Naihu Wu

Sucrose Regulates Elongation of Carrot Somatic Embryo Radicles as a Signal Molecule

Elongation of carrot somatic embryo radicles was inhibited by sucrose at or above 5%(145 mM). This effect would not be released until the sucrose concentration was lowered again. Morphological and cytological studies as well as determination of ABA content and analysis of the expression mode of a Lea gene, all point to its similarity to natural dormancy and germination of seeds. Use of monosaccharides (glucose and fructose), other disaccharide (maltose), and isomolar concentration of osmotica (mannitol and sorbitol), did not show similar regulatory effect. It is thus clear that the regulatory effect is not a result of simple osmotic stress. Hexokinase inhibitors such as glucosamine and N-acetyl-glucosamine did not exert any influence on the regulation–deregulation effects of sucrose. Mannose, which inhibits germination of Arabidopsis seeds, did not prevent carrot somatic embryo radicles from elongating. It is thus inferred that this sucrose-signaling pathway may be independent of hexokinase. As a first step to understand the molecular mechanism of this process, a carrot sucrose transporter gene (cSUT) expressed in the embryos and roots specifically was isolated. Studies on transformed yeast mutant with cSUT cDNA identified its sucrose transport activity. Northern hybridization and gel retardation experiment revealed that there is a marked increase in expression of cSUT at the beginning of somatic embryo germination, and this is attributed to regulation on the level of transcription. This suggested the possibility that cSUT has an important role in this sucrose signal regulation system.

Fertility analysis of the Arabidopsis transformed with antisense rice osRACD gene

Abstract The full length osRACD cDNA sequence was subcloned into the pBI121 plasmid in the antisense orientation under the control of the CaMV35S promoter to construct the expression vector pBID, and the constructs were introduced into Arabidopsis plants by using the vacuum infiltration method. The siliques of the transformants stopped growing after anthesis, and they turned yellow or died later; and the siliques from the control plants transformed by the pBI continued growing after anthesis and matured normally. In vitro pollen germination demonstrated that the growth and elongation process of the pollens of the transgenic plants was inhibited, the pollen tubes were shorter and slightly fatter than the tubes of the control plants, which grew normally with long cyclindrical tubes. The above results suggest the function of osRACD gene involved in regulation of the growth and elongation process of pollen tube, its encoding protein may be one of the important factors in regulation of fertility transition of ...

Structural feature of sorghum chloroplastpsbA gene and regulation effects of its 5'-noncoding region.

Comparative analysis reveals remarkahle homology between the sequences of bothpsbA gene nucleotides and the inferred amino acids of sorghum, a C4 plant, and those of rice, a C3 plant. The 5′-noncoding region of sorghumpsbA gene contains the conservative promoter elements, “—35” element and “—10” element, like the prokaryote and the promoter element, TATA box, like the eukaryote. As compared with that of the rice, an extra sequence of 7 bp is found in the leader sequence of the mRNA in the former. Using anin vitro system, it has been demonstrated that protein factor exists in sorghum chloroplast protein extract which specifically hinds to the 5′-noncoding region ofpsbA gene. Measurement of the expression of luciferase shows a 2–5 time greater reaction of the expression plasmids pALqs which contain leader region of sorghumpsbA gene than that of the expression plasmids pALqr which contain leader region of ricepsbA gene inE. coli.

Cloning and characterization of a new actin gene from Oryza sativa L.

Abstract Using Rho family member osRACD as bait, a new member of actin gene family - Act was isolated from Oryza sativa by yeast two-hybrid system. The full-length cDNA was cloned with 5′ RACE technology, which contains an open reading frame of 1134 bp with a predicted protein of 377 amino acids. Sequence alignment revealed 96% to 81.8% identities with some known actin proteins in plants. The method of bioinformatics was used to analyze the protein modification sites, structure and evolution of the gene. Southern blot analysis showed that Act is a single-copy gene in the genome. The result of RT-PCR showed it is ubiquitously expressed in root, shoot, callus and panicle in a temporal fashion. The relationship between Rho family and actin family in evolution and function was also studied.

Biochemical characterization of the protein encoded by rice osRACD gene

Abstract A recombinant plasmid pET- racd was first constructed by cloning osRACD, the development-regulating gene that controls photoperiod fertility transformation in the photoperiod sensitive genic male sterile rice Nongken 58S, into a prokaryote expression vector pET28a(+). It was then transformed into E. coli BL-21. Cutting with thrombin of the fusion protein extracted from transformants and PAGE separation yielded pure osRACD protein, which was further concentrated using ultrafiltration and renatured using glutathione oxidation/reduction refolding system for later functional study. As demonstrated by in vitro functional assay, the osRACD protein expressed in E. coli B-21 shows remarkable activity in binding GTP specifically and hydrolyzing it.

Cloning and expression of DnaJ homolog in carrot somatic embryo

Abstract As the co-chaperone of DnaK/Hsp70 protein, DnaJ/Hsp40 protein influences the synthesis and assembly of the protein complex by regulating ATPase activity of DnaK/Hsp70 protein. By employing the modified method of cDNA representational difference analysis, a homologous fragment of DnaJ was isolated from the deregulated carrot somatic embryos and it was further used as the probe to screen the cDNA library of carrot somatic embryo deregulated for 12 h. As the result. DcJ1 gene, the homologous gene of DnaJ, was isolated from carrot. Sequence analysis showed that its coding region is 1257 bp, which codes 418 amino acids and comprises 3 highly-conserved characteristic domains. Southern blot analysis suggested that the DcJ1 gene seems to be a single copy in the genome, while Northern blot result indicated that DcJ1 expresses only in roots and its degree of expression changes obviously with the regulation-deregulation process. These results suggest that DcJ1 is correlated with the early development of car...

Isolation of development-related genes in somatic embryo radicle of carrot (Daucus carota L.) using modified cDNA RDA

Using modified cDNA RDA capitalizing on the high affinity of streptavidin for biotin and magnetic-absorption-based separation, we have obtained four bands of specifically expressed cDNA in the carrot somatic embryo deregulated for 12 h, which were designated as NR-1, NR-2, NR-3 and NR-4, respectively. As revealed by homology analysis of their DNA sequences after cloning them into pBS, remarkable homology was demonstrated in NR-2, NR-3 and NR-4 with the genes coding for LEA (late embryogenesis abundant protein), Dna J and xyloglucan endo-transglycosylase in plants. On the other hand, NR-1 showing no homology with any known sequence may have come from unknown genes. Using32P-labeled NR-1 as probe, hybridization with cDNA fragment population has shown that we have actually cloned a new gene fragment related to radicle development. As shown by further Southern hybridization, these genes may be present in carrot genome in the form of single or low copies.

Expressional regulationin vitro of psbA mRNA 3′ untranslated region in rice and sorghum

To study the relationship between the structures of rice and sorghum psbA 3′UTR and gene expressional activity, six chimeric luc genes that encode luciferase under control of rice or sorghum psbA 5′UTR and 3′UTR or only 5′UTR were constructed. The levels of LUC accumulation inE. coli and the transcript stability in soluble protein extracts of rice, sorghum chloroplast andE. coli were examined respectively, and a detailed analysis about the function of these two 3′UTR has been carried out. Here the regulation effect of these 3′UTR are reported: (i) When having the same promoter, the chimeric genes with rice 3′ UTR produce LUC much more than that with sorghum 3′UTR; (ii) elimination of 3′UTR results in the fluctuation of LUC accumulation no matter whether it is under control of rice or sorghum psbA 5′ UTR; (iii) rice psbA 3′ UTR exhibits a greater effect on stabilizing transcripts; (iv) psbA 3′IR-RNAs are more stable in chloroplast protein extracts than in E.coli protein extracts.