Cloning and expression of <Emphasis Type="Italic">AtPLC6</Emphasis>, a gene encoding a phosphatidylinositol-specific phospholipase C in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

A full-length cDNA clone corresponding to a putative phosphatidylinositol-specific phospholipase C(PIPLC) was isolated from Arabidopsis thaliana by screening a cDNA library and using RT-PCR strategy.The cDNA,designated AtPLC6,encodes a putative polypeptide of 578 amino acid residues with a calculated molecular mass of 66251.84 D and a pI of 7.24. The sequence analysis indicates that the polypeptide contains X, Y, EF-hand and C2 domains.The overall structure of putative AtPLC6 protein, like other plant PI-PLCs,is most similar to that of mammalian PLCδ The recombinant AtPLC6 protein expressed in E. coil was able to hydrolyze phosphatidylinositol 4,5-biophosphate (PIP2) to generate inositol 1,4,5-trisphate (IP3) and 1,2-diacylglycerol (DAG).The protein hydrolyzes PIP2 in a Ca^2 -dependent manner and the optimum concentration of Ca^2 is 10μmol/L.These results suggested that AtPLC6 gene encodes a genuine PIPLC.Northern blot analysis showed that the AtPLC6 gene is expressed at low level in all examined tissues, such as roots,stems,leaves,flowers,siliques and seedlings under normal growth conditions.The gene is strongly induced under low temperature and weakly induced under various stresses,such as ABA, high-salt stress and heat. These results suggested that AtPLC6 might be involved in the signal-transduction pathways of cold responses of the plants.     

Ethylene-induced nitric oxide production and stomatal closure in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> depending on changes in cytosolic pH

We investigated changes in cytosolic pH and nitric oxide (NO) during ethylene-induced stomatal closure in Arabidopsis thaliana using pharmacological, laser scanning confocal microscopy (LSCM), and spectrophotography techniques. Treatment with ethephon (a direct source of ethylene when applied to plants) and 1-aminocycloaminopropane-1-carboxylic acid (ACC, an ethylene precursor) resulted in a rapid accumulation of NO and cytosolic alkalinization in guard cells. Acetic acid (a weak acid) and sodium orthovanadate (NaVO3; a plasmalemma H+-ATPase inhibitor) reduced stomatal closure induced by ethylene and blocked ethylene-induced activity of nitrate reductase. However, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), a NO scavenger, had no effect. These results suggest that NO production is downstream of the rise in cytosolic pH in A. thaliana.     

Characterization of a calmodulin binding protein kinase from<Emphasis Type="Italic">Arabidopsis thalian</Emphasis>

A full-length calmodulin binding protein kinase cDNA ,AtCBK1 ,from Arabidopsis has been isolated by screening of an Arabidopsis cDNA library and by 5′-RACE-Northern blot and in situ hybridization indicated that the expression of AtCBK1 was more abundant in the vascular bundles and the meristems than in other tissues,The phylogenetic analyses revenl that AtCBK1 is different from animal CaMKs and it falls into CRK subgroup,indicating that they may come from different ancestors,The result suggests that AtCBK1 encoldes a CaM-binding serine/threonine protein kinase.     

The initiation and connection of vessel elements in Arabidopsis thaliana （Brassicaceae） seedlings

This research used confocal laser scanning microscopy to examine the initiation and connection of vessel elements in Arabidopsis thaliana （L.） Heynh seedlings. The results indicated that vessel elements differentiated first in the lower portion of cotyledon-node zone （CNZ） 2 h after germination, and then extended downwards to the hypoeotyl and root and upwardly to the middle portion of CNZ. 10 h after germination, vessel elements developed in the lower portion of cotyledon blade, the second initiating site, connecting gradually with that initiated in cotyledon node and forming pinnate-marginal venation. The connection of vessel elements occurred between the upper portions of CNZ and epieotyl-shoot zone 7 d after germination. By then, the complete connection of vessel elements in the axial and lateral organs had formed.     

<Emphasis Type="Italic">Cis</Emphasis>-regulatory change and expression divergence between duplicate genes formed by genome duplication of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

As an index of functional divergence, expression divergence between duplicate gene copies has been observed and correlated with protein coding sequence divergence and bias in gene functional classes. However, the changes in the cis-regulatory region of the duplicate genes which is thought to have important role in expression divergence, has not been explored on the genome-wide scale. We analyzed functional genomics data for a large number of duplicated gene pairs formed by ancient polyploidy events in Arabidopsis thaliana. The divergence in cis-regulatory regions between two copies is positively correlated with the magnitude difference of expression. Moreover, we find that highly expressed duplicate gene pairs have a more diverged cis-regulatory region than weakly expressed gene pairs. We also show that the correlation between expression functional constraint and protein functional constraint is different in old and young duplicate pairs. Our results suggest that cis-regulatory sequence divergence contributes to the expression divergence of duplicate genes formed by genome-wide du-plication. Cis-regulatory region diverges faster in highly expressed duplicate pairs. The diversify selection strengths that act on cis-regulatory region and protein coding region are negatively correlated in young duplicate pairs under expression con-straint.     

Agrobacterium tumefaciens-mediated Transformation of CryⅠA(b) gene to Trichoderma harzianum

In this study, Cry ⅠA(b) gene was successfully transferred into the biocontrol fungus Trichoderma harzianum with an efficiency of 60-180 transformants per 10^6 spores by using Agrobacterium tumefaciens-mediated transformation. Putative transformants were analyzed to test the presence of Cry ⅠA(b) gene by Southern blot. Most transformants contained a single T-DNA copy. RT-PCR analysis showed that the Cry ⅠA(b) gene was transcribed. Antifungal activities and insecticidal activities of the transformants were examined. There was no obvious difference in antifungal activities between the transformants and their wild strains. The modified mortalities of the transformants T1 and T2 were 69.57% and 91.30%, respectively. The tranformation system mediated by A. tumefaciens proved to be a powerful tool for the filamentous fungi transformation and functional genomic study with its high transformation frequency, simplicity of T-DNA integration, and genetic stability of transformants.     

Ubi1 intron-mediated enhancement of the expression of Bt cry1Ah gene in transgenic maize （Zea mays L.）

The cry1Ah gene was one of novel insecticidal genes cloned from Bacillus thuringiensis isolate BT8. Two plant expression vectors containing cry1Ah gene were constructed. The first intron of maize ubiqutinl gene was inserted between the maize Ubiquitin promoter and cry1Ah gene in one of the plant expressing vectors （pUUOAH）. The two vectors were introduced into maize immature embryonic calli by microprojectile bombardment, and the reproductively plants were acquired. PCR and Southern blot analysis showed that foreign genes had been integrated into maize genome and inherited to the next generation stably. The ELISA assay to T1 and T2 generation plants showed that the expression of CrylAh protein in the construct containing the ubil intron （pUUOAH） was 20% higher than that of the intronless construct （pUOAH）. Bioassay results showed that the transgenic maize harboring cry1Ah gene had high resistance to the Asian corn borers and the insecticidal activity of the transgenic maize containing the ubil intron was higher than that of the intronless construct. These results indicated that the maize ubil intron can enhance the expression of the Bt cry1Ah gene in transgenic maize efficiently