Biomedical subjects

Zhen Zhu

Publications and source records attributed to Zhen Zhu.

33 records · Page 2Linked to original sources

PubMed2003

Transformation of tobacco with genes encoding Helianthus tuberosus agglutinin (HTA) confers resistance to peach-potato aphid (Myzus persicae).

The effects of the hta gene encoding Helianthus tuberosus agglutinin (HTA) on an insect in the order Homoptera were investigated. Homologous cDNAs of hta-a, hta-b, hta-c and hta-d with CaMV35S as promoter were introduced into tobacco via Agrobacterium tumefaciens. Southern blot results showed that the exogenous hta gene was inserted into the genome of host plants, and northern blot analysis confirmed that hta was expressed in transgenic plants. A bioassay with peach-potato aphid (Myzus persicae) demonstrated that transgenic plants had deleterious effects on the insect. The average population of aphids fed on transgenic T0 plants during an 11-day assay decreased by 70%, compared controls. In transgenic plants of T1 generation, aphid fecundity inhibitions were 53.0% (hta-b) and 64.6% (hta-c), respectively. The development of aphids was notably retarded. We conclude that hta could be a novel and promising candidate for plant transgenic engineering against homopteran insect pests.

Agglutinins↗

PubMed2003

Isolation and identification of a super strong plant promoter from cotton leaf curl Multan virus.

The activity of the C1 and the V1 gene promoter of cotton leaf curl Multan virus (CLCuMV) was investigated in transgenic plants with the gus gene as a reporter gene. Quantitative GUS activity analysis of the transgenic plant leaves showed the average activity of the CLCuMV C1 gene promoter was 3- to 5-fold higher than that of the CaMV 35S promoter, with maximal expression being 10-fold higher. CLCuMV V1 gene promoter activity was only about 1/10th that of the CaMV 35S promoter in the absence of trans-activator C2. Histochemical GUS staining of the transgenic plants indicated that the CLCuMV C1 gene promoter was active in leaves, stems, roots and almost all reproductive organs. Functional analysis of promoter 5'-deletion series indicated that promoter activity of a 257 nucleotide fragment (-257 to the transcription initiation site) and a 241 nucleotide fragment (-241 to the transcription initiation site) were 5-fold and 2-fold stronger than that of the full-length CLCuMV C1 promoter respectively. These results demonstrate that the CLCuMV C1 promoter is a super-strong near-constitutive promoter in plants and has great application potential for plant genetic engineering studies.

Base Sequence↗

PubMed2003

[Post-transcriptional gene silencing--a plant defense strategy to viruses invasion].

Gene silencing is a kind of epigenetic phenomenon discovered in transgenic plants in recent years. Gene silencing can be divided into two types: position effect and homology dependent gene silencing (HdGS). Homology-dependent gene silencing (HdGS), which is the generic term for transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS) and RNA-mediated virus-resistance (RmVR) have been shown to frequently occur in transgenic plants. Post-transcriptional gene silencing (PTGS) is an antiviral defense system of plants, which has led to a new understanding of the relationship between viruses and plants. And it's a co-evolution result between plants and viruses. A few hypothesis models have been proposed to elucidate the mechanism of gene silencing, but they could not explain all the phenomena in gene silencing. In this paper, we reviewed the relationship between PTGS and the plants defense mechanism to virus, and proposed a model of gene silencing based on our experiments results.

Gene Silencing↗

PubMed2003

Purification and immunity analysis of recombinant 6His-HPT protein expressed in E. coli.

OBJECTIVE: To obtain HPT protein (Hygromycin B Phosphotransferase), a kind of plant selective maker gene product expressed from E. coli and to prepare the polyclonal antibody (pAbs) against it. METHODS: HPT cDNA fragment was obtained by PCR and was inserted into the prokaryotic expressing vector pBV222. Then the constructed recombinant plasmid pBV222-HPT was transferred into E. coli DH5alpha for HPT expression. The recombinant expressing system was confirmed by restriction endonuclease digestion, DNA sequencing and protein expression. E. coli cells were lysed by sonication and detergent dissolution. After cell membrane was extracted, the inclusion bodies were denatured by 8 mol/L Urea and purified with metal chelate affinity chromatography on Ni-NTA agarose under denaturing condition. The purified 6His-HPT was characterized by SDS-PAGE, and used to immunize rabbit. The titer and specificity of antisera were detected by ELISA and Western blot respecitively. RESULTS: Analysis of DNA sequence and restricted enzymes showed that the sequence of PBV222-HPT plasmid was correct. The amount of recombinant HPT expressed in E. coli accounted for 30% of total cellular proteins. From 1 liter of fermentative bacteria about 22 milligrams of pure recombinant HPT was isolated with purity above 95%. The recombinant HPT protein could produce high titer antiserum in rabbits and show good immunity activity. Western blot showed specific binding reaction between the antiserum to the purified 6His-HPT protein and their expressed products (plants protein and bacterial protein). CONCLUSION: HPT protein can be expressed and purified from E. coli by a relatively simple method, which has high immunity activity.

DNA, Bacterial↗

PubMed2003

[Study on transgenic elite indica container line D297B containing foreign insect-resistant gene gna].

It was reported in this article that the transgenic elite indica container line D297B containing snowdrop lectin (Galanthus nivalis agglutinin, GNA) gene gna was obtained by biolisties. PCR, Southern blotting and Western blotting indicated that the transgenes were integrated into the genome of D297B and expressed in transgenic plants. Analysis of protein activity showed that product of transgene had activity of agglutinin. Resistance of transgenic seeds to Hygromycin B suggested that the transgenes were integrated in a single locus and inherited according to the pattern of 3:1 in most of transgenic plants. In addition, the PCR analysis revealed that the insect-resistant gene gna and selective marker gene hpt were co-integrated and co-inherited.

Animals↗

PubMed2003

[Study on the preparing of polyclonal antibodies against plant-selected maker gene hpt expression protein by DNA immunization].

AIM: To prepare the polyclonal antibodies (pAbs) against HPT, a kind of plant-selected maker gene encoding protein, by DNA immunization technique and explore the influencing factors for this gene immunization. METHODS: The coding sequence of hpt was cloned into eukaryotic expression vector pCDNA3. The sequence of the plasmid pCDNA3-HPT was demonstrated by restricting enzyme digestion analysis and DNA sequencing. The sequence-correct recombinant plasmids were purified and used to immunize BALB/c mice. The titer and specificity of antisera were detected by ELISA and Western blot, respecitively. RESULTS: No pAb against HPT was detected following three immunization with hpt genes. Then mice were divided into three groups when the forth booster immunization was carried out: 1st group (immunization with the endotoxin-free recombinant plasmids), 2nd group (immunization with (His)(6)-HPT fusion protein expressed in E.coli) and 3rd group (immunization with the same plasmids as before mentioned). As a result, the pAb titer of the 1st group mice increased to 1:200, and the that of 2nd group was up to 1:2 000. Yet the 3rd group detected no anti-HPT antibody. Western blot analysis had proved that antisera of the first two groups could produce specific binding reaction to the purified GST-HPT, (His)(6)-HPT protein and their expressed product (bacterial protein). CONCLUSION: We have got successfully the specific pAb against HPT by DNA immunization, but its titer is yet unsatisfactory, inferring that the character of the hpt gene self and its expression level may play an important role. In order to raise level of serum antibody prepared by DNA immunization, the farther study on various influencing factors still need to be performed.

Animals↗

PubMed2003

[Fusion expression, purification and bioactivity assay of CpTI in Escherichia coli].

CpTI (Cowpea Trypsin Inhibitor) is a widely used insect resistance gene in the plant genetic engineering for its high insecticidal activity and the minimal ability of the insects to evolve resistance to it. To facilitate the safety assessment of genetically modified foods (GMFs) with CpTI protein, we need to produce gram quantities of this protein in microbes. With the pGEX fusion expression system, we expressed the GST-CpTI protein in E. coli BL21, which accounted for approximately 40% of germ proteins. By Glutathione Sephrose 4B affinity chromatography, GST-CpTI was obtained with the purity up to 90%. Overnight incubate the fusion proteins with Thrombin protease, we got the CpTI proteins cleavage of GST tag. Both of the GST-CpTI and CpTI proteins showed notable trypsin inhibitor activity. Immunization of rabbits with purified fusion protein generated high titer antibodies (> 20000), measuring by ELISA. Western Blotting also showed specific Ag-Ab binding band between the antiserum and the CpTI proteins no matter in the whole supersonic germ proteins or purified from the column. All these made a good ground for the further safety assessment of CpTI protein.

Animals↗

PubMed2003

[Relativity betweeen expression level of potato virus X coat protein and the codon usage of wobble codon].

The relationship between the quantities of rare codons and the expression levels of foreign genes is very important in gene engineering. But up to now, there is few experiment on it in multi-cellular eukaryotes, especially in plants. To study the effect of rare codons on the expression level of potato virus X (PVX) coat protein (CP) in transgenic tobacco, some preferred codons in PVX coat protein gene were substituted with synonymous rare codons without changing the encoded amino acid sequence using site-specific mutation. The modified coat protein gene (cpm) and non-modified coat protein gene (cpw) were inserted into binary vector under the control of CaMV35S promoter, and these two plant expression constructs were transferred into tobacco (Nicotiana tabacum cv. Xanthi) genomes via Agrobacterium mediated method and transgenic plants were generated. Western blot and ELISA of these transgenic tobaccos showed that the expression level of modified CP is about one third to one sixth of that of non-modified CP. Northern blot analysis of RNAs from some transformants indicated that the transcriptional levels of cpm are the same as that of cpw, which implicated that the quantity of rare codons in foreign gene was a possible limited factor in foreign gene expression process. Changing the quantity of rare codons maybe an effective method for organism to control gene expression.

Amino Acid Sequence↗

PubMed2002

Relaxed alignment tolerance of monolithic ring lasers.

An investigation of relaxed alignment tolerance in monolithic ring lasers is presented. It was found that the relaxed alignment tolerance is strongly dependent on the out-of-plane angle. A preferred region for the out-of-plane angle is proposed. In this region the resonator has greatly relaxed alignment tolerances, and the unidirectional operation can be obtained under a much-lower magnetic field. Two lasers have been constructed in the region, and satisfactory performance was found, in good agreement with the theoretical analysis.

Journal Article↗

PubMed2002

A draft sequence of the rice genome (Oryza sativa L. ssp. indica).

We have produced a draft sequence of the rice genome for the most widely cultivated subspecies in China, Oryza sativa L. ssp. indica, by whole-genome shotgun sequencing. The genome was 466 megabases in size, with an estimated 46,022 to 55,615 genes. Functional coverage in the assembled sequences was 92.0%. About 42.2% of the genome was in exact 20-nucleotide oligomer repeats, and most of the transposons were in the intergenic regions between genes. Although 80.6% of predicted Arabidopsis thaliana genes had a homolog in rice, only 49.4% of predicted rice genes had a homolog in A. thaliana. The large proportion of rice genes with no recognizable homologs is due to a gradient in the GC content of rice coding sequences.

Arabidopsis↗

PubMed2002

[Obtaining stem borer-resistant homozygous transgenic lines of Minghui 81 harboring novel cry1Ac gene via particle bombardment].

A modified cry1Ac gene was generated by fusing with Lys-Asp-Glu-Lue (KDEL), an endoplasmic reticulum retention signal at the 3'-ends, with signal peptide coding sequence of Soybean kunitz trypsin inhibitor (SKTI) at the 5'-ends. Vector containing the modified cry1Ac gene coding region flanked by the corn ubiquitin 1 promoter and the nopaline synthase gene (nos) terminator with Hygromycin Phosphotransferase (hpt) gene as a plant selection marker was constructed. The modified cry1Ac gene in which toxic protein targeted to endoplasmic retention was successfully transferred into Minghui 81 (Oryza sativa L. subsp. indica), an elite restoring line of commercial CMS indica hybrid rice, through particle bombardment and obtained fertile transformants. Homozygous transgenic rice lines were obtained in the third generation exploiting self-seed set reproduction and HygromycinB selection. These transgenic lines were confirmed with polymerase chain reaction (PCR) amplification, Southern blotting and ELISA detection. Pest insect-resistant bioassay indicated that some of the homozygous cry1Ac-transgenic rice plants of T2 progeny showed high-level resistance against striped stem borer (Chilo suppressalis) at field trials.

Animals↗

PubMed2002

[Anther culture generated stem borer-resistance DH lines of Minghui 81(Oryza sativa L. subsp. indica) expressing modified cry1Ac gene].

2600 Anthers from T0 modified cry1 Ac-transgenic rice lines of Minghui 81, an elite restoring line of commercial CMS indica hybrid rice, were cultured on SK3 media. 83 green plantlets were recovered, 43 double haploid (DH) and 40 haploid among them. Results of PCR analyzes indicated that 55 plants of 83 were harbored the cry1Ac gene, and the ratio of cry1Ac-positive against cry1Ac-negative was 2:1 (55/28). 36 putative transgenic DH plants were further confirmed by Southern blot. ELISA detection showed that Cry1Ac level in different transgenic rice plants of the same cry1Ac-DH clone was almost equal and the highest one amount to 0.25% of the total soluble protein. Pest insect-resistant bioassay at field trials demonstrated that some of the homozygous cry1Ac-transgenic rice plants not only showed high-level resistance against striped stem borer (Chilo suppressalis) but also retained elite agronomy characters. These results demonstrated that rice anther culture has a great value in rice molecular breeding.

Animals↗

PubMed2002

[Transformation of Bt-CpTi fusion protein gene to cabbage (Brassica oleracea var. capitata) mediated by Agrobacterium tumefaciens and particle bombardment].

Bt-CpTI fusion protein gene was transferred to the explants of hypocotyl, cotyledon with petiole and shoot apex of cabbage (Brassica oleracea L. var. capitata) variety "Zhonggan No 8" via Agrobacterium tumefaciens and particle bombardment, and 13 kanamycin-resistant plants were obtained. PCR and Southern blotting hybridization verified that all these plants of the kanr plants of type I regenerated from hypocotyl and cotyledon with petiole mediated by A. tumefaciens were transgenic plants, 2 karr plants of type II stemed from shoot apex mediated by A. tumefaciens were "false-positive"plants and one of 2 kanr plants of type III regenerated from shoot apex via particle bombardment was non-transformed plants. It was showed that part of transgenic plants had high activity of trypsin inhibitor and strong resistance to resist common cabbage worm through the analysis of CpTI relative capacity and insect-resistant test.

Agrobacterium tumefaciens↗

PubMed2002

[Advances in the studies of plant basic leucine zipper (bZIP) proteins (B)--DNA-binding property, gene expression, function and application].

Plant basic leucine zipper (bZIP) proteins play an important role in the expression and regulation of higher plant genes. The DNA-binding properties of plant bZIP protein are introduced first in this article. Then their expression and function are discussed. Finally, their application to the studies of molecular biology and genetic engineering is reviewed.

English Abstract↗

PubMed2002

[Plant lectin and its application in insect-resistant plant genetic engineering].

Plant lectins are proteins possessing at least one non-catalytic domain that binds reversibly to specific mono-or oligosaccharides. They distinguish themselves from other plant proteins by the ability of carbohydrate binding. Most plant lectins are directed to bind foreign polysaccharide. Plant lectin is believed to take part in the defense responses against invader. In this paper we presented a review on the classification, characters, functions, crystal structure and,functions related to the carbohydrate binding activity. The status and prospect of plant lectins utilization were also discussed.

English Abstract↗