene expression performed by using an A. thaliana whole genome Affymetrix gene chip revealed SDD1 tends to be significantly lower in tetraploid Isatis indigotica than in diploid ones. To intensively investigate different SDD1 expression in response to polyploidy, a full-length cDNA clone (IiSDD1) encoding SDD1 was isolated from the traditional Chinese medicinal herb I. indigotica cDNA library. IiSDD1 shared a high level of identity with that from A. thaliana, containing some basic features of subtilases: D, H and S regions, as well as a substrate-binding site. Real-time quantitative PCR analysis indicated that IiSDD1 was constitutively expressed in all tested tissues, including roots, stems and leaves, both in tetraploid and diploid I. indigotica, and with the highest expression in leaves. In addition, IiSDD1 was also found to be down-regulated by signalling molecules for plant defence responses, such as abscisic acid (100 μM) and gibberellin (100 mg/L), as well as by environmental stresses including salt, darkness, coldness and drought. Our study, for the first time, indicates SDD1 participates not only in the defense/stress responsive pathways, but also probably involves in plants polyploidy evolution.     

Cloning and Drought Resistance Analysis of Soybean <i>GmHsps_p23-like</i> Gene

Soybean (Glycine max (L.) Merr.) is an important cultivated crop, which requires much water during its growth, and drought seriously affects soybean yields. Studies have shown that the expression of small heat shock proteins can enhance drought resistance, cold resistance and salt resistance of plants. In this experiment, soybean GmHsps_p23-like gene was successfully cloned by RT-PCR, the protein encoded by the GmHsps_p23-like gene was subjected to bioinformatics analysis, and the pCAMBIA3301-GmHsps_p23-like overexpression vector and pCBSG015-GmHsps_p23-like gene editing vector were constructed. Agrobacterium-mediated method was used to transform soybeans to obtain positive plants. RT-PCR detection, rehydration experiment and drought resistance physiological and biochemical index detection were performed on the T₂ generation positive transgenic soybean plants identified by PCR and Southern hybridization. The results showed that the overexpression vector plant GmHsps_p23-like gene expression increased. After rehydration, the transgenic overexpression plants returned to normal growth, and the damage to the plants was low. After drought stress, the SOD and POD activities and the PRO content of the transgenic overexpression plants increased, while the MDA content decreased. The reverse was true for soybean plants with genetically modified editing vectors. The drought resistance of the overexpressed soybeans under drought stress was higher than that of the control group, and had a stronger drought resistance. It showed that the expression of soybean GmHsps_p23-like gene can improve the drought resistance of soybean. The cloning and functional verification of soybean GmHsps_p23-like gene had not been reported yet. This is the first time that PCR technology has been used to amplify the soybean GmHsps_p23-like gene and construct an expression vector for this gene. This research has laid the foundation for transgenic technology to improve plant drought resistance and cultivate new drought-resistant transgenic soybean varieties.     

Molecular cloning and characterization of an F-box family gene <Emphasis Type="Italic">CarF</Emphasis>-<Emphasis Type="Italic">box1</Emphasis> from chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.)

F-box protein family has been found to play important roles in plant development and abiotic stress responses via the ubiquitin pathway. In this study, an F-box gene CarF-box1 (for Cicer arietinum F-box gene 1, Genbank accession no. GU247510) was isolated based on a cDNA library constructed with chickpea seedling leaves treated by polyethylene glycol. CarF-box1 encoded a putative protein with 345 amino acids and contained no intron within genomic DNA sequence. CarF-box1 is a KFB-type F-box protein, having a conserved F-box domain in the N-terminus and a Kelch repeat domain in the C-terminus. CarF-box1 was localized in the nucleus. CarF-box1 exhibited organ-specific expression and showed different expression patterns during seed development and germination processes, especially strongly expressed in the blooming flowers. In the leaves, CarF-box1 could be significantly induced by drought stress and slightly induced by IAA treatment, while in the roots, CarF-box1 could be strongly induced by drought, salinity and methyl jasmonate stresses. Our results suggest that CarF-box1 encodes an F-box protein and may be involved in various plant developmental processes and abiotic stress responses.     

Characterization of an Oxidative Stress Inducible Nonspecific Lipid Transfer Protein Coding cDNA and its Promoter from Drought Tolerant Plant <Emphasis Type="Italic">Prosopis juliflora</Emphasis>

Abiotic stress is the principal cause of crop failure worldwide. Prosopis juliflora is a hardy plant reported to be tolerant to drought, salinity, extremes of soil pH, and heavy metal stress. Here, we report the characterization of a cDNA clone for a putative nonspecific lipid transfer protein (Pj LTP1) found abundantly in a drought stressed leaf cDNA library of P. juliflora and its promoter. A multiple sequence analysis of Pj LTP1 with well-established and officially designated nonspecific lipid transfer proteins allergens revealed high similarity at the amino acid level. Northern analysis of Pj LTP1 in P. juliflora leaves under oxidative stress revealed steady upregulation at the time points analyzed. A 929-bp fragment was isolated from the 5′ end of Pj LTP1, and transient reporter gene expression studies revealed it to be a functional promoter. Several cis-acting elements previously reported to function in stress response were found in this promoter. The possible reasons for changes in gene expression during stress in relation to the host plant’s stress tolerance mechanisms are discussed.     

陆地棉GhCDPK1基因响应干旱胁迫的功能初探

钙依赖性蛋白激酶(CDPKs)是一类重要的钙信号感受蛋白和响应蛋白,在植物干旱、低温、盐碱等非生物胁迫应答中起着重要的调控作用。为探讨陆地棉GhCDPK1基因在干旱胁迫下所起的作用,该研究利用实时荧光定量PCR技术分析了PEG模拟干旱胁迫下该基因的表达量,发现GhCDPK1基因受干旱胁迫诱导。通过构建植物表达载体pCAMBIA2300-GhCDPK1,采用农杆菌介导的叶盘法转化模式植物烟草,发现干旱胁迫下转基因植株保水能力明显高于野生型植株,叶绿素、脯氨酸、可溶性蛋白含量及POD、SOD活性也高于野生型植株,而丙二醛含量低于野生型植株。研究结果表明,GhCDPK1基因作为正向调控因子响应干旱胁迫诱导,过表达GhCDPK1基因可以使植株积累更多的渗透调节物质、增强抗氧化系统酶的活性和维持细胞膜的稳定性来提高植物抵御外界干旱胁迫的能力。