Expression of. Arabidopsis tryptophan biosynthetic pathway genes: effect of the 5'' coding region of phosphoribosylanthranilate isomerase gene

There are three non-allelic isogenes encoding phosphoribosylanthranilate isomerase (PAI) in Arabidopsis thaliana. The expression plasmids were constructed by fusion of the GUS reporter gene to the three PAI promoters with or without the 5' region encoding PAI N-terminal polypeptides and transferred into Arabidopsis plants by Agrobacterium tumefaciens. Analysis of GUS activity revealed that the PAI 5' coding region was necessary for high expression of GUS activity. GUS activity in transgenic plants transformed with the expression plasmids containing the 5' coding region of PAI1 or PAI3 was 60—100-fold higher than that without the corresponding 5' region. However, the effect of 5' coding region of PAI2 gene on the GUS activity was very small (only about 1 time difference). The GUS histochemical staining showed a similar result as revealed by GUS activity assay. It was expressed in the mesophyll cells and guard cells, but not in the epidermic cells, indicating that the N-terminal polypeptides encoded by t     

Molecular mechanism of diapause in insect (I) --Expression of diapause hormone gene and incubation temperature in embryonic stage of Bombyx mori

The embryonic diapause of the silkworm, Bombyx mori, is induced by the diapause hormone (DH) which is secreted from the suboesophageal ganglion of pupae. The diapause nature of bivoltine strains uses environmental stimuli as the initial signal to determine the diapause nature. The experiments showed that DH gene expression is a direct response to the environmental stimulus, such as high incubation temperature. The cDNA from the embryonic stage was cloned and sequence analysis showed the cDNA encoding DH. Expression patterns of the DH gene in embryonic stage are different at incubation temperatures 15℃ and 25℃, suggesting that the incubation temperature as an environmental signal is kept within the body to control the DH gene expression at the pupal stage, so that the embryonic diapause of next generation can be determined.     

Novel alleles among soybean Bowman-Birk proteinase inhibitor gene families

Trypsin inhibitors have been found in various animals, plants and microorganisms.There were two types of trypsin inhibitors in soybean including Bowman-Birk protease inhibitors(BBI) and Kunitz in-hibitors(KTI).The different BBI genes from wild soybean(G.soja) and cultivated soybean(G.max) formed a multigene family.We constructed a cDNA library of cultivar 'SuiNong 14' seed at the R7 growth stage using the SMART Kit.Seventeen contigs or singletons were highly homologous to soy-bean protease inhibitors.Contigs of 5, 35, 8 and 9 were highly homologous to BBI family members BBI-A1, BBI-A2, BBI-C and BBI-D, respectively.Sequence analyses showed there were novel allelic varia-tions among the 4 BBI members in SuiNong 14.Based on the comparison of soybean seed cDNA li-braries from different developmental stages, it was apparent that the expression of trypsin inhibitors increased during seed development in soybean.Phylogenetic analysis of BBI gene sequences among dicotyledonous and monocotyledonous plants demonstrated that these genes shared a common pro-genitor.     

Establishing a gene trap system mediated by T-DNA(GUS) in rice

Two plasmids, p13GUS and p13GUS2, were constructed to create a gene trap system containing the promoterless β-glucuronidase (GUS) reporter gene in the T-DNA region. Transformation of these two plasmids into the rice variety Zhonghua 11 (Oryza sativa ssp. japonica cv.), mediated by Agrobacterium tumefaciens, resulted in 942 independent transgenic lines. Histochemical GUS assays revealed that 31 To plants had various patterns of the reporter gene expression, including expression in only one tissue, and simultaneously in two or more tissues. Hygromycin-resistant (hygr) homozygotes were screened and the copy number of the T-DNA inserts was determined in the GUS-positivs transgenic plants. The flanking sequences of the T-DNA were isolated by inverse-polymerase chain reaction and the insert positions on the rice genome of T-DNA were determined by a basic local alignment search tool in the GUS-positive transgenic plants transformed with plasmid p13GUS. Moreover, calii induced from the seeds of the T1 generation of 911 GUS-negative transgenic lines were subjected to stress and hormone treatments. Histochemical GUS assays were carried out on the calli before and after treatment. The results revealed that calli from 21 lines displayed differential GUS expression after treatment. All of these data demonstrated that this trap system is suitable for identifying rice genes, including those that are sensitive to induction.     

诱导根癌土壤杆菌vir区基因表达信号分子的构效研究

以根癌土壤杆菌为介导的外源基因转化系统是目前应用最为广泛和有效的植物基因转化系统。根癌土壤杆菌D质粒毒性区。irA、virB、。irC、。irD。。irE、。irG6个基因位点,在外源诱导性信号分子的作用下,表达出转移DNA（T－DNA）复合物,整合人植物细胞核基因组中。利用根癌土壤杆菌这种天然的转化机制,已获得双子叶转基因植株。但单子叶植物难以被根癌土壤杆菌转化,某些研究者认为这是由于单子叶植物缺乏促使根癌土壤杆菌产生趋化运动以及诱导。ir区基因表达的信号分子［‘」。我们从幼穗分化期至抽穗扬花期水稻中分离获得2种高效…     

Structure activity Research of Signal Factors Inducing Agrobacterium vir Genes

Four rice ( Oryza sativa L. cv. IR72) signal factors with the functions of inducing Agrobaterium tumefaciens (Smith et Townsend) Corm vir genes were constructed. They were 5,7,4'-trihydmxy-3', 5'- dimethoxy-flavone (Fl) and its structure analogues F2, F3 and F4. These four signal factors were detected by Agrobacterium vir:: lacZ fusion gene system as to compare their functions of inducing virA, virB, virC, virD, virE and virG genes. The structure characters and stmcture-activity relationship of these signal factors with the best inducing effects to Agrobacterium exogenous gene transformation system were defined which may provide some basic information for foreign gene transformation in rice.     

Absence in monocotyledonous plants of the diffusible plant factors inducing T-DNA circularization and vir gene expression in Agrobacterium

Summary T-DNA circularization is one of the molecular events specifically induced in agrobacterial cells upon their infection of dicotyledonous plant cells. We developed a seedling co-cultivation procedure to determine whether or not monocotyledonous plants have the ability to induce T-DNA circularization and vir gene expression. Co-cultivation of Agrobacterium tumefaciens with seedlings of dicotyledonous plants showed that the circularization event takes place efficiently. The exudates and extracts of the seedlings also effectively induced T-DNA circularization and vir gene expression, indicating that dicotyledonous seedlings contain diffusible factors capable of inducing these molecular events. In contrast, neither T-DNA circularization nor vir gene expression was detectable when Agrobacterium was incubated with seedlings of monocotyledonous plants. Supplementing with acetosyringone, a known inducer of vir gene expression and T-DNA circularization, resulted in the induction of circularization during co-cultivation with monocotyledonous seedlings. These results indicate that the seedlings of monocotyledonous plants have no detectable amounts of diffusible inducers, unlike dicotyledonous seedlings. Therefore, it is unlikely that the vir genes are expressed in Agrobacterium inoculated in monocotyledonous plants. This may be one of the blocks in tumorigenesis of monocotyledonous plants by Agrobacterium.     

The regulatory VirA protein of Agrobacterium tumefaciens does not function at elevated temperatures.

Previous studies have shown that Agrobacterium tumefaciens causes tumors on plants only at temperatures below 32 degrees C, and virulence gene expression is specifically inhibited at temperatures above 32 degrees C. We show here that this effect persists even when the virA and virG loci are expressed under the control of a lac promoter whose activity is temperature independent. This finding suggests that one or more steps in the signal transduction process mediated by the VirA and VirG proteins are temperature sensitive. Both the autophosphorylation of VirA and the subsequent transfer of phosphate to VirG are shown to be sensitive to high temperatures (> 32 degrees C), and this correlates with the reduced vir gene expression observed at these temperatures. At temperatures of 32 degrees C and higher, the VirA molecule undergoes a reversible inactivation while the VirG molecule is not affected. vir gene induction is temperature sensitive in an acetosyringone-independent virA mutant background but not in a virG constitutive mutant which is virA and acetosyringone independent. These observations all support the notion that the VirA protein is responsible for the thermosensitivity of vir gene expression. However, an Agrobacterium strain containing a constitutive virG locus still cannot cause tumors on Kalanchoe plants at 32 degrees C. This strain induces normal-size tumors at temperatures up to 30 degrees C, whereas the wild-type Agrobacterium strain produces almost no tumors at 30 degrees C. These results suggest that at temperatures above 32 degrees C, the plant becomes more resistant to infection by A. tumefaciens and/or functions of some other vir gene products are lost in spite of their normal levels of expression.     

Sugar-mediated induction of Agrobacterium tumefaciens virulence genes: structural specificity and activities of monosaccharides.

The virulence genes of Agrobacterium tumefaciens are induced by specific plant phenolic metabolites and sugars (G. A. Cangelosi, R. G. Ankenbauer, and E. W. Nester, Proc. Natl. Acad. Sci. USA, in press). In this report, monosaccharides, derivatives, and analogs which induce the vir regulon have been identified and the structural requirements for monosaccharide-mediated induction have been determined. Pyranose sugars with equatorial hydroxyls at C-1, C-2, and C-3 displayed strong vir gene-inducing activity; the C-4 hydroxyl could be epimeric and a wide variety of substitutions at C-5 were permissible. The acidic monosaccharide derivatives D-galacturonic acid and D-glucuronic acid were the strongest inducers among the monosaccharides tested. Eight of the 11 inducing compounds are known plant metabolites, and 7 are monomers of major plant cell wall polysaccharides. A role for monosaccharides and plant phenolic compounds as wound-specific plant metabolites which signal the ChvE/VirA/VirG regulatory system is proposed.     

Both wound-inducible and tuber-specific expression are mediated by the promoter of a single member of the potato proteinase inhibitor II gene family

A chimeric gene consisting of 1.3 kb of the 5' regulatory region of a member of the potato proteinase inhibitor II gene family, the coding region of the bacterial β-glucuronidase (GUS) gene and 260 bp of the proteinase inhibitor II 3'-untranslated region containing the poly(A) addition site was introduced into potato and tobacco by Agrobacterium tumefaciens mediated transformation. Analysis of transgenic plants demonstrates systemic, wound-inducible expression of this gene in stem and leaves of potato and tobacco. Constitutive expression was found in stolons and tubers of non-wounded potato plants. Histochemical experiments based on the enzymatic activity of the GUS protein indicate an association of the proteinase inhibitor II promoter activity with vascular tissue in wounded as well as in systemically induced non-wounded leaves, petioles, potato stems and in developing tubers. These data prove that one single member of the proteinase inhibitor II gene family contains cis-active elements, which are able to respond to both developmental and environmental signals. Furthermore they support the hypothesis of an inducing signal (previously called proteinase inhibitor inducing factor), which is released at the wound site and subsequently transported to non-wounded parts of the plant via the vascular system from where it is released to the surrounding tissue.     

单、双子叶植物的代谢物调节农杆菌Vir区基因表达的研究

本文研究了六种植物（三种单子叶植物,三种双子叶植物）愈伤组织的 渗出物和抽提物对农杆菌Vir基因表达的调节作用,其调节水平植物的不同而明显不同,但单,双子叶植物的代谢物对Vir基因表达的调节作用并非截然分开,即使在双子叶植物（如大豆）的抽提物与渗出物中也存在着抑制Vir基因表达的因子,而在单子叶植物（如玉米等）的抽提物与渗出物中也存在着促进Vir基因表达的调节因子,Vir位点的调节反应随渗出物与抽提物的种类不同而明显不同,不同Vir位点对同类渗出物或抽提物的反应也不同,渗出物对Vir基因表达的正调节效应优于抽提物,植物渗出物与AS对Vir区基因表达的调节并不表现简单的累加效应或协同作用,相反,在渗出物中还存在着不同程度阻抑AS对Vir基因表达正调节的因子。