小麦WPBF与高分子量谷蛋白基因上游Prolamin-Like box的特异结合

【目的】克隆普通小麦胚乳特异表达转录因子基因WPBF,对其原核表达条件进行优化,并验证其编码蛋白和小麦高分子量谷蛋白亚基基因(HMW-GS)上游Prolamin-Like box的结合特性,为小麦HMW-GS表达调控机制的阐明奠定基础。【方法】利用RT-PCR扩增得到WPBF编码区,将其连接到pET-21a-MBP载体中,并分别在E.coliT7 Express和Origami B(DE3)菌株中诱导表达。依次利用Amylose和Ni-NTA亲和层析柱纯化重组蛋白,最后通过凝胶迁移阻滞试验(EMSA)对重组蛋白的DNA结合特性进行分析。【结果】在33℃的Origami B(DE3)菌株中表达含MBP和His双标签的重组WPBF大部分以可溶性形式存在,其表达量占细菌总蛋白的48.0%。重组WPBF和含HMW-GS上游Prolamin-Like box的两类DNA探针能发生特异结合,但重组WPBF和含"TGCAAAG"基序DNA探针的结合强度高于其和含"TGCAAG"基序DNA探针的结合。【结论】建立了优化的WPBF原核表达系统,并获得具有生物活性的重组蛋白;重组WPBF能与HMW-GS启动子来源的两种Prolamin-Like box特异性结合,但结合能力存在差异。

哺乳动物Jun和Fos基因增加水稻prolamin 4a基因启动子的表达

本文通过共转化和瞬间表达的方法,报道了哺乳动物的转录调节因子Jun和Fos基因对天然的水稻prolamin 4a基因启动子(该启动子含有Apl结合位点)的调节作用。这是哺乳动物的转录因子作用于水稻启动子的首次报道。

Genetic Diversity and Classification of Chinese Elite Foxtail Millet [Setaria italica (L.) P. Beauv.] Revealed by Acid-PAGE Prolamin

Arid and semi-arid regions of China account for more than half of the country. Because of drought resistance and high nutritive value, elite foxtail millet (Setaria Italica (L.) P. Beauv.) is one of the most important cereal crops in China. Evaluation of germplasm and genetic diversity of foxtail millet is still in its infancy, but prolamin could play an important role as a protein marker. To investigate the genetic diversity and population structure of foxtail millet from different ecological zones of China, 90 accessions of foxtail millet were collected from three major ecological areas: North, Northwest, and Northeast China. The prolamin contents were examined by acid polyacrylamide gel electrophoresis (acid-PAGE). Five to twenty-two prolamin bands appeared in tested varieties, of which were polymorphic, so prolamin patterns of foxtail millet varieties can be used in variety identification and evaluation. Structure analysis identified six groups, which matches their pedigree information but not their geographic origins. This indicated a high degree (87.78%) of consistency with a phylogenetic classification based on SSR. The results showed prolamin banding patterns were an effective method for analyzing foxtail millet genetic variability.

Disulphide Linkages Occur in Many Polypeptides of Rice Protein Fractions: a Two-dimensional Gel Electrophoretic Study

Most of the work on rice storage proteins focused on the major fraction - glutelin. In contrast to previous reports of the occurrence of single legumin-like polypeptide pair in glutelin, two-dimensional gel electrophoresis revealed many glutelin bands, with some having and some lacking disulphide-linkages. Five legumin-like polypeptide pairs exhibited wide heterogeneity over a range of molecular weight （Mr） 25 to 60 kDa, each having a large subunit （Mr ranged from 18 to 40.5 kDa） disulphide-bonded to a small subunit （Mr ranged from 16.5 to 18.0 kDa）. A band of 49 kDa was homodimeric with two subunits of 29 kDa each; a polypeptide of 51 kDa which altered position to ones corresponding to 53 kDa and 57 kDa on 2-D gels contained intrapolypeptide linkages. Polypeptides of 65 kDa and 60 kDa occurred as aggregates of 110 kDa. The number of polypeptides in other seed protein fractions albumins, globulins and prolamins, varied from four to ten. One of the albumin bands had intrapolypeptide disulphide linkages （20 kDa） and the globulins contained two such bands （13.5 and 20 kDa）. Thus, the present study provides a description of the polypeptide composition of different rice protein fractions that is finely resolved with respect to the occurrence of disulphide linkages.

Studies on the rice prolamine compositions and contents

Polamines are the major seed storage proteinsin most of the sereals. The rice prolaminescontain a high mole percentage of glutamineresidues and low levels of lysine, histidine,cystine, and methionine. Among the fourcomponent of rice prolamines, 16KDa and10KDa contain more sulfate-riched amino acidresidues. So the total prolamine content andthe percentages of each component were im-portant to parameters for the rice storage pro-tein and its nutrient value. Prolamines of 36rice lines were studied with SDS-PAGE, andrelative quantities of 16 KDa, 13 a, 13 b, and

Amplification and sequencing of a sulfur-rich 10kd prolamin gene from rice seeds

Nutritious value of seed storage protein is low due to deficiency in essential amino acid contents. Cereals are mainly deficient in lysine and legumes in sulfur-containing amino acids (methionine and cysteine). So far, several sufur-rich seed protein genes have been isolated and the essential amino acid contents of seed proteins were increased in transgenic tobacco and Brassica napus. In this paper we report the isolation and sequencing of the 10kd prolamin gene from seeds. Poly(A) RNA were prepared from the immature endosperms of japonica rice, Sachiminori, 10 d after flowering. Complementary DNAs were synthesized according to Promega Instruction Manual. Two primers were synthesized and their sequences were Primer Ⅰ: CGTCTACACCATCTGGAATC, Primer Ⅱ: GTGTTTGCAGATAGTATGC. The amplified fragraents were inserted into the Sma I site of pGEM-7zf(+) and was used to transform E. coli JM101 after PCR reaction. DNA sequence were determined by Sanger’s Chain-termination method. Synthesis of cDNA. Using mRNAs as

Rice seed storage proteins:Biosynthetic pathways and the effects of environmental factors

Rice(Oryza sativa L.)is the most important food crop for at least half of the world’s population.Due to improved living standards,the cultivation of high-quality rice for different purposes and markets has become a major goal.Rice quality is determined by the presence of many nutritional components,including seed storage proteins(SSPs),which are the second most abundant nutrient components of rice grains after starch.Rice SSP biosynthesis requires the participation of multiple organelles and is influenced by the external environment,making it challenging to understand the molecular details of SSP biosynthesis and improve rice protein quality.In this review,we highlight the current knowledge of rice SSP biosynthesis,including a detailed description of the key molecules involved in rice SSP biosynthetic processes and the major environmental factors affecting SSP biosynthesis.The effects of these factors on SSP accumulation and their contribution to rice quality are also discussed based on recent findings.This recent knowledge suggests not only new research directions for exploring rice SSP biosynthesis but also innovative strategies for breeding high-quality rice varieties.

5'-flanking Region Responsible for Endospermspecific Expression of Rice Prolamin Chimeric Gene in Transgenic Tobacco

The 5' upstream region (—680—+40) containing the complete signal peptide coding sequence of the rice seed storage prolamin gene was amplified in vitro with polymerase chain reaction from the genome of Chinese super rice cultivar Zhonghua 8. Physical map and DNA sequence analysis showed strong homology with the 5'-flanking region of rice prolamin gene reported by Kim in 1988. No changes in the signal peptide coding sequence and a long leader sequence with several small ORFs were found. Chimeric gene containing 5'-flanking region of the prolamin gene has been transcriptionally fused with the β-glucuronidase reporter gene. The fusion junction was confirmed by both physical map and DNA sequence analysis. The resultant chimeric gene was used to transform the tabacco explants by Ti binary system of Agrobacterium tumefaciens LBA4404. With dot and Southern blotting hybridization, three transgenic tobacco plants with the copies of chimeric GUS genes as many as 20 were obtained. Histochemical analysis revealed that the GUS activity in the endosperm tissues of tobacco seeds at the developmental stage was about 20 DAP. No GUS activity was found in leaves, stems, roots and flowers of the transgenic tobacco plants. Therefore, we concluded that the 5'-upstream cis-elements from—680 to—18 were enough to confer the endosperm-specific and temporal expression of rice prolamin gene.

水稻种子贮存蛋白Prolamin 4a基因的组织特异性表达调控

本文分离了水稻中花8号种子贮存蛋白Prolamin基因的5′端调控区域,对其DNA的全序列结构作了分析,表现启动子DNA的典型序列结构。构建了GUS融合基因,利用农杆菌LBA4404双元载体系统转化烟草,分子杂交获得转基因烟草植株,在转基因烟草开花20天时,对烟草种子作GUS基因表达的组织化学分析。实验结果表明Prolamin基因的启动子在种子的胚乳中激活下游GUS基因的表达,这是水稻Prolamin基因5′端调控序列功能的首次报道。

Specialized endoplasmic reticulum-derived vesicles in plants:Functional diversity,evolution,and biotechnological exploitation

A central role of the endoplasmic reticulum(ER)is the synthesis,folding and quality control of secretory proteins.Secretory proteins usually exit the ER to enter the Golgi apparatus in coat protein complex II(COPII)-coated vesicles before transport to different subcellular destinations.However,in plants there are specialized ER-derived vesicles(ERDVs)that carry specific proteins but,unlike COPII vesicles,can exist as independent organelles or travel to the vacuole in a Golgi-independent manner.These specialized ERDVs include protein bodies and precursor-accumulating vesicles that accumulate storage proteins in the endosperm during seed development.Specialized ERDVs also include precursor protease vesicles that accumulate amino acid sequence KDEL-tailed cysteine proteases and ER bodies in Brassicales plants that accumulate myrosinases that hydrolyzes glucosinolates.These functionally specialized ERDVs act not only as storage organelles but also as platforms for signal-triggered processing,activation and deployment of specific proteins with important roles in plant growth,development and adaptive responses.Some specialized ERDVs have also been exploited to increase production of recombinant proteins and metabolites.Here we discuss our current understanding of the functional diversity,evolutionary mechanisms and biotechnological application of specialized ERDVs,which are associated with some of the highly remarkable characteristics important to plants.