大叶落地生根SAHH基因功能鉴定  

Functional Identification of SAHH Gene from Kalanchoe daigremontiana

作　　者：李珊珊[1] 朱晨[1] 滕珂[1] 刘成兰[1] 曾会明[1] 

机构地区：[1]北京林业大学,北京100083

出　　处：《农业生物技术学报》2016年第4期538-547,共10页Journal of Agricultural Biotechnology

基　　金：国家高技术研究发展计划(863计划)项目(No.2013AA102607)

摘　　要：S-腺苷-L-高半胱氨酸水解酶(S-adenosyl-L-homocysteine hydrolase,SAHH)是已知的唯一能够水解S-腺苷高半胱氨酸(S-adenosylhomocysteine,SAH)成半胱氨酸和腺苷的酶。SAH作为转甲基反应的抑制剂,与S-腺苷甲硫氨酸(S-adenosyl-L-methionine,SAM,体内甲基供体)的比值常用来作为衡量植物整体甲基化水平的重要指标。为鉴定大叶落地生根(Kalanchoe daigremontiana)SAHH的功能,本研究根据已知的大叶落地生根SAHH基因(Kd SAHH)序列(Gen Bank登录号:KF953475)设计引物,对其进行干旱胁迫下的表达分析、亚细胞定位分析以及烟草(Nicotiana tabacum)转化,并对T1代苗用20%PEG6000模拟干旱处理,测定0、12和24 h的相对含水量(relative water content,RWC)、叶绿素(chlorophyll,Chl)含量、丙二醛(malondialdehyde,MDA)含量、过氧化物酶(peroxidase,POD)含量、超氧化物歧化酶(superoxide dismutase,SOD)含量等与抗旱相关的生理指标。结果表明,Kd SAHH基因在轻度(20 d)、中度(35 d)、重度(50 d)干旱胁迫下表达量依次升高,表明该基因与抗旱性相关;亚细胞定位结果显示,该蛋白定位于细胞质与细胞核;对转基因烟草进行PCR检测,获得9株阳性植株;测定干旱胁迫下的相关生理指标,结果显示,与野生型烟草相比,转Kd SAHH烟草在干旱胁迫下能够保持相对稳定的RWC、Chl含量以及相对较低的MDA、POD含量。与野生型相比,转Kd SAHH烟草受伤害轻,抗旱性增强,表明Kd SAHH基因能够提高烟草植株的抗旱性。研究结果丰富了Kd SAHH基因的功能,为后续研究该基因提供了借鉴和参考。S-adenosyl-L-homocysteine hydrolase （SAHH） is the only known enzyme to catalyze the breakdown of S-adenosylhomocysteine （SAH） into homocysteine and adenosine. SAH is the inhibitor of most transmethylation reactions which is often compared with S-adenosyl-L-methionine （SAM, one of methyl donor） to evaluate the overall methylation level in plant. In order to identify the function of SAHH in Kalanchoe daigremontiana （KdSAHH）, primers were designed according to the full length of KdSAHH cDNA in GenBank （Accession No. KF953475）. The KdSAHH gene expression pattern under drought stress （light drought stress（20 d）; middle drought stress （35 d）; high drought stress （50 d））, subcellular location in cell and ectopic transformation in tobacco （Nicotiana tobacum） were done to unveil the basic biological function. Drought stress was simulated with 20% PEG6000 in the seedling stage between transgenic and wild type tobacco and drought-related physiological indexes like leaf relative water content （RWC）, chlorophyll （Chl） content, malondialdehyde （MDA） content, peroxidase （POD） content, superoxide dismutase （SOD） content were measured at 3 different time points （0, 12 and 24 h）. The semiquantitative RT-PCR analysis showed that the highest expression level of KdSAHH gene was measured in high drought stress （50 d）, followed by middle drought stress （35 d） and light drought stress （20 d）. The result of subcellular location indicated that KdSAHH gene was located both in nucleus and cytoplasm. Nine positive tobacco transgenic line were obtained by PCR. Physiological indicators of transgenic and wild type tobacco under 3 time periods （0, 12 and 24 h） of 20% PEG6000 treatment showed that both of them showed obvious water loss symptoms at 24 h, but the transgenic tobacco of KdSAHH gene had lower wilting degree compared with wild type tobaccos, and some leaves at the upper end of the transgenic plant were still standing, while all leaves of wild type tobaccos

关 键 词：大叶落地生根 S-腺苷-L-高半胱氨酸水解酶基因(SAHH) 亚细胞定位 表达分析 烟草转化 

分 类 号：Q78[生物学—分子生物学]