Biomedical subjects

Zan-Min Hu

Publications and source records attributed to Zan-Min Hu.

6 recordsLinked to original sources

PubMed2006

Transgenic wheat progeny resistant to powdery mildew generated by Agrobacterium inoculum to the basal portion of wheat seedling.

To improve the transformation efficiency of wheat (Triticum aestivum L.) mediated by Agrobacterium tumefaciens, we explored the possibility of employing the basal portion of wheat seedling (shoot apical meristem) as the explants. Three genotypes of wheat were transformed by A. tumefaciens carrying beta-1, 3-glucanase gene. After vernalization, the seeds to be transformed were germinated. When these seedlings grew up to 2 approximately 5 cm, their coleoptile and half of the cotyledon were cut out, and the basal portions were infected by A. tumefaciens. A total 27 T(0) transgenic plants were obtained, and the average transformation efficiency was as high as 9.82%. Evident segregation occurred in some of the T(1) plants, as was indicated by PCR and Southern blotting analysis. Investigation of the T(2) plants revealed that some transformed plants had higher resistance to powdery mildew than the controls. Northern blotting revealed that beta-1, 3-glucanase gene was normally expressed in the T(2) plants, which showed an increased resistance to powdery mildew. The results above indicate that the exogenous gene has been successfully integrated into the genome of wheat, transmitted and expressed in the transgenic progeny. From all the results above, it can be concluded that Agrobacterium inoculum to the basal portion of wheat seedling is a highly efficient and dependable transformation method. It can be developed into a practicable method for transfer of target gene into wheat.

Agrobacterium tumefaciens↗

PubMed2006

Chloroplast transformation.

In this chapter we briefly review the developmental history and current research status of chloroplast transformation and introduce the merits of chloroplast transformation as compared with the nuclear genome transformation. Furthermore, according to the chloroplast transformation achieved in oilseed rape (Brassica napus), we introduce the preparation of explants, transformation methods, system selection, identification methods of the transplastomic plants, and experimental results. The technical points, the bottleneck, and the further research directions of the chloroplast transformation are discussed in the notes.

Biological Assay↗

PubMed2006

[Regeneration of transgenic Lespedeza thunbergii expressing levansucrase gene (Sac B) from Bacillus subtilis].

The Bacillus subtilis Sac B gene with the vacuolar targeting signal sequence driven by 35S promotor was transferred into Lespedeza thunbergii by Agrobacterium mediated method. Total 62 Kan-resistant plants were obtained, of which 5 plants were proved to be transgenic plants. The transgenic plants were characterized by PCR amplification, PCR-Southern hybridization and RT-PCR. The physiological assay results showed that the transgenic plants were more tolerant to stress than the controls under the condition of 200mmol/L NaCl and 5% PEG, respectively, and that the content of soluble sugar in trnsgenic plants was significantly higher than that of controls in the period of tests (5-15 days) under salt and PEG stress.

Bacillus subtilis↗

PubMed2005

Cloning of resistance gene analogs located on the alien chromosome in an addition line of wheat-Thinopyrum intermedium.

Homology-based gene/gene-analog cloning method has been extensively applied in isolation of RGAs (resistance gene analogs) in various plant species. However, serious interference of sequences on homoeologous chromosomes in polyploidy species usually occurred when cloning RGAs in a specific chromosome. In this research, the techniques of chromosome microdissection combined with homology-based cloning were used to clone RGAs from a specific chromosome of Wheat-Thinopyrum alien addition line TAi-27, which was derived from common wheat and Thinopyrum intermedium with a pair of chromosomes from Th. intermedium. The alien chromosomes carry genes for resistance to BYDV. The alien chromosome in TAi-27 was isolated by a glass needle and digested with proteinase K. The DNA of the alien chromosome was amplified by two rounds of Sau3A linker adaptor-mediated PCR. RGAs were amplified by PCR with the degenerated primers designed based on conserved domains of published resistance genes (R genes) by using the alien chromosome DNA, genomic DNA and cDNA of Th. intermedium, TAi-27 and 3B-2 (a parent of TAi-27) as templates. A total of seven RGAs were obtained and sequenced. Of which, a constitutively expressed single-copy NBS-LRR type RGA ACR 3 was amplified from the dissected alien chromosome of TAi-27, TcDR 2 and TcDR 3 were from cDNA of Th. intermedium, AcDR 3 was from cDNA of TAi-27, FcDR 2 was from cDNA of 3B-2, AR 2 was from genomic DNA of TAi-27 and TR 2 was from genomic DNA of Th. intermedium. Sequence homology analyses showed that the above RGAs were highly homologous with known resistance genes or resistance gene analogs and belonged to NBS-LRR type of R genes. ACR 3 was recovered by PCR from genomic DNA and cDNA of Th. intermedium and TAi-27, but not from 3B-2. Southern hybridization using the digested genomic DNA of Th. intermedium, TAi-27 and 3B-2 as the template and ACR 3 as the probe showed that there is only one copy of ACR 3 in the genome of Th. intermedium and TAi-27, but it is absent in 3B-2. The ACR 3 could be used as a specific probe of the R gene on the alien chromosome of TAi-27. Results of Northern hybridization suggested that ACR 3 was constitutively expressed in Th. intermedium and TAi-27, but not 3B-2, and expressed higher in leaves than in roots. This research demonstrated a new way to clone RGAs located on a specific chromosome. The information reported here should be useful to understand the resistance mechanism of, and to clone resistant genes from, the alien chromosome in TAi-27.

Amino Acid Sequence↗

PubMed2004

Screening and analysis of differentially expressed genes from an alien addition line of wheat Thinopyrum intermedium induced by barley yellow dwarf virus infection.

The alien addition line TAI-27 contains a pair of chromosomes of Thinopyrum intermedium that carry resistance against barley yellow dwarf virus (BYDV). A subtractive library was constructed using the leaves of TAI-27, which were infected by Schizaphis graminum carrying the GAV strain of BYDV, and the control at the three-leaf stage. Nine differentially expressed genes were identified from 100 randomly picked clones and sequenced. Two of the nine clones were highly homologous with known genes. Of the remaining seven cDNA clones, five clones matched with known expressed sequence tag (EST) sequences from wheat and (or) barley whereas the other two clones were unknown. Five of the nine differentially expressed sequences (WTJ9, WTJ11, WTJ15, WTJ19, and WTJ32) were highly homologous (identities >94%) with ESTs from wheat or barley challenged with pathogens. These five sequences and another one (WTJ18) were also highly homologous (identities >86%) with abiotic stress induced ESTs in wheat or barley. Reverse Northern hybridization showed that seven of the nine differentially expressed cDNA sequences hybridized with cDNA of T. intermedium infected by BYDV. Three of these also hybridized with cDNA of line 3B-2 (a parent of TAI-27) infected by BYDV. The alien chromosome in TAI-27 was microdissected. The second round linker adaptor mediated PCR products of the alien chromosomal DNA were labeled with digoxygenin and used as the probe to hybridize with the nine differentially expressed genes. The analysis showed that seven differentially expressed genes were homologous with the alien chromosome of TAI-27. These seven differentially expressed sequences could be used as ESTs of the alien chromosome of TAI-27. This research laid the foundation for screening and cloning of new specific functional genes conferring resistance to BYDV and probably other pathogens.

Blotting, Northern↗

PubMed2003

Chloroplast transformation in oilseed rape.

The chloroplast transformation vector pNRAB carries two expression cassettes for the spectinomycin resistance gene aadA and the insect resistance gene cry1Aa10. The two cassettes are sited between the rps7 and ndhB targeting fragments. Biolistic delivery of the vector DNA, followed by spectinomycin selection, yielded chloroplast transformants at a frequency of four in 1000 bombarded cotyledon petioles. PCR analysis and Southern blot of PCR products confirmed the site-specific integration of aadA and cry1Aa10 into the chloroplast genomes of transgenic oilseed rape. When transgenic oilseed rape leaves were fed to second instar Plutella xylostera larvae, 47% mortality was observed against this insect and the surviving larvae had significantly lower weight than the control. This is the first report of chloroplast transformation in oilseed rape and the introduction of novel genes between the rps7 and ndhB genes in the chloroplast genome. This offers an opportunity for improvement of oilseed rape by chloroplast genetic engineering.

Anti-Bacterial Agents↗