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Molecular Biological and Biochemical Studies Reveal New Pathways Important for Cotton Fiber Development

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Journal of Integrative plant Biology 2007年第1期49卷 69-74页

作者： Yu Xu Hong-Bin Li Yu-Xian Zhu national laboratory of protein engineering and plant genetic engineering Peking University Beijing 100871 China

As one of the longest single-celled seed trichomes, fibers provide an excellent model for studying fundamental biological processes such as cell differentiation, cell expansion, and cell wall biosynthesis. In this review, we summarize recent progress in cotton functional genomic studies that characterize the dynamic changes in the transcriptomes of fiber cells. Extensive expression profilings of cotton fiber transcriptomes have provided comprehensive information, as quite a number of transcription factors and enzyme-coding genes have been shown to express preferentially during the fiber elongation period. Biosynthesis of the plant hormone ethylene is found significantly upregulated during the fiber growth period as revealed by both microarray analysis and by biochemical and physiological studies. It is suggested that genetic engineering of the ethylene pathway may improve the quality and the productivity of cotton lint. Many metabolic pathways, such as biosynthesis of cellulose and matrix polysaccharides are preferentially expressed in actively growing fiber cells. Five gene families, including proline-rich proteins （PRP）, arabinogalactan proteins （AGP）, expansins, tubulins and lipid transfer proteins （LTP） are activated during early fiber development, indicating that they may also be needed for cell elongation. In conclusion, we identify a few areas of future research for cotton functional genomic studies.

关键词： cotton fiber functional genomics plant hormone transcriptome transcription factors

Analysis of Five Differentially Expressed Gene Families in Fast Elongating Cotton Fiber

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Acta Biochimica et Biophysica Sinica 2004年第1期36卷 51-56页

作者： Jian-Xun FENG, Sheng-Jian JI, Yong-Hui SHI, Yu XU, Gang WEI, and Yu-Xian ZHU( national laboratory of protein engineering and plant genetic engineering, College of Life Sciences, Peking University, Beijing 100871, China ) national laboratory of protein engineering and plant genetic engineering College of Life Sciences Peking University Beijing 100871 China

Using the suppression subtractive hybridization method, we isolated five gene families, including proline-rich proteins (PRPs), arabinogalactan proteins (AGPs), expansins, tubulins and lipid transfer proteins (LTPs), from fast elongating cotton fiber cells. Expression profile analysis using cDNA array technology showed that most of these gene families were highly expressed during early cotton fiber developmental stages (0-20 days post anthesis, DPA). Many transcripts accumulated over 50-fold in 10 DPA fiber cells than in 0 DPA samples. The entire gene family -AGP, together with 20 individual members in other 4 gene families, are reported in cotton for the first time. Accumulation of cell wall proteins, wall loosening enzymes, microtubules and lipid transfer proteins may contribute directly to the elongation and development of fiber cells.

关键词： cotton fiber cDNA array cell elongation arabinogalactan protein lipid transfer protein

Studies on gene structure, enzymatic activity and regulatory mechanism of acetohydroxy acid isomeroreductase from G2 pea

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Science China(Life Sciences) 2003年第4期46卷 389-398页

作者：徐云剑顾雪松李珺李晴 PeterJ.Davies 朱玉贤 The national laboratory of protein engineering and plant genetic engineering Peking University Department of plant Biology Cornell University Ithaca

The AAIR genomic DNA of G2 pea (Pisum sativum L.) was amplified by PCR method. Sequence analysis showed that it was composed of 8 introns and 9 exons with three of the introns containing specific A/T-rich endogenous promoter regions. Molecular hybridization experiments revealed that the expression of AAIR remained at a high level before and after flowering if grown in short day growth chambers. However, when grown under long day conditions, the level of AAIR expression declined very rapidly after flowering. This variation of AAIR expression is consistent with the change of enzymatic activity of acetohydroxy acid isomeroreductase. Functional complementation experiments carried out using an acetohydroxy acid isomeroreductase deficient E. coli strain showed that these cells could not grow on M9 medium without addition of branched-chain amino acids unless they were transformed with the AAIR expression vector. Further study revealed that overexpression of the pea AAIR cDNA in acetohydroxy acid isomeroreductase deficient E. coli strain enhanced significantly its branched-chain amino acid biosynthetic capacity. Results from gel shift experiments showed that fractions of pea nuclear protein extracts could bind specifically to some A/T rich regions present in introns of the AAIR gene. The A/T-rich-region-binding proteins remained at a steady level in the non-senescing apical buds of short-day grown G2 pea. In the rapid-senescing apical buds of long-day grown G2 pea, the levels of these proteins declined rapidly after flower initiation. Therefore, the nuclear protein binding capacities to endogenous promoter regions may constitute an important mechanism to regulate AAIR gene expression.

关键词： AAIR G2 pea A/T rich region enzymatic activity senescence. D

Recombinant scorpion insectotoxin AaIT kills specifically insect cells but not human cellss

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Cell Research 2002年第2期12卷 143-150页

作者： SHENG JIAN JI, FENG LIU, ER Qiu LI, Yu XIAN ZHUThe national laboratory of protein engineering and plant genetic engineering, College of Life Sciences, Peking University, Beijing 100871, China 1The national laboratory of protein engineering and plant genetic engineering College of Life Sciences Peking University Beijing 100871 China 1The national laboratory of protein engineering and plant genetic engineering College of Life Sciences Peking University Beijing 100871 China 1The national laboratory of protein engineering and plant genetic engineering College of Life Sciences Peking University Beijing 100871 China 1The national laboratory of protein engineering and plant genetic engineering College of Life Sciences Peking University Beijing 100871 China

The nucleotide sequence deduced from the amino acid sequence of the scorpion insectotoxin AaIT was chemically synthesized and was expressed in Escherichia coli. The authenticity of this in vitro expressed peptide was confirmed by N-terminal peptide sequencing. Two groups of bioassays, artificial diet incorporation assay and contact insecticidal effect assay, were carried out separately to verify the toxicity of this recombinant toxin. At the end of a 24 h experimental period, more than 60% of the testing diamondback moth （Plutella xylostella） larvae were killed in both groups with LC50 value of 18.4 μM and 0.70 μM respectively. Cytotoxicity assay using cultured Sf9 insect cells and MCF-7 human cells demonstrated that the toxin AaIT had specific toxicity against insect cells but not human cells. Only 0.13 μM recombinant toxin was needed to kill 50% of cultured insect cells while as much as 1.3 μM toxin had absolutely no effect on human cells. Insect cells produced obvious intrusions from their plasma

关键词： Scorpion toxin AaIT prokaryotic expression cytotoxicity.

Cloning and Sequencing of Trichosanthin Gene and Its Expression in Escherichia coli and Tobacco plant

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Science China Chemistry 1993年第6期36卷 669-676页

作者：鲍一明储瑞银韩晋华张会潘乃穟顾孝诚陈章良 The national laboratory of protein engineering and plant genetic engineering Department of Biology Peking University Beijing 100871 PRC

A Trichosanthin gene was cloned from Trichosanthes kirilowii genomic DNA by polymerase chain reaction (PCR). Nucleotide sequence data indicated that we obtained the coding region of the mature Trichosanthin peptide as well as its signal peptide at the N-terminus. Comparisons of our sequence with the previously reported nucleotide sequences of this gene showed 99.25% homology, yet there were notable differences between the previously reported amino acid sequence and our deduced result. This gene was subcloned into a highlevel expression plasmid (pJLA502) of E. coli under the control of a P_RP_L promoter, and we observed the gene product after temperature induction. The gene was further cloned into plant intermediate vector pE3 under the control of a CaMV 35S promoter, and transferred into a tobacco genome using the agrobacterium-mediated gene transfer system. Western blotting analysis of the protein extracted from Escherichia coli and transgenic tobacco plants proved that the Trichosanthin gene has been expressed in both organisms.

关键词： Trichosanthin polymerase chain reaction nucleotide sequence analysis gene expression transgenic plant

genetic Basis of Ethylene Perception and Signal Transduction in Arabidopsis

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Journal of Integrative plant Biology 2008年第7期50卷 808-815页

作者： Ziqiang Zhu Hongwei Guo national laboratory of protein engineering and plant genetic engineering College of Life Sciences Peking University Beijing 100871 China

Ethylene is a simple gaseous hormone in plants. It plays important roles in plant development and stress tolerance. In the presence of ethylene treatment, all ethylene receptors are in an activated form, which can physically interact with CTR1 and consequently recruit CTR1 protein to endoplasmic reticulum membrane to activate it. Activated CTR1 suppresses the downstream signal transduction by an unknown mechanism. Upon binding to its receptors, ethylene will inactivate the receptor/CTR1 module and in turn alleviate their inhibitory effect on two positive regulators acting downstream of CTR1： EIN2 and EIN3. genetic study reveals that EIN2 is an essential component in the ethylene signaling pathway but its biochemical function remains a mystery. EIN3 is a plant-specific transcription factor and its protein abundance in the nucleus is rapidly induced upon ethylene treatment. In the absence of ethylene signal, EIN3 protein is degraded by an SCF complex containing one of the two F-box proteins EBF1/EBF2 in a 26S proteasome-dependent manner. EIN3 can bind to the promoter sequences of a number of downstream components, such as ERFs, which in turn bind to a GCC box, a c/s-element found in many ethylene-regulated defense genes. Ethylene has been shown to also regulate many other hormones＇ signaling pathways including auxin, abscisic acid and jasmonic acid, implying the existence of complicated signaling networks in the growth, development and defense responses of various plants.

关键词： Arabidopsis crosstalk EIN3 ethylene signal transduction.

Identification and Quantitative Analysis of Significantly Accumulated proteins During the Arabidopsis Seedling De-etiolation Process

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Journal of Integrative plant Biology 2006年第1期48卷 104-113页

作者： Bai-Chen Wang Ying-Hong Pan Da-Zhe Meng Yu-Xian Zhu The national laboratory of protein engineering and plant genetic engineering College of Life Sciences Peking University Beijing 100871 China

Proteomic analysis was performed on seedlings after different light treatments. A total of （1 350±31） protein spots was separated and visualized on each silver nitrate-stained two-dimensional gel using protein samples prepared from light-grown or etiolated seedlings with or without 6-9 h light treatment. Twenty-five protein spots （encoded by 19 genes） that were significantly accumulated upon light treatment were identified using the matrix-assisted laser desorption ionization time-of-flight mass spectrometry method. Functional proteomics indicated that these proteins involved mainly in chloroplast development, energy metabolism, cell cycle progression and membrane electron transport. For 18 of the protein-coding genes we identified through an internet search, the transcript levels of 17 genes matched roughly with their protein content in etiolated and green seedlings, suggesting that these genes were regulated by light mainly at the transcriptional level. Despite a very significant increase in the amount of proteins upon light treatment, similar RNA levels were found in dark-grown or green seedlings for the carbonic anhydrase gene At3g05100, indicating a possible post-transcriptional regulatory mechanism. Elucidation of light-induced protein accumulation will undoubtedly enhance our understanding of plant photomorphogenesis.

关键词： Arabidopsis de-etiolation light two-dimensional electrophoresis.

Cloning, sequencing and structural analysis of a pea cDNA encoding EF-1α

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Chinese Science Bulletin 2000年第4期45卷 338-343页

作者： Xiangyu Cong Shiyun Ling Yuxian Zhu national laboratory of protein engineering and plant genetic engineering College of Life Science Peking University Beijing China

A cDNA library with the capacity of 2.0×106 was constructed using mRNA extracted, from gibberellic acid (GA)-treated G2 pea seedlings and a 1.6 kb cDNA with 100 nucleotides of 5’ non-coding region and 223 nucleotides of 3’ non-coding region was obtained by random screening. The DNA fragment contains an open reading frame of 1 344 nucleotides and encodes 447 amino acids. Seguence analysis of DNA and deduced polypeptide demonstrated that it encoded for a pea elongation factor 1-alpha (EF-1α). The functional domains of EF-1α is well conserved and EF-1α can be a good candidate for studying molecular evolution.

关键词： pea cDNA library EF-1α sequence analysis.

Posttranslational modulation on the biological activities of molecular chaperones

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Science China(Life Sciences) 2009年第6期52卷 515-520页

作者： CHANG ZengYi Center for protein Science,School of Life Science,national laboratory of protein engineering and plant genetic engineering,Peking University,Beijing 100871,China Center for protein Science School of Life Science National Laboratory of Protein Engineering and Plant Genetic Engineering Peking University Beijing 100871 China

Molecular chaperones are a family of proteins that were first noticed to exist about 45 years ago from their increased transcription under heat shock *** a result,the regulation of their encoding genes has been subject to extensive *** studies revealed that the biological activities of molecular chaperones can also be effectively modulated at the protein *** ways of modulation so far elucidated include allosteric effect,covalent modification,protein-protein interaction,and con-formational alteration induced by such macro-environmental conditions as temperature and *** latter aspects were reviewed *** here is the importance of such immediate structural alterations that lead to an immediate activity increase,providing the immediate protection needed for the cells to survive the stress conditions.

关键词： molecular chaperone activity modulation sHsp Hsp60 Hsp70 Hsp90 Hsp100 HdeA DegP