Liquan Zhang

Induced Expression of DREB Transcriptional Factor and Study on Its Physiological Effects of Drought Tolerance in Transgenic Wheat

Expression vector pBAC128F, which carries DREB transcriptional factor gene driven by drought inducing promoter rd29B and bar gene driven by CaMV 35S promoter and maize Adh1 gene first intron, was transferred into the explants of immature inflorescence and immature embryos of hexaploid winter wheat cv. 8901, 5-98, 99-92 and 104 by particle bombardment. More than 70 resistant transgenic plants were obtained. Genomic PCR and RNA dot blotting analyses showed that DREB gene had been integrated into wheat genome of the transgenic plants (T0 and T1) and was well expressed in offspring seed of different transgenic lines. The content of proline in leaves and seeds of T2 transgenic lines was analyzed. Among 16 tested transgenic lines, 10 transgenic lines exhibited more than two fold of proline level in leaves as compared with CK plants. Under drought condition, after stopping water for 15 days the leaves of transgenic lines were still green, while CK were faded. After rewatering for 10 days, the leaves of transgenic lines maintained their green, while all CK plants were dead. Our research suggested that introducing a novel DREB transcriptional factor into wheat is an effective way to improve its drought-tolerance ability.

Transgene inheritance and quality improvement by expressing novel HMW glutenin subunit (HMW-GS) genes in winter wheat

The expression vector pBPC30, which carries the high molecular weight glutenin subunit (HMW-GS)1Dx5 and1Dy10 genes, was transferred into hexaploid winter wheat cv. Jinghua No. 1, Jing411 and Jingdong No. 6 explants of immature embryos and immature inflorescence by particle bombardment. A. large number of resistant transgenic plants were obtained under the selection of herbicide bialaphos or phosphinothricin (PPT). Confirmed transgenic plants of T0 generation showed successful integration of HMW-GS genes and bar gene into the wheat genome. T1 generation of transgenic plants can resist 20–150 mg/L PPT. Protein analysis of T2 seed by SDS-PAGE showed that HMW-GS1Dx5 and1Dy10 genes were well expressed in offspring seed of transgenic lines by co-expression with or substitution of endogenous1Dx2 or1Dy10. In one transgenic line, TG3-74, a new protein band between endogenous protein subunits 7 and 8 (marked as 8*) of glutenin appeared, but endogenous subunit 8 (encoded by lBy8 gene) was absent. Analysis of gluten rheological quality on seed proteins of 102 T3 plants showed that the sedimentation value of 5 transgenic lines (44.2–49.0 mL) was remarkably improved, 59.6%–64.3% higher than that of wild type Jinghua No. 1 and Jingdong No. 6, similar to bread wheat Cheyenne (48.0 mL). Analysis of dough rheological properties of transgenic lines showed that the dough stable time of 5 transgenic lines range from 16 to 30 min, whereas the dough stable time of wild type was only between 3–7 min. Our research suggests that introducing novel HMW-GS genes into wheat is an efficient way to improve its bread-making quality.The expression vector pBPC30, which carries the high molecular weight glutenin subunit (HMW-GS)1Dx5 and1Dy10 genes, was transferred into hexaploid winter wheat cv. Jinghua No. 1, Jing411 and Jingdong No. 6 explants of immature embryos and immature inflorescence by particle bombardment. A. large number of resistant transgenic plants were obtained under the selection of herbicide bialaphos or phosphinothricin (PPT). Confirmed transgenic plants of T0 generation showed successful integration of HMW-GS genes and bar gene into the wheat genome. T1 generation of transgenic plants can resist 20–150 mg/L PPT. Protein analysis of T2 seed by SDS-PAGE showed that HMW-GS1Dx5 and1Dy10 genes were well expressed in offspring seed of transgenic lines by co-expression with or substitution of endogenous1Dx2 or1Dy10. In one transgenic line, TG3-74, a new protein band between endogenous protein subunits 7 and 8 (marked as 8*) of glutenin appeared, but endogenous subunit 8 (encoded by lBy8 gene) was absent. Analysis of gluten rheological quality on seed proteins of 102 T3 plants showed that the sedimentation value of 5 transgenic lines (44.2–49.0 mL) was remarkably improved, 59.6%–64.3% higher than that of wild type Jinghua No. 1 and Jingdong No. 6, similar to bread wheat Cheyenne (48.0 mL). Analysis of dough rheological properties of transgenic lines showed that the dough stable time of 5 transgenic lines range from 16 to 30 min, whereas the dough stable time of wild type was only between 3–7 min. Our research suggests that introducing novel HMW-GS genes into wheat is an efficient way to improve its bread-making quality.

A maize bundle sheath defective mutation mapped on chromosome 1 between SSR markers umc1395 and umc1603

The bsd-pg (bundle sheath defective pale green) mutant is a novel maize mutation, controlled by a single recessive gene, which was isolated from offspring of maize plantlets regenerated from tissue callus of the maize inbred line 501. The characterization was that the biogenesis and development of the chloroplasts was mainly interfered in bundle sheath cells rather than in mesophyll cells. For mapping the bsd-pg, an F2 population was derived from a cross between the mutant bsd-pg and an inbred line Xianzao 17. Using specific locus amplified fragment sequencing (SLAF-Seq) technology, a total of 5 783 polymorphic SLAFs were analysed with 1 771 homozygous alleles between maternal and paternal parents. There were 49 SLAFs, which had a ratio of paternal to maternal alleles of 2:1 in bulked normal lines, and three trait-related candidate regions were obtained on chromosome 1 with a size of 3.945 Mb. For the fine mapping, new simple sequence repeats (SSRs) markers were designed by utilizing information of the B73 genome and the candidate regions were localized a size of 850 934 bp on chromosome 1 between umc1603 and umc1395, including 35 candidate genes. These results provide a foundation for the cloning of bsd-pg by map-based strategy, which is essential for revealing the functional differentiation and coordination of the two cell types, and helps to elucidate a comprehensive understanding of the C4 photosynthesis pathway and related processes in maize leaves.

Expression of the intact C4 typepepc gene cloned from maize in transgenic winter wheat

Maize intactC4-pepc gene was amplified through LA-PCR and successfully sub-cloned into modified vector pGreen0029 to form a stable expression construct named as pBAC214 (12 kb), which contains CaMV 35S promoter drivenbar gene as selection marker. Comparing the cloned DNA sequences (6.7 kb) with published maizeC4-pepc gene (GenBank accession E17154) sequences, the identity of DNA sequence alignment is 98.96%. There are only 49 differences between these two intact DNA sequences, of which 13 occur in the region of promoter, 18 in introns, and 18 in exons. The homology of mRNA sequence alignment is 99.38%, and the putative amino acids sequence identity is 99.38% There are only 15 differences between these two mRNA, and these differences bring 4 sites mutant on the putative amino acids of PEPC protein. Through biolistic bombardment of PDS1000/He system, expression vector pBAC214 has been transformed into winter wheat. Southern blotting results show that the intactC4pepc gene has been integrated into genome of winter wheat. SDS-PAGE analysis of leaf soluble protein in transgenic wheat showed that the intactC4-pepc gene was well transcribed, spliced and translated as in maize. The enzyme activity of leaf PEPC in transgenic wheat has been detected. The activities of leaf PEPC increased over 3–5 times in some transgenic plants. The data of photosynthesis rate and transpiration rate of transgenic wheat flag leaves showed that theC4-pepc gene can increase the photosynthesis rate and transpiration rate of transgenic wheat.

Producing Transformed Maize with Dehydration-Responsive Transcription Factor CBF4 Gene

Abstract The CBF4 gene was cloned from Arabidopsis thaliana and driven by the promoter of preceding stress responsive gene rd29A. By means of biolistic bombardment, this gene was transferred into immature embryo and embryogenic callus of several elite maize (Zea mays L.) inbred lines via expression vector pBAC146. A total of 64 transgenic plants were obtained following callus induction, resistance selection, differentiation, and plant regeneration. The transgene proved to be integrated into maize genome by PCR, PCR-Southern, and Southern blotting analyses. Under the simulated drought condition, one of the transgenic lines showed twice the contents of proline and chlorophyll compared to the wild type. This suggests that drought resistance is improved to some extent in this transgenic line.