橡胶树蛋白激酶HbBIN2基因的克隆、蛋白表达纯化及酶活分析

橡胶树是天然橡胶的唯一材料来源。寒害是橡胶树面临的主要自然灾害之一,严重限制了橡胶树的生长发育和种植区域分布,克隆和鉴定橡胶树低温应答基因尤为重要。蛋白激酶BIN2（brassinosteroid insensitive 2）是一种丝氨酸/苏氨酸蛋白激酶,在调控植物应对低温胁迫中发挥重要作用,但橡胶树蛋白激酶HbBIN2还未被克隆与鉴定。本研究从橡胶树cDNA中成功克隆出HbBIN2,序列分析表明：HbBIN2的开放阅读框为1143 bp,编码380个氨基酸,蛋白的分子量为42.9 kDa,理论等电点为8.74,是亲水性蛋白且无跨膜结构域。将HbBIN2构建到原核表达载体pET28a上,转化大肠杆菌BL21（DE3）表达菌株,摸索合适的诱导条件后成功诱导表达出HbBIN2蛋白。比较HbBIN2在不同的诱导温度（16、28、37℃）、诱导时间（3、6、12 h）和IPTG浓度（0.1、0.3、0.5 mmol/L）下的表达量,结果表明,HbBIN2在37℃,0.3 mmol/L IPTG诱导12 h的表达量最大。对HbBIN2蛋白体外纯化条件进行探索,结果表明,100 mmol/L咪唑能将目的蛋白完全洗脱。纯化HbBIN2蛋白并进行激酶活性鉴定,结果表明,该蛋白具有激酶活性。该研究为后续HbBIN2蛋白功能分析和橡胶树应对低温胁迫的调控机制研究提供参考,为橡胶树耐寒品种分子育种提供重要的基因资源。

Cloning,Expression, Purification and Enzyme Activity Analysis of Protein Kinase HbBIN2 from Hevea brasiliensis

Rubber tree (Hevea brasiliensis Muell. Arg.) is the exclusive commercial source of natural rubber because of its high productivity and superior product quality. Low temperature is a major abiotic stress that disadvantageously affect rubber tree growth and development, and ultimately limit ecological distribution. However, the underlying mechanisms of H. brasiliensis cold stress response remain elusive. Identifying cold response genes in rubber tree and understanding the mechanisms regulating cold stress is of great potential value. BIN2 (Brassinosteroid Insensitive 2), a serine/threonine protein kinase, plays an important role in plant responses to cold stress, but HbBIN2 in H. brasiliensis has not been cloned and characterized. In this study, HbBIN2 gene was successfully cloned from H. brasiliensis. The open reading frame of HbBIN2 included 1143 nucleotides and encoded 380 amino acids, with predicted molecular mass of 42.9 kDa. The theoretical isoelectric point (pI) of HbBIN2 was 8.74. HbBIN2 was a hydrophilic protein, which had no transmembrane domain. The full-length CDS of HbBIN2 was inserted into the polyclone sites between BamHⅠ and XhoⅠ of the prokaryotic expression vector pET-28a for generating the recombinant plasmid pET28a-HbBIN2. The recombinant plasmid pET28a-HbBIN2 was transformed into E. coli BL211 (DE3) strain to induce HbBIN2 protein expression. By comparing the expression levels of HbBIN2 at different induction temperatures (16, 28, 37℃), induction time (3, 6, 12 h) and IPTG concentration (0.1, 0.3, 0.5 mmol/L), The results demonstrated that the optimal induction expression condition of HbBIN2 was at 37℃ for 12 h with 0.3 mmol/L IPTG. In addition, the purification conditions of HbBIN2 protein in vitro were then explored, and the optimal concentration of imidazole for purifying HbBIN2 was 100 mmol/L. The kinase activity assay demonstrated that the purified HbBIN2 had kinase activity in vitro. This study would lay a foundation for further research on the functional analysis of HbBIN2 protein and the regulatory mechanism of H. brasiliensis cold stress response, and provide elite gene resource for rubber tree breeding for cold resistance.