Erwinia carotovora ssp. carotovora Infection Induced ＂Defense Lignin＂ Accumulation and Lignin Biosynthetic Gene Expression in Chinese Cabbage （Brassica rapa L. ssp. pekinensis）

Erwinia carotovora subsp, carotovora （Ecc） infects and causes soft rot disease in hundreds of crop species including vegetables, flowers and fruits. Lignin biosynthesis has been implicated in defensive reactions to injury and pathogen infection in plants. In this work, variations of lignin content and gene expression in the molecular interaction between Chinese cabbage and Ecc were investigated. H2O2 accumulation and peroxidase activity were detected by 3, 3＇- Dimethoxybenzidine staining at mocked and Ecc-inoculated sites of Chinese cabbage leafstalks. Klason lignin content in inoculated plants increased by about 7.84%, 40.37%, and 43.13% more than that of the mocked site at 12, 24 and 72 h after inoculation, respectively. Gas chromatography detected more p-coumaryl （H） and less coniferyl （G） and sinapyl （S） monolignins in leafstalks of Chinese cabbage. All three monomers increased in Ecc-infected leafstalks, and the Ecc-induced ＂defense lignin＂ were composed of more G and H monolignins, and less S monolignin. After searching the expressed sequence tags （EST） data of Chinese cabbage, 12 genes putatively encoding enzymes involved in lignin biosynthesis were selected to study their expression. All of these genes could be induced by mock inoculation and Ecc infection, while the gene expression lasted for several more hours in the infected samples than in mocked and untreated plants. Our results indicated that ＂defense lignin＂ was different from the developmental lignin in composition; G and S monolignins were significantly induced in plants in response to the soft rot Ecc; thus, lignin biosynthesis was differentially regulated and played a role in plant response to the soft rot Ecc.     

Expression of two types of acetylcholinesterase gene from the silkworm, Bombyx mori, in insect cells

Complementary DNAs encoding two types of acetylcholinesterase （ACHE） were isolated from the silkworm, Bombyx mori. The type 1 （Bmacel） and type 2 （Bmace2） ORFs are 2052 and 1917 bp in length, respectively. Both the complete ORFs of the Bmaces and C- terminal truncated forms were recombined into the Bacmid baculovirus vector under the control of the polyhedrin promoter and expressed in Trichoplusia ni （Tn-5B 1-4） cells. The resulting products exhibited ACHE activity and glycosylation of the expressed proteins. An inhibition assay indicated that the ace2-type enzyme was more sensitive than the acel-type enzyme to inhibition by eserine and paraoxon.     

Cloning and biological activity of an anti-tumor peptide of Tumstatin

To obtain an anti-tumor peptide of Tumstatin and detect its biological activity, the nucleotide sequence encoding 185–203 amino acids (19peptide) of Tumstatin was synthesized and inserted into the fusion protein vector pTYB2. After identification by sequencing and restriction endonucleases, the recombined vector was transformed into BL-21 (DE3) E. coli competent cells. Transformed E. coli BL-21 (DE3) were induced by isopropyl-β-thiogalactopyranoside (IPTG), and then expressed. By 1,4-dithiothreitol (DTT) reduction, the soluble 19peptide was obtained from a chitin affinity chromatograph. The biological activity of 19peptide was determined by 3-[4,5-dimethylthiazol-2-y1]-2,5-diphenytetrazolium bromide (MTT) assay, cell growth curve, the effect of the ascitic fluid transfevent H22 hepatoma on mice and via histopathological slices. The purified 19peptide directly inhibited proliferation and migration of murine B16 melanoma cells, SMMC-7721hepatoma carcinoma cells and human umbilical vein endothelial cells (HUVEC). The tumor inhibition rate of mice ascitic fluid transfevent H22 hepatoma was 48.46%. Histopathological slices showed that it could promote tumor tissue necrosis and decrease the density of blood vessels. With higher anti-tumor activity, 19peptide has the potential to become a novel, potent anti-tumor agent. Translated from Chinese Journal of Biochemistry and Molecular Biology, 2005, 21(3): 322–328 [译自: 中国生物化学与分子生物学学报]     

Cloning, sequence analysis and identification of a nonsense mutation-mediated mRNA decay of porcine GSTM2 gene

The glutathione S-transferase mu 2 gene （GSTM2） encodes a GST functioning in the elimination of electrophilic compounds and the regulation of cell growth. In this study, the sequence of porcine GSTM2 gene that contains the complete sequence encoding a protein of 218 amino acids was cloned. The deduced amino acid sequence shared 76%, 78% and 76% identity with that of human, mouse and rat, respectively, mRNA expression analysis showed that the porcine GSTM2 gene was expressed at a high level in liver and testis, at a medium level in longissimus dorsi muscle, adipose tissue, spleen and lung, at a low level in kidney, and at a very low level in heart and embryo. A nonsense mutation （CGA→TGA） resulted from C27T substitution in the fifth exon to produce a premature translation termination codon was identified, and it was discovered that nonsense-mediated mRNA decay might have an effect on the regulation of porcine GSTM2 gene expression. This polymorphism was analyzed in Large White, Landrace, Meishan and Qingping pig populations using the Taq I-polymerase chain reaction-restriction fragment length polymorphism method. The result showed that allele C had a higher frequency than allele T in each population.     

cDNA微阵列技术研究干旱胁迫下柽柳基因的表达

运用cDNA微阵列技术研究干旱胁迫下柽柳（Tamarix androssowii）基因的表达。分别将Cy5和Cy3两种荧光染料标记在干旱处理和对照的柽柳cDNA上,并与载有柽柳基因的微阵列进行杂交,通过计算机对芯片进行扫描和分析研究干旱胁迫下基因的表达。共获得了47个下调表达和62个上调表达的基因。Blastx分析表明这些基因按功能可以分为脱水保护、信号传导与调控、活性氧清除、光合作用、代谢、核糖体蛋白、蛋白质的分解与再生等几大类别。同时,发现了一些与干旱胁迫相关的功能未知基因和新基因。从而揭示了柽柳具有活性氧消除、代谢调节、脱水保护、蛋白质降解与再生等抗旱途径,并阐述了干旱胁迫前后柽柳基因的差异表达。     

发菜中超氧化物歧化酶基因的克隆及在大肠杆菌中的表达

采用基因工程技术从发菜总DNA中克隆了一段的基因序列,该序列与基因库中已公布的编码地木耳（Nostoc commnue）超氧化物歧化酶的氨基酸序列同源性为97%。将该基因插入含T7启动子质粒pET-32中构建表达质粒pET-sod,然后将该表达质粒转入大肠杆菌BL21中进行蛋白表达,表达菌株用1 mmol·L^-1 IPTG诱导表达数小时后,产生较多的重组的蛋白,且该蛋白以可溶性蛋白形式存在。SDS-PAGE分析表明,在相对分子量约为22 kd的位置有一条明显蛋白质带。将诱导表达后的蛋白通过亲和层析的方法进行蛋白纯化;NBT光还原法测定表达产物的比活力,每毫克纯化蛋白约为2 550 U。对纯化后的蛋白进行高温胁迫研究,将该纯化蛋白在60℃高温下胁迫90 min后,其活性为原（未经胁迫）蛋白活性的85%。     

绝经对血液单核细胞基因表达谱的影响:基因芯片初步研究

绝经是女性一生中很重要的生理现象之一,它能增加一系列复杂免疫、神经退化、新陈代谢和心血管方面的疾病。血液单核细胞能分化成各种各样的细胞,这些细胞在组织形态发生和免疫应答方面起着很重要的作用。本研究中采用了包含大约14,500个基因探针的Affymetrix Human U133A基因芯片来研究健康的绝经前和绝经后女性外周血液单核细胞中的基因表达谱。样本之间的对比分析表明有20个基因上调,20个基因下调。其中的28个基因根据它们的生物过程如细胞繁殖、免疫应答、细胞代谢等等被分成了6个主要的GO类别;剩下的12个基因其生物学功能还没有被鉴定。研究结果支持了我们的假设:血液单核细胞的功能状态确实受到绝经的影响,而且由此带来的改变可能是由全基因组范围的基因表达谱而决定的。本研究中鉴定的一些差异表达基因有可能作为以后研究与绝经相关的系统免疫、神经退化和心血管疾病的候选基因研究。此工作是这个研究方向的第一次尝试,为将来的进一步研究奠定了基础。     

microRNA在小鼠乳腺不同发育时期差异表达谱及作用

microRNA是一类大小约22个核苷酸的非编码RNA分子,是一种广泛存在的对基因表达进行微调的分子。microRNA可以通过与靶基因mRNA的特定位点结合,抑制该蛋白的合成或诱导该mRNA的降解,从而参与基因的表达调控。一般来源于染色体的非编码区域,由大约70个核苷酸大小的可形成发夹结构的前体经Dicer酶加工而来。这类小RNA在表达上具有组织和时间的特异性,是调节其他功能基因表达的重要调控分子,在生物的生长发育过程中发挥着重要作用。因此,虽然microRNA的研究仅有很短的历史,但已成为基因表达调控研究的热点领域。以中国昆明小鼠不同发育时期的乳腺组织为实验材料,应用芯片技术及荧光定量PCR技术,分析发育不同时期的乳腺组织microRNA差异表达图谱。本文研究发现microRNA在乳腺不同的发育时期表达图谱不同;与青春期、退化期比较,妊娠期、哺乳期有十余种microRNAs表达上调,20余种microRNAs表达下调;microRNAs在乳腺发育和泌乳周期中发挥重要的作用。     

小立碗藓叶绿体分裂相关基因PpMinE的克隆及其功能与进化分析

用RT-PCR技术从小立碗藓中(Physcomitrella patens)克隆了核编码的MinE基因,命名为PpMinE,并克隆了该基因的基因组DNA。序列比对显示该基因编码的蛋白质与真细菌和绿藻叶绿体编码的MinE蛋白具有较高的相似性。pMinE-EGFP融合蛋白在烟草中的瞬时表达证明该蛋白定位于叶绿体内。在大肠杆菌中过量表达PpMinE导致细胞不正常分裂,产生无染色体的小细胞,这表明MinE的功能在进化上是保守的。在系统发育树中,PpMinE和高等陆生植物有较近的亲缘关系。在已知的陆生植物的叶绿体基因组中没有找到MinE的同源蛋白,这暗示在进化过程中MinE从叶绿体到细胞核的水平转移可能发生在陆生植物发生以前。