大叶落地生根SAHH基因功能鉴定

发布时间：2018-05-17 16:47

本文选题：大叶落地生根 + S-腺苷-L-高半胱氨酸水解酶基因(SAHH)　；参考：《农业生物技术学报》2016年04期

【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。
[Abstract]:S-adenosyl-L-homocysteine hydrolase (S-adenosyl-L-homocysteine hydrolase SAHH) is the only known enzyme that can hydrolyze S-adenosylhomocysteine to cysteine and adenosylhomocysteine SAH as an inhibitor of transmethylation. The ratio of S-adenosyl-L-methionine (SAM) to S-adenosyl-L-methionine (S-adenosyl-L-methionine) is often used as an important index to evaluate the overall methylation level of plants. In order to identify the function of Kalanchoe daigremontiana)SAHH, primer was designed according to the known sequence of SAHH gene (Kd SAHH), Genin Bank accession number: KF953475), and the expression of KF953475 was analyzed under drought stress. Subcellular localization analysis and tobacco Nicotiana tabacum) transformation were performed. The relative water content, chlorophyll chlorophyllChl, malondialdehyde (malondialdehyde) and peroxidase (POD) contents in T1 generation seedlings were determined by 20%PEG6000 simulated drought treatment at 12 and 24 h, respectively. Superoxide dismutase (SOD) content and other physiological indexes related to drought resistance. The results showed that the expression of Kd SAHH gene increased under drought stress at 20 d, 35 d, 50 d, respectively, indicating that the gene was related to drought resistance, and the subcellular localization showed that the protein was located in cytoplasm and nucleus. Nine positive plants were obtained by PCR detection of transgenic tobacco, and the relative physiological indexes of transgenic tobacco under drought stress were determined. The results showed that compared with wild type tobacco, Under drought stress, the transgenic Kd SAHH tobacco could maintain a relatively stable content of RWCN Chl and a relatively low content of MDA-POD. Compared with wild type, transgenic Kd SAHH tobacco was less damaged and more drought-resistant, indicating that KD SAHH gene could improve the drought resistance of tobacco plants. The results enrich the function of KD SAHH gene and provide reference for further study of KD SAHH gene.
【作者单位】：北京林业大学;
【基金】：国家高技术研究发展计划(863计划)项目(No.2013AA102607)
【分类号】：S682.33

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