Molecular characterization of <Emphasis Type="Italic">Arabidopsis</Emphasis> PHO80-like proteins,a novel class of CDKA;1-interacting cyclins

Cyclins are regulatory proteins that interact with cyclin-dependent kinases (CDKs) to control progression through the cell cycle. In Arabidopsis thaliana, 34 cyclin genes have been described, grouped into five different types (A, B, D, H, and T). A novel class of seven cyclins was isolated and characterized in Arabidopsis, designated P-type cyclins (CYCPs). They all share a conserved central region of 100 amino acids (cyclin box) displaying homology to the corresponding region of the PHO80 cyclin from Saccharomyces cerevisiae and the related G1 cyclins from Trypanosoma cruzi and T. brucei. The CYCP4;2 gene was able to partially re-establish the phosphate-dependent expression of the PHO5 gene in a pho80 mutant strain of yeast. The CYCPs interact preferentially with CDKA;1 in vivo and in vitro as shown by yeast two-hybrid analysis and co-immunoprecipitation experiments. P-type cyclins were mostly expressed in proliferating cells, albeit also in differentiating and mature tissues. The possible role of CYCPs in linking cell division, cell differentiation, and the nutritional status of the cell is discussed.Received 9 February 2004; received after revision 18 March; accepted 19 April 2004     

拟南芥悬浮培养细胞外钙结合蛋白的初步检测

钙离子通过与其结合的蛋白质在植物细胞信号转导中起着重要的作用,已知植物细胞外空间钙离子浓度高达毫摩尔级,而与钙结合的胞外蛋白有待进一步的分析研究.采用丙酮沉淀法分别制备了拟南芥悬浮培养细胞壁盐提蛋白及培养介质蛋白,制备的蛋白经SDS-PAGE电泳分离后,电转移至硝酸纤维素膜,45Ca2 覆盖结合,放射自显影,结果表明悬浮细胞外可能存在多条可与钙离子结合的蛋白条带,其分子量约为17,30,40,68 ku,推测这些蛋白可能参与胞外钙离子相关的生物学过程.     

Structure and function of eukaryotic NAD(P)H:nitrate reductase

Pyridine nucleotide-dependent nitrate reductases (NRs; EC 1.6.6.1–3) are molybdenum-containing enzymes found in eukaryotic organisms which assimilate nitrate. NR is a homodimer with an ∼100 kDa polypeptide which folds into stable domains housing each of the enzyme's redox cofactors—FAD, heme-Fe molybdopterin (Mo-MPT) and the electron donor NAD(P)H—and there is also a domain for the dimer interface. NR has two active sites: the nitrate-reducing Mo-containing active site and the pyridine nucleotide active site formed between the FAD and NAD(P)H domains. The major barriers to defining the mechanism of catalysis for NR are obtaining the detailed three-dimensional structures for oxidized and reduced enzyme and more in-depth analysis of electron transfer rates in holo-NR. Recombinant expression of holo-NR and its fragments, including site-directed mutagenesis of key acative site and domain interface residues, are expected to make large contributions to this effort to understand the catalytic mechanism of NR.     

Cloning the Promoter of BcNA1 from Brassica napus and Fad2 Gene from Arabidopsis thaliana and Construction of the Plant Expression Vector

The upstream regulatory region of a seed-specific gene was isolated from the genomic DNA of Brassica napus by PCR amplification. The cloned fragment contained 1755 nucleotides, and shared a sequence homology of 99.6% with the reported data. The coding region of oleic acid desaturase gene was then cloned from Arabidopsis thaliana. The sequencing analysis indicated that the sequence of the PCR product was just the same as reported before. In addition, the plant expression vector harboring the seed-specific promoter and trans-Fad2 gene was constructed.     

盐胁迫下盐芥和拟南芥内源激素质量分数变化的研究

采用酶联免疫吸附(ELISA)法测定了盐胁迫下盐芥(Thellungiella halophila)和拟南芥(Arabidopsis thaliana)叶片中生长素(IAA)、赤霉素(GA)、细胞分裂素(ZR)和脱落酸(ABA)等4种内源激素的质量分数,分析了2种植物的4种内源激素在盐胁迫下的响应特征.结果表明:48 h盐胁迫下拟南芥中的ABA质量分数增加2～2.7倍,是主要的胁迫响应激素;盐芥中呈积累增加的是IAA、ZR、GA,皆为促进生长的激素.说明盐胁迫下2种植物的激素应答方式不同.     

拟南芥PAL基因家族鉴定及表达模式分析

为研究苯丙氨酸解氨酶（PAL）作为苯丙烷类代谢途径的关键酶,在植物响应环境胁迫过程中的重要作用,本研究利用HMMER、Pfam与SMART等工具,从拟南芥全基因组中筛选获得 9 个PAL成员,分析其编码酶蛋白的理化性质、二级结构与保守基序等信息.同时,结合转录组与定量PCR分析PAL成员在干旱与盐胁迫条件下的表达模式.结果表明,拟南芥PAL成员的二级结构主要由α-螺旋和无规则卷曲组成;亮氨酸和丙氨酸为含量较高的氨基酸残基;拟南芥PAL成员含有 20 种不同的保守基序,其中motif6含有ASG活性位点,为所有PAL成员所共有;较多PAL成员具有光响应、激素响应、胁迫响应和生长发育相关的顺式调控元件.转录组学与定量PCR分析发现,AT3G53260.1、AT3G53260.2与AT3G47660.1的mRNA表达水平在干旱与盐胁迫后发生显著变化,推测这 3 条基因可能参与拟南芥对干旱及盐胁迫的响应过程,表明PAL基因在调控植物生长发育与非生物胁迫响应过程中发挥重要作用.