小麦脱水素TaDHN-2基因功能与结构分析

Functional and Structural Analysis of Dehydrin Gene TaDHN-2 in Wheat

脱水素是一类在逆境胁迫过程中诱导表达的重要功能蛋白。为了探讨脱水素基因的生物学功能,本研究以洛旱2号为研究材料,利用RT-PCR技术克隆了小麦脱水素基因TaDHN-2,qRT-PCR分析了其在ABA诱导、PEG、NaCl胁迫处理下的表达模式,检测了TaDHN-2体外活性。结果表明,TaDHN-2可编码152个氨基酸,N端包含Y片段(VDEYGNP)及S片段(SSSSSEDDGMGGRRKK),C端包含两个保守的K片段(KIKEKLPG),属于脱水素的YnSKn亚类;表达特性分析显示,TaDHN-2受植物激素ABA诱导,属于ABA依赖的基因表达调控通路。渗透胁迫过程中,该基因在中国春和洛旱2号两个品种中具有不同的诱导表达模式,在中国春根系中的诱导表达幅度显著高于洛旱2号,而在洛旱2号叶片中的诱导表达量更高;体外活性鉴定发现,60℃高温条件下,浓度为100μg·mL^(-1)的TaDHN-2可使α-淀粉酶活性提高2倍;在4℃低温条件下,TaDHN-2在浓度大于20μg·mL^(-1)时可以提高α-淀粉酶的活性,当浓度增加到100μg·mL^(-1)时,可将α-淀粉酶活性提高为对照的1.6倍。以上结果说明,TaDHN-2蛋白具有显著的酶活性保护功能。

Dehydrin is a kind of functional proteins,which is induced by abiotic stresses.In order to characterize the function of dehydrin genes in wheat,and provide the theoretical basis for further molecular breeding of stress resistance,with Luohan 2 as material,a new Dehydrin gene TaDHN-2 was cloned by using RT-PCR;the expression profile under ABA,PEG and NaCl treatments were analyzed,and the function was detected in vitro.The results showed that TaDHN-2 encodes 152 amino acids,including a Y(VDEYGNP)and S(SSSSSEDDGMGGRRKK)segment in N-terminus,as well as,two conserved K segments(KIKEKLPG)in C-terminus.These characteristics indicated that TaDHN-2 belongs to subclass YnSKn of dehydrin family.The expression profiling revealed that TaDHN-2 was induced by plant hormone ABA,which implied this dehydrin is regulated by ABA dependent signal pathway.Under the osmotic stress,the dehydrin gene demonstrated different expression patterns between two wheat cultivars,CS(Chinese Spring)and LH(Luohan 2).In root tissues,TaDHN-2 was up-regulated when induced by osmotic stress,significantly higher in CS than that in LH,while in leaf tissues,the expression level is higher in LH.In order to validate the activity of TaDHN-2,the protein was expressed in E.coli and purified by affinity method.Interestingly,it was found that TaDHN-2 with the concentration of 100μg·mL^-1 effectively increased the activity ofα-amylase by 2 times at 60℃.Additionally,under the low temperature condition of 4℃,the activity ofα-amylase was not increased until the concentration of TaDHN-2 over 20μg·mL^-1,and the enzyme activity was increased by 1.6 times when the concentration of TaDHN-2 reached to 100μg·mL^-1.The results indicated that TaDHN-2 has protective effects on enzyme activity.