《张荣娟差异基因》PPT课件.ppt

1、cDNA-AFLP-差异基因,所有挑选的差异均为间作处理中特异存在,总结-6个差异基因,Q-PCR验证部分差异基因,RJ4 secA RJ6 CgtA RJ6b ProB1 RJ7 prkA RJ9 gltA RJ14 thiC,总结,a,b,a,a,b,c,b,a,c,b,a,b,a,a,a,a,b,b,b,b,b,a,c,c,c,JY9（gltA,文献报道： 根瘤菌突变体中：不能结瘤或形成无效瘤，类菌体减少，胞外多糖比野生型多三倍，推测破坏了侵染线，缺乏能量，对结瘤固氮必须； 大肠杆菌中：突变体生长速度减慢,JY14-thiC,文献报道： Putative thiamine biosynt

2、hesis protein ； 突变体会推迟结瘤； Thiamine is a role during the transition from aerobic to fermentative-like metabolism,b,a,JY4（secA,文献报道： 细菌特有的一种蛋白质，具有ATP酶活性，是S e c蛋白质转运途径中的“ 动力泵”，通过 ATP的水解循环驱使多肽穿过通道,c,b,a,JY7（prkA,文献报道： 丝氨酸蛋白激酶；仅在芽孢杆菌中有报到；是一种cAMP依赖的蛋白激酶作用,c,b,a,JY6B和JY6（下调,Disruption of the cgtA gene was

3、lethal, demonstrating that this gene is essential for cell growth,secA,E.coli and Bacillus 功能已经明确， hydrolyzes ATP and an essential component of the protein translocation ATPase ； SecA Protein的ATP-binding site已经确定； SecA 识别并结合 preprotein-SecB complex, 结合到 inner-membrane anionic phospholipids, 插入 the m

4、embrane bilayer 以后，再与the preprotein translocator, SecY/SecE结合，从而将蛋白运输过膜,putative transport protein transmembrane,hypothetical protein,c,b,a,SecB,thiC,文献分布： Sinorhizobium meliloti (2) R.etli(2) E.coli and else(15) Sinorhizobium 突变体库中有关于这个突变体的信息； Rhizobium etli：在其中表达thiCOGE，将增加其固氮效率； Thiamin biosynthe

5、sis in prokaryotes：其中涉及12个基因（ thiFSGH, I, and dxs， thiC， thiE， thiD, thiM, thiL, and pdxK,突变体库查询内容,prkA(1,hypothetical protein SMc01266,Bacillus subtilis ：prkA gene encoding a novel serine protein kinase,c,b,a,SMc00367（假定蛋白,Protein:putative oxidoreductase protein,putative oxidoreductase protein,JY6B

6、和JY6（下调,obgE,obgE/proB1,50S ribosomal protein L21,hypothetical protein,gamma-glutamyl kinase,gamma-glutamyl phosphate reductase,nicotinic acid mononucleotide adenylyltransferase,obgE (15篇E.coli most) (cgtA ，yhbZ, obgE,coding for an essential GTP-binding protein， 染色体复制、分裂等过程中起调节作用，可能作用于核糖体合成的最后一步,pro

7、B1（ Escherichia coli,Glutamate-5-kinase： proline（脯氨酸） 由 glutamate（谷氨酸盐）合成需要三步： catalyzed by glutamate 5-kinase (G5K), glutamyl 5-phosphate reductase (G5PR) and pyrroline 5-carboxylate reductase； 脯氨酸plays an important role as an osmoprotectant in microorganisms Glutamate-5-kinase 的active site residue

8、s已确定,The EMBO Journal vol.8 no.3 pp.961 -966, 1989 SecA protein hydrolyzes ATP and is an essential component of the protein translocation ATPase of Escherichia coli Characterization of a Bacillus subtilis SecA mutant protein deficient in translocation ATPase and release from the membrane Histidine R

9、esidues Are Involved in Translocation-Coupled ATP Hydrolysis by the SecA Protein Mol Microbiol. 1993 Apr;8(1):31-42. Characterization of a Bacillus subtilis SecA mutant protein deficient in translocation ATPase and release from the membrane-It is concluded that the GKT motif in the amino-terminal do

10、main of SecA is part of the catalytic ATP-binding site. This site may be involved in the ATP-driven protein recycling function of SecA which allows the release of SecA from its association with precursor proteins, and the phospholipid bilayer,secA,SecA protein: autoregulated ATPase catalysing prepro

11、tein insertion and translocation across the Escherichia coli inner membran,Recent insight into the biochemical mechanisms of protein translocation in Escherichia coli indicates that SecA ATPase is required both for the initial binding of preproteins to the inner membrane as well as subsequent transl

12、ocation across this structure. SecA appears to promote these events by direct recognition of the preprotein or preprotein-SecB complex, binding to inner-membrane anionic phospholipids, insertion into the membrane bilayer and association with the preprotein translocator, SecY/SecE. ATP binding appear

13、s to control the affinity of SecA for the various components of the system and ATP hydrolysis promotes cycling between its different biochemical states. As a component likely to catalyse a rate-determining step in protein secretion, SecA synthesis is co-ordinated with the activity of the protein exp

14、ort pathway. This form of negative regulation appears to rely on SecA protein binding to its mRNA and repressing translation if conditions of rapid protein secretion prevail within the cell. A precise biochemical scheme for SecA-dependent catalysis of protein export and the details of secA regulatio

15、n appear to be close at hand. The evolutionary conservation of SecA protein among eubacteria as well as the general requirement for translocation ATPases in other protein secretion systems argues for a mechanistic commonality of all prokaryotic protein export pathways,prkA,Cloning and characterizati

16、on of the Bacillus subtilis prkA gene encoding a novel serine protein kinase. Cloning, expression, purification and characterization of the stress kinase YeaG from Escherichia coli,Characterization of the Bacillus subtilis thiC Operon Involved in Thiamine Biosynthesis,Expression of Thiamin Biosynthe

17、tic Genes (thiCOGE) and Production of Symbiotic Terminal Oxidase cbb3 in Rhizobium etli These data show a direct relationship between expression of thiC and production of the cbb3 terminal oxidase. This is consistent with the proposition that a purine-related metabolite, 5-aminoimidazole-4-carbox- a

18、mide ribonucleotide, is a negative effector of the production of the symbiotic terminal oxidase cbb3 in R. etli,Thiamin biosynthesis in prokaryotes Twelve genes involved in thiamin biosynthesis in prokaryotes have been identified and overexpressed. Of these, six are required for the thiazole biosynt

19、hesis (thiF- SGH, I, and dxs), one is involved in the pyrimidine biosynthesis (thiC), one is required for the linking of the thiazole and the pyrimidine (thiE), and four are kinases (thiD, thiM, thiL, and pdxK,A conserved RNA structure (thi box) is involved in regulation of thiamin biosynthetic gene

20、 expression in bacteria(R. etli strain CE3,thiC,Thiamine limitation determines the transition fromaerobic to fermentative-like metabolism in Rhizobium etli CE3,ThiC Is an Fe-S Cluster Protein That Requires AdoMet To Generate the -Amino-5-hydroxymethyl-2-methylpyrimidine Moiety in Thiamin Synthesis,R

21、eaction of AdoMet with ThiC generates a backbone free radical,Role of the cgtA gene function in DNA replication of extrachromosomal elements in Escherichia coli(2003) It seems that DNA synthesis per se is not affected by CgtA, and that this protein might control replication initiation indirectly, by

22、 regulation of function(s) or production of one or more replication factors. In fact, we found that level of the host-encoded replication protein DnaA is significantly decreased in the cgtA mutant. This indicates that CgtA is involved in the regulation of dnaA gene expression,Overexpression of the c

23、gtA (yhbZ, obgE) gene, coding for an essential GTP-binding protein, impairs the regulation of chromosomal functions in Escherichia coli.(2002,DNA replication defect in the Escherichia coli cgtA(ts) mutant arising from reduced DnaA levels(2006,Obg/CtgA, a Signaling Protein That Controls Replication,

24、Translation, and Morphological Development?(2006) The recent finding that the ObgE GTPase acts as a ated with Obg: the effects on bacterial replication (Foti replication checkpoint protein in Escherichia coli has et al., 2005). A new E. coli obgE mutant was isolated important implications,Involvemen

25、t of the cgtA gene function in stimulation of DNA repair in Escherichia coli and Vibrio harveyi(2003,The Escherichia coli GTPase CgtAE Is Involved in Late Steps of Large Ribosome Assembly(2006) Mutations in CgtAE cause both polysome and rRNA processing defects; small- and large-subunit precursor rRN

26、As accu-mulate in a cgtAE mutant,The ObgE/CgtA GTPase inuences the stringent response to amino acid starvation in Escherichia coli(2009,Functional analysis of the GTPases EngA and YhbZ encoded by Salmonella typhimurium(2007) Abstract The S. typhimurium genome encodes proteins, designated EngA and Yh

27、bZ, which have a high sequence identity with the GTPases EngA/Der and ObgE/CgtAE of Escherichia coli. The wild-type activity of the E. coli proteins is essential for normal ribosome maturation and cell viability. In order to characterize the potential involvement of the Salmonella typhimurium EngA a

28、nd YhbZ proteins in ribosome biology, we used high stringency affinity chromatography experiments to identify strongly binding ribosomal partner proteins. A combination of biochemical and microcalorimetric analysis was then used to characterize these protein:protein interactions and quantify nucleot

29、ide binding affinities. These experiments show that YhbZ specifically interacts with the pseudouridine synthase RluD (KD 2 mM and 1:1 stoichiometry), and we show for the first time that EngA can interact with the ribosomal structural protein S7. Thermodynamic analysis shows both EngA and YhbZ bind GDP with a higher affinity than GTP (20-fold difference for EngA and 3.8-fold for YhbZ), and that the two nucleotide