Hongmei Qian

Expression and analysis of thymosin α1 concatemer in Escherichia coli

Tα1 (thymosin α 1) is important in treating immunodeficiency and other diseases. In order to study the feasibility of expressing Tα1 in plants, as the first attempt, we designed and synthesized the Tα1 gene according to the plant codon usage preference and constructed the 4×Tα1 concatemer (four copies of a DNA sequence arranged end‐to‐end in tandem). The latter was inserted into Escherichia coli expression vector pQE30, resulting in a recombinant plasmid that was subsequently transformed into E. coli M15. The 4×Tα1 concatemer protein was successfully expressed in E. coli in a soluble form. The expressed protein was purified and its bioactivity was analysed by MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide] assay. Preliminary results showed that the 4×Tα1 concatemer protein could stimulate the mice spleen lymphocyte proliferation. This is the first report on the expression of 4×Tα1 concatemer that was synthesized according to plant codon usage preference in an E. coli expression system. The present study provides the basis for expressing the synthesized active Tα1 gene in plants in the future.

Transgenic tobacco plants expressingBoRS1 gene fromBrassica oleracea var.acephala show enhanced tolerance to water stress

Water stress is by far the leading environmental stress limiting crop yields worldwide. Genetic engineering techniques hold great promise for developing crop cultivars with high tolerance to water stress. In this study, theBrassica oleracea var.acephala BoRS1 gene was transferred into tobacco throughAgrobacterium- mediated leaf disc transformation. The transgenic status and transgene expression of the transgenic plants was confirmed by polymerase chain reaction (PCR) analysis, Southern hybridization and semi-quantitative one step RT-PCR analysis respectively. Subsequently, the growth status under water stress, and physiological responses to water stress of transgenic tobacco were studied. The results showed that the transgenic plants exhibited better growth status under water stress condition compared to the untransformed control plants. In physiological assessment of water tolerance, transgenic plants showed more dry matter accumulation and maintained significantly higher levels of leaf chlorophyll content along with increasing levels of water stress than the untransformed control plants. This study shows thatBoRS1 is a candidate gene in the engineering of crops for enhanced water stress tolerance.

Molecular cloning and promoter analysis of the specific salicylic acid biosynthetic pathway gene phenylalanine ammonia‐lyase (AaPAL1) from Artemisia annua

Phenylalanine ammonia‐lyase (PAL) is the key enzyme in the biosynthetic pathway of salicylic acid (SA). In this study, a full‐length cDNA of PAL gene (named as AaPAL1) was cloned from Artemisia annua. The gene contains an open reading frame of 2,151 bps encoding 716 amino acids. Comparative and bioinformatics analysis revealed that the polypeptide protein of AaPAL1 was highly homologous to PALs from other plant species. Southern blot analysis revealed that it belonged to a gene family with three members. Quantitative RT‐PCR analysis of various tissues of A. annua showed that AaPAL1 transcript levels were highest in the young leaves. A 1160‐bp promoter region was also isolated resulting in identification of distinct cis‐regulatory elements including W‐box, TGACG‐motif, and TC‐rich repeats. Quantitative RT‐PCR indicated that AaPAL1 was upregulated by salinity, drought, wounding, and SA stresses, which were corroborated positively with the identified cis‐elements within the promoter region. AaPAL1 was successfully expressed in Escherichia. coli and the enzyme activity of the purified AaPAL1 was approximately 287.2 U/mg. These results substantiated the involvement of AaPAL1 in the phenylalanine pathway.

Molecular cloning, characterization, and promoter analysis of the isochorismate synthase (AaICS1) gene from Artemisia annua

Isochorismate synthase (ICS) is a crucial enzyme in the salicylic acid (SA) synthesis pathway. The full-length complementary DNA (cDNA) sequence of the ICS gene was isolated from Artemisia annua L. The gene, named AaICS1, contained a 1710-bp open reading frame, which encoded a protein with 570 amino acids. Bioinformatics and comparative study revealed that the polypeptide protein of AaICS1 had high homology with ICSs from other plant species. Southern blot analysis suggested that AaICS1 might be a single-copy gene. Analysis of the 1470-bp promoter of AaICS1 identified distinct cis-acting regulatory elements, including TC-rich repeats, MYB binding site (MBS), and TCA-elements. An analysis of AaICS1 transcript levels in multifarious tissues of A. annua using quantitative real-time polymerase chain reaction (qRT-PCR) showed that old leaves had the highest transcription levels. AaICS1 was up-regulated under wounding, drought, salinity, and SA treatments. This was corroborated by the presence of the predicted cis-acting elements in the promoter region of AaICS1. Overexpressing transgenic plants and RNA interference transgenic lines of AaICS1 were generated and their expression was compared. High-performance liquid chromatography (HPLC) results from leaf tissue of transgenic A. annua showed an increase in artemisinin content in the overexpressing plants. These results confirm that AaICS1 is involved in the isochorismate pathway.摘要目 的研究青蒿异分支酸合酶的表达模式, 评价其对青蒿素含量的影响。创新点该研究首次克隆了青蒿异分支酸合酶基因(AaICS1), 并发现AaICS1 影响青蒿素的合成, 为更有效地开发利用青蒿提供了新思路。方 法根据青蒿转录组数据, 利用聚合酶链式反应(PCR)克隆AaICS1 基因和启动子, 并进行多重序列分析和启动子作用元件预测。通过实时定量PCR( qRT-PCR ) 对AaICS1 进行表达分析, 用Southern 杂交分析AaICS1 的拷贝数。构建AaICS1过表达载体和干扰表达载体, 转化青蒿获得转基因植株, 用高效液相色谱法(HPLC)分析青蒿素含量。结 论AaICS1 含一个总长为1710 bp 的完整阅读框, 编码570 个氨基酸, 与其它植物的ICS 基因具有较高的相似性。Southern 杂交结果表明AaICS1 为单拷贝(图4), qRT-PCR 结果显示该基因能够响应伤害、干旱、盐胁迫和水杨酸的处理, 处理后基因表达量提高(图6), 和启动子作用元件预测相符。qRT-PCR 结果显示过表达转基因青蒿中AaICS1 表达量提高, 干扰转基因青蒿中该基因表达量降低(图7)。HPLC 显示过表达AaICS1 转基因植株中青蒿素含量提升, 最高可达对照的1.9倍(图8)。