分子生物学外文文献汇报1

Fig4 Fluence dependencies in phosphorylation of the Ser-851 in response to blue light.
Etiolated seedlings were irradiated by a pulse of blue light for 30 sec at the indicated fluence rates. The seedlings were disrupted 1 min after the start of blue light, and microsomal membranes were immediately isolated from the seedlings. The membranes of 40 and 20 μ g of protein were used for the immunoblots of pSer-851 (Upper) and phot1(Lower), respectively.
The S849D S851D construct mimicked the phosphorylation of the Ser residues in the activation loop. However, the construct did not induce any responses without blue light in planta . This is explained by a recent report that LOV2 acts as a kinase inhibitor by binding to the kinase domain and that the kinase becomes able to phosphorylate the substrate by dissociation of LOV2 via blue light. it is most likely that LOV2 prevents both activation loop autophosphorylation and substrate transphosphorylation in the dark; the blue light-induced dissociation of LOV2 from the kinase domain allows the phosphorylation of the activation loop.
Fig 4、5表 明：Ser-851 自我磷酸化 是蓝光依赖 性。
Fig5 Time courses of phosphorylation and dephosphorylation of the Ser-851 in response to blue light
The seedlings were irradiated by blue light at 100 mol m21 for 30 sec and were disrupted at indicated times after the start of blue light. Microsomal membranes were immediately isolated, and used for immunoblots of pSer-851 (Upper) and phot1 (Lower).
Ⅴ、Functional Role of Phosphorylation in Phot1 in H＋ Pumping.
Fig7 Blue light-dependent H+ pumping in guard cell protoplasts, determined by pH decrease
Table1 List of transgenic plants with various phot1 constructs
Ⅱ、 Preparation of Phot1 from Etiolated Seedlings Stimulated by Blue Light
1.从32P标记的黄化幼苗中提取质膜微囊；
Results and discussion
Ⅰ、Plant Materials：
◆Etiolated seedlings of Arabidopsis glabrous1 (gl1) as a Control； ◆ phot1-5 phot2-1 double mutant； ◆ all transformants：transforming the phot1 phot2 double mutant with the mutated phot1 constructs. in which the identified Ser or Thr residues had been substituted with Ala or Asp.
Fig1 Identification of the phosphorylation of Arabidopsis phot1
Etiolated seedlings labeled by 32P were kept in the dark(DK) or illuminated with blue light at 100μmol·m-2·s-1 for 1 min(BL).The phot1 protein was isolated by immunoprecipitation from the microsomes of the seedlings,and separated by SDS/PAGE.An autoradiogram is illustrated.
1、 N-末端敏感区域:具有能与FMN结合的两个 LOV(light, oxygen, voltage)－LOV1与LOV2；
2、C-末端的Ser/Thr蛋白激酶区域。
作者首先测定了拟南芥保卫细胞中phot1在蓝 光照射下发生磷酸化，进而对磷酸化位点进 行定位，然后用Ala置换这些磷酸化位点的 Aa残基，通过检测不同突变型phot1调节的 气孔开放、叶绿体聚集、叶片伸展、向光性， 分析这些位点磷酸化对蓝光诱导的信号传导 所起的作用。
Guard cell protoplasts (100 μ g of proteins) were irradiated with red light at 600 μmol·m-2·s-1and superimposed with blue light at 100 μmol·m-2·s-1 for 30 sec. The protoplasts were added by 10 μ M fusicoccin. Measure ments were done at 24℃. BL, blue light; FC, fusicoccin.
此实验说明： Ser-851在蓝光下 发生了自我磷酸 化。
Fig3 Autophosphorylation of the Ser-851 in the activation loop of phot1 in vivo by blue light
Phot1 was immunopurified from 200 μ g of proteins of microsomal membranes. The obtained phot1 was visualized with anti-pSer-851 antibodies (Upper) and immunodetected (Lower).
Ⅲ、Mapping of in Vivo Phosphorylation Sites of Phot1.
Fig2 A typical case of a phosphorylation site mapping in phot1 by LCMS/MS.
Phot1 protein was digested with trypsin in gel. A MS/MS spectrum of a phosphorylated peptide 408KSpSLSFMGLK417 is represented. pS indicates phosphorylated Ser. Mascot database
蓝光对植物生长发育的影响是至关重要的，涉及多 种生理过程的调节作用，如脱黄化、开花、昼夜节 律、向光性、叶绿体运动、气孔运动等。 (Lin2000;Briggs和Huala-999)
Phototropin作为一种蓝光受体激酶能够调节植物的 向光性弯曲、叶绿体运动、气孔开放和叶片伸展。
人们已经在拟南芥、水稻、玉米等植物中发现了基 因PHOT1、PHOT2分别编码两种向光素phot1和phot2 蛋白，phot1和phot2都含有两个重要的多肽区域：
Fig6 Stomatal opening in the epidermis. Epidermal peels were irradiated by red light (60 μmol·m-2·s-1 ; RL) or red (50 μmol·m-2·s-1 ) and blue (10 μmol·m-2·s-1;RL＋BL) light for 3 h. Values are means of three independent experiments with standard deviations, with 45 stomatas measured in each experiment.
Salomon et al. determined eight phosphorylation sites in vitro in Avena phot1a. However, those sites did not include any of the sites in the kinase domain or in the C terminus shown in this study. The difference in results is probably due to the phosphorylation condition of the phot1 protein. We phosphorylated Arabidopsis phot1 in planta by an endogenous phot1 kinase, but Salomon et al. phosphorylated the recombinant Avena phot1a by PKA in vitro. Furthermore, the techniques used to identify phosphorylation site differed between the two experiments. We may have missed phosphorylation sites that exist in too-long or -short peptides produced by tryptic digestion,because these peptides were beyond the detection range of LC-MS/MS.
PNAS April 8, 2008 vol. 105 no. 14
Blue light-induced autophosphorylation of phototropin
is a primary step for signaling 蓝光诱导的向光素自我磷酸化 是信号传导的重要步骤
2012.10.20
Introduction Results and Discussion Conclusion
Intr1)吸收红光/远红光(波长为 600-750nm)的光敏色素(phytochrome)；(2)吸收蓝 光/UV-A(波长为300-500nm)的隐花色素 (eryptoehrome)和向光素(phototropin)；(3)UVB(波长为282-320nm)受体，但此受体迄今尚未分离 得到。
Ⅳ、Functional Role of Phosphorylation in Phot1 in Stomatal Opening.
此实验表明：a.活化 环中两个Ser残基是 气孔开放必需的， Ser-851比Ser-849更 重要；b.活化环磷酸 化不足以引起气孔打 开，必须有蓝光诱导。 c. 蓝光诱导的气孔 开放与活化环的自我 磷酸化有关。
2.phot1蛋白分离：immunoprecipitation,antibodies against phot1
3.磷酸化检测：Coomassie brilliant blue染色，放射自显 影检测
蓝光照射下野生型 phot1磷酸化水平比 黑暗条件下高， D806N-2型磷酸化没 有发生变化。 说明体内实验蓝光 诱导了野生型phot1 自我磷酸化，黑暗 条件下少量的磷酸 化可能是其它激酶 的作用。