两个南荻抗逆NAC基因MlNAC9和MlNAC10的功能解析

QIBEBT-IR > 资源植物与环境工程研究组

	两个南荻抗逆NAC基因MlNAC9和MlNAC10的功能解析
	赵珣
导师	周功克 ; 胡瑞波
	2016-05
学位授予单位	中国科学院研究生院
学位授予地点	北京
学位专业	生物工程
关键词	南荻非生物胁迫 Nac转录因子抗逆性活性氧
摘要	NAC(NAM、ATAF1/2、CUC2)类转录因子在植物的非生物胁迫响应中发挥着重要的调控作用。南荻是我国特有的多年生草本纤维类芒属能源植物，具有生物量高、抗逆性强、适应性广等优点，已成为今后纤维素燃料乙醇生产的重要原料来源之一。南荻能在边际土地种植，抗逆性是一个非常重要的指标，但是目前关于南荻NAC基因的文献报道较少。本文主要对南荻MlNAC9基因在非生物胁迫响应中的功能进行了研究。首先通过序列分析和进化分析，确定了MlNAC9与拟南芥的LOV1、ANAC034/035同源。使用hiTAIL-PCR技术克隆了MlNAC9的启动子序列，序列分析发现其中含有多种胁迫响应相关的顺式作用元件。构建了MlNAC9启动子驱动的GUS表达载体并侵染拟南芥，GUS染色结果表明，pMlNAC9::GUS主要在幼苗的子叶和真叶的叶脉、下胚轴以及叶柄中表达。构建MlNAC9蛋白C端融合YFP的表达载体，通过注射烟草叶片的瞬时表达，确定了MlNAC9定位于细胞核中。将MlNAC9基因的全长、N端和C端序列构建到酵母表达载体（pGBKT7）中，转化AH109进行转录激活实验，结果表明MlNAC9为转录激活因子，其转录激活域位于C端。构建过表达载体（CaMV 35S启动子驱动）并转化野生型拟南芥，获得过表达转基因植株。在正常生长条件下，MlNAC9过表达植株和WT的表型无差异。通过ABA和NaCl处理的种子萌发和根伸长实验，表明MlNAC9过表达的种子在萌发和根伸长过程中对NaCl耐受，对ABA敏感。在冷害、盐分和干旱胁迫处理下，MlNAC9过表达植株抗寒、耐盐和耐旱显著增强。通过qRT-PCR分析冷、干旱、ABA和NaCl胁迫下逆境响应相关基因的表达量，结果表明所有胁迫响应基因的表达量均上调，且上调倍数都显著高于野生型。在干旱、盐以及冷等逆境胁迫下，MlNAC9过表达的超氧化物歧化酶（SOD）、过氧化物酶（POD）、过氧化氢酶（CAT）等抗氧化酶的酶活显著高于野生型，而丙二醛（MDA）的含量则低于野生型，表明MlNAC9过表达植株增强了对活性氧的清除能力。此外，对MlNAC10的功能特性进行了初步的解析。通过hiTAIL-PCR技术克隆了MlNAC10的启动子序列，经序列分析发现多种胁迫响应相关的顺式作用元件。构建了MlNAC10启动子驱动的GUS表达载体并侵染拟南芥，构建了MlNAC10蛋白融合YFP表达载体，酵母表达载体以及过表达载体。通过烟草叶片细胞的瞬时表达，表明MlNAC10定位于细胞核中。酵母的转录激活实验，表明MlNAC10为转录激活因子，其转录激活域位于C端。过表达载体侵染野生型拟南芥，获得的过表达转基因植株在正常生长条件下与WT的表型并无差别。
其他摘要	NAC (NAM, ATAF1/2, CUC2) transcription factors are known to play important roles in response to abiotic stresses in plants. Currently, little information regarding the functional roles of NAC genes in stress tolerance is available in Miscanthus lutarioriparius, a promising bioenergy plant for lignocellulosic feedstock production. In this study, a detailed functional characterization of MlNAC9 in abiotic stress tolerance was carried out. Phylogenetic analysis revealed that MlNAC9 was clustered together with Arabidopsis LONG VEGETATIVE PHASE ONE (LOV1, ANAC034/035). The MlNAC9 promoter was amplified by hiTAIL-PCR. Sequence analysis revealed that a number of stress-responsive related cis-acting elements were present in the MlNAC9 promoter. The MlNAC9 promoter fragment was inserted upstream of GUS reporter gene in pCXGUS-P vector and transformed into Arabidopsis Col-0. The results indicated that pMlNAC9::GUS was mainly expressed in the hypocotyl, euphylla, hypocotyl and petiole. The C-terminus YFP fusion construct of MlNAC9 gene driven by CaMV 35S promoter were transiently expressed in tobacco epidermal cells. Subcellular localization of MlNAC9-YFP fusion protein indicated that MlNAC9 is localized in the nucleus. The entire coding region, N-terminal domain and C-terminal domain of MlNAC9 gene was separately cloned into pGBKT7. The constructs and empty vector pGBKT7 (negative control) were transformed into yeast strain AH109. Transactivation assay in yeast cells demonstrated that MlNAC9 function as a transcription activator and its activation domain is located in the C-terminus. Under the normal growth condition, overexpression of MlNAC9 lines displayed no differences with Arabidopsis Wild type (WT) in growth phenotypes. The effects of ABA and NaCl treatment on seed germination and root elongation of in MlNAC9 overexpression lines were examined, the results showed that MlNAC9 overexpression confers enhanced salinity tolerance and ABA hypersensitivity at both stages of seed germinationand root elongation. Overexpression of MlNAC9 in Arabidopsis significantly enhanced drought, salt and cold tolerance. qRT-PCR analysis showed that the stress-responsive marker genes involved in cold, drought, ABA and NaCl stresses were significantly up-regulated in transgenic lines compared to WT. Further more, the activities of antioxidant enzymes superoxidedismutase (SOD), peroxidase (POD) and catalase (CAT) activities were significantly increased and the malondialdehyde (MDA) was lower accumulated in MlNAC9 overexpression lines under drought, salt and cold treatments. Correspondingly, MlNAC9 overexpression lines enhanced the scavenging ability of reactive oxygen species (ROS). In addition, we carried out a preliminary study about the functional role of MlNAC10 in abiotic stress tolerance. The MlNAC10 promoter was amplified by hiTAIL-PCR. Sequence analysis revealed that a number of stress-responsive related cis-acting elements were present in the MlNAC10 promoter. The MlNAC10 promoter fragment was inserted upstream of GUS reporter gene in pCXGUS-P vector and transformed into Arabidopsis Col-0. The C-terminus YFP fusion constructs, yeast transactivation vectors and overexpression vector under the control of CaMV 35S promoter were constructed. Subcellular localization of MlNAC10-YFP fusion protein indicated that MlNAC10 is a nuclear protein. Transactivation assay in yeast cells demonstrated that MlNAC10 function as a transcription activator and its activation domain is located in the C-terminus. Under the normal growth conditions, overexpression of MlNAC10 displayed no differences with Arabidopsis Col-0 in growth phenotypes.
作者部门	植物代谢工程
学科领域	植物代谢工程
公开日期	2017-12-31
学位类型	硕士 ; 学位论文
语种	中文
文献类型	学位论文
条目标识符	http://ir.qibebt.ac.cn/handle/337004/9787
专题	资源植物与环境工程研究组
作者单位	中国科学院青岛生物能源与过程研究所
推荐引用方式 GB/T 7714	赵珣. 两个南荻抗逆NAC基因MlNAC9和MlNAC10的功能解析[D]. 北京. 中国科学院研究生院,2016.

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