Cloning and characterization of PdDHN2b gene from Populus deltoides×Populus nigra.
-
摘要: 以欧美杨为材料,通过RT-PCR方法从欧美杨叶片中克隆到了抗旱脱水素DHN2b基因(命名为PdDHN2b),该基因开放阅读框为1 440 bp,编码379个氨基酸,绝大多数氨基酸组成属于亲水性氨基酸,无明显跨膜结构域。亚细胞定位表明PdDHN2b基因定位在细胞质或细胞膜上。荧光定量PCR分析表明:该基因在顶端中表达量最高,功能叶、根和茎中则无表达。同时该基因受NaCl、ABA和干旱等诱导表达。另外,构建了原核表达载体pET-PdDHN2b,并在大肠杆菌BL21中表达融合蛋白,诱导纯化后免疫小白鼠,制备抗体。SDS-PAGE电泳结果表明,融合蛋白分子量为58 ku。最后利用Ni-NTA纯化的PdDHN2b-his重组蛋白用于Western blot分析。Abstract: With a RT-PCR method we cloned a drought tolerant DHN2b gene (designated PdDHN2b) from Populus deltoides×Populus nigra. The open reading frame of PdDHN2b is 1 440 bp and encodes 379 amino acids of which most are hydrophilic, without an obvious transmembrane domain. A subcellular localization assay showed that PdDHN2b is localized in the membrane orcytoplasm. Real time fluorescence quota PCR expression analysis suggests that PdDHN2b is strongly expressed in top leaves, but not in functional leaves, roots or stems. PdDHN2b is induced by NaCl, ABA and drought. A recombinant pET-PdDHN2b vector was constructed for prokaryotic expression and expressed in E. coli BL21. Fusion proteins were purified and used to immunize white mice to obtain antiserum. SDS-PAGE electrophoresis showed that the molecular weight of the fusion protein was 58 ku. In the end, we used the Ni-NTA purified recombinant PdDHN2b-his protein for Western blot analysis.
-
Key words:
- Populus deltoides×Populus nigra /
- PdDHN2b /
- clone /
- express /
- stress
-
[1] ABBA S, GHIGNONE S, BONFANTE P. A dehydration-inducible gene in the truffle Tuber borchii identifies a novel group of dehydrins[J]. BMC Genomics, 2006, 7: 39- 54. [2] ZHAO S J. Experimental guide for plant physiology[M]. Beijing: Agricultural Science and Technology Press of China, 2004. [3] LIU R L, LIU M Q, LIU J, et al. Heterologous expression of a Ammopiptanthus mongolicus late embyogenesis abundant protein gene (AmLEA) enhances Escherichia coli viability under cold and heat stress[J]. Plant Growth Regul, 2010, 60:163- 168. [4] ABEBE T, GUENZI A C, MARTIN B, et al. Tolerance of mannitol-accumulating wheat to water stress and salinity[J]. Plant Physiol, 2003, 131: 1748- 1755. [5] RIERA M, FIGUERAS M, LOPEZ C, et al. Protein kinase CK2 modulates developmental functions of the abscisic acid responsive protein Rab17 from maize[J]. Proc Natl Acad Sci USA, 2004, 101: 9879- 9884. [6] EWA M K, STANISLAWA P. Possible roles of LEA proteins and sHSPs in seed protection: a short review[J]. Biotechnology Letters, 2007, 44(3): 3- 16. [7] ESSPELUND M, SABOE-LARSSEN S, HUGHES D W, et al. Late embryogenesis abundant genes encoding proteins with different numbers of hydrophilic repeats are regulated differentially by abscisic acid and osmotic stress[J]. Plant J, 1992, 2: 241- 252. [8] BRAY E A. Molecular responses to water deficit[J]. Plant Physiol, 1993, 103 (4): 1035- 1040. [9] XU L. A repeating 11-mer amino acid motif and plant desiccation[J]. Plant J, 1993, 3:363- 369. [10] XU L, BRAWLEY S H , CLOSE T J. Proteins immunologically related to dehydrins in fucoid algae[J]. J Phycol, 1998, 34: 642- 650. [11] 赵世杰. 植物生理学实验指导[M]. 北京: 中国农业科学技术出版社,2004. [12] LEVI A, PANTA G R, PARMENTIER C M, et al. Complementary DNA cloning, sequencing and expression of an unusual dehydrin from blueberry floral buds[J]. Physiol Plant, 1999, 107: 98- 109. [13] KIM H S, LEE J H, KIM J J, et al. Molecular and functional characterization of CaLEA6, the gene for hydropobic LEA protein from Capsicum annuum[J]. Gene, 2005, 344: 115- 1231. [14] MARTIN J, GEROMANOS S, TEMPST P, et al. Identification of nucleotide-binding regions in the chaperonin proteins GroEL and GroES[J]. Nature, 1993, 366: 279- 282. [15] ZHU J K. Salt and drought stress signal transduction in plants[J]. Annu Rev Plant Biol, 2002, 53: 247- 273. [16] LISSE T, BARTELS D, KALBITZER H R, et al. The recombinant dehydrin-like desiccation stress protein from the resurrection plant Craterostigma plantagineum displays no defined three-dimensional structure in its native state[J]. Biol Chem, 1996, 377: 555- 561. [17] LIU Y, ZHENG Y Z. PM2, a group 3 LEA protein from soybean, and its 22-mer repeating region confer salt tolerance in Escherichia coli[J]. Res Commum, 2005, 331: 325- 332. [18] RIERA M, PERACCHIA G, NADAL E, et al. Maize protein kinase CK2: regulation and functionality of three β regulatory subunits[J]. Plant J, 2001, 25: 365- 374. [19] MANFRE A J, LANNIL M, MARCOTTEWR J R. The Arabidopsis group 1 late embryogenesis abundant protein AIEM6 is required for normal seed development[J]. Plant Physio1, 2006, 140: 140- 149. -
点击查看大图
计量
- 文章访问数: 392
- HTML全文浏览量: 30
- PDF下载量: 9
- 被引次数: 0
下载:
