Expression Analysis of HMW-GS 1Bx14 and 1By15 in Wheat Varieties and Transgenic Research of 1By15 Gene

High-molecular-weight glutenin subunits （HMW-GSs）, one class of seed storage proteins in wheat, play an important role in determining bread-making quality of flour. More and more proves support that HMW-GS- 1Bx 14 and - 1Bx 15 subunits are strongly positively associated with good bread-making and excellent noodle-making quality. The two subunits are encoded by two genes, Glu-1Bx14 and Glu-1Bx15, which are tightly linked and located on the 1BL. Protein assay by SDS-PAGE indicated that the expression of Glu-1Bx14 gene was always much stronger than that of Glu-1By15 in the same variety. But, variation of expression level for Glu-1By15 gene existed among varieties, such as in Xiaoyan 54, Xiaoyan 6, Yanzhan 1 and Shanyou 225. We also investigated the transcription difference of Glu-1By14 and Glu-1By15 genes in Xiaoyan 54 and Shanyou 225 by semi-quantitative RT-PCR method. The Glu-1By14 always transcripts much more than the Glu-1By15. This was basically consistent with the translation difference between the two genes. Promoters of 1Bx14, 1By15, 1By8, 1Dx2 and 1Dy12 were cloned from Xiaoyan 54, Chinese Spring and Aegilops tauschii. Sequence analysis indicated that the HMW-GS genes had high homology at their promoter regions. However, significant difference existed between sequence of 1Bx14 promoter and those of other HMW-GS genes. The transient expression experiment showed that the promoter of 1By15 has lower activity than that of 1Bx14, which was consistent with their transcription level of the two genes in varieties. In addition, transient expression of the gus driven by the promoter （P2） of HMW-GS 1Dx2 gene was higher than by other HMW-GS promoters. Therefore, we constructed 1By15 gene expression vector driven by the 1Dx2 promoter, and transformed the 1By15 gene into wheat commercial variety, Jimai 20 by pollen tube method. Of 45 independent transgenic lines identified by PCR, 3 were confirmed to contain the HMW-GS 1By15 gene via Southern hybridization. The delivered 1By15 gene expressed the expected HMW-GS protein in the seeds of transgenic plants.

Assessment of Lipid Transfer Protein (LTP1) Gene in Wheat Powdery Mildew Resistance

This study is to investigate the role of lipid transfer protein （LTP1） gene of wheat （Triticum aestivum L.） in powdery mildew （Blumeria graminis f.sp. tritici, Bgt） resistance. A pair of primers based on the full length cDNA of wheat LTP1 was used for amplifying the coding regions of LTP in hexaploid （AABBDD） wheat and its diploid donors T. urartu （AA）, Ae. speltoides ssp speltoide （SS） and Ae. tauchii ssp strangulate （DD）. LTP1 and LTP2 of wheat were isolated from the tested two hexaploid （ABD） materials： powdery mildew resistance near isogenic line （NIL） Mardler/7 × Bainong 3217 and its susceptible parent Bainong 3217 at the same time, while only one kind ofLTP gene was found in the tested three diploid materials respectively by using the above PCR primer pairs. Two peaks of the expression of LTP1 and LTP2 induced by powdery mildew were observed [one occurred at 3 h after inoculation （hai）; the other occurred at 10 hai] in resistant NIL Mardler/7 × Bainong3217 in comparison with a steady transcript level of LTP1 and LTP2 in susceptible Bainong3217. Transient over-expression result showed that LTP1 reduced the penetration efficiency （PE） of powdery mildew in susceptible cultivar by about 28.3%. This result indicated an obvious effectiveness of LTP1 in powdery mildew resistance. Expression analysis also showed that LTP1 and LTP2 of wheat are generally involved in salt/drought, but not in low temperature stress early responses.

A screening assay for thyroid hormone signaling disruption based on thyroid hormone-response gene expression analysis in the frog Pelophylax nigromaculatus

Amphibian metamorphosis provides a wonderful model to study the thyroid hormone（TH）signaling disrupting activity of environmental chemicals, with Xenopus laevis as the most commonly used species. This study aimed to establish a rapid and sensitive screening assay based on TH-response gene expression analysis using Pelophylax nigromaculatus, a native frog species distributed widely in East Asia, especially in China. To achieve this, five candidate TH-response genes that were sensitive to T3 induction were chosen as molecular markers, and T3 induction was determined as 0.2 nmol/L T3 exposure for 48 hr. The developed assay can detect the agonistic activity of T3 with a lowest observed effective concentration of 0.001 nmol/L and EC50 at around 0.118–1.229 nmol/L, exhibiting comparable or higher sensitivity than previously reported assays. We further validated the efficiency of the developed assay by detecting the TH signaling disrupting activity of tetrabromobisphenol A（TBBPA）, a known TH signaling disruptor. In accordance with previous reports, we found a weak TH agonistic activity for TBBPA in the absence of T3,whereas a TH antagonistic activity was found for TBBPA at higher concentrations in the presence of T3, showing that the P. nigromaculatus assay is effective for detecting TH signaling disrupting activity. Importantly, we observed non-monotonic dose-dependent disrupting activity of TBBPA in the presence of T3, which is difficult to detect with in vitro reporter gene assays. Overall, the developed P. nigromaculatus assay can be used to screen TH signaling disrupting activity of environmental chemicals with high sensitivity.