摘要
【目的】探讨枳氮素含量在干旱条件下的变化,以及氮代谢途径枳亚硝酸还原酶基因(NiR)和NLP转录因子的响应表达,分析确定枳NLP转录因子与NiR启动子区域硝酸盐响应顺式作用元件(nitrate-responsive cis-element,NRE)位点绑定互作。【方法】以一年生的砧木枳为试验材料,进行干旱处理直至植株叶片开始萎蔫,并设置对照。利用凯氏定氮仪检测干旱条件下枳叶片和侧根的全氮含量,并同步利用实时荧光定量PCR(RT-qPCR)和2-ΔΔCT法分析PtNiR、PtNLP2、PtNLP4、PtNLP7及PtNLP8在枳叶片和侧根的表达情况。通过基因克隆并检索与比对分析,获取PtNiR启动子区域的硝酸盐响应顺式作用元件序列,之后利用同源克隆方法构建用于酵母单杂交的pHIS2-4×NRE和p GADT7-Rec-NLP载体,将构建的pHIS2-4×NRE和p GADT7-Rec-NLP载体质粒共同转化到酵母菌株Y187中,在氨基酸缺失培养基(SD/-Trp/-Leu,以及SD/-His/-Trp/-Leu/+120 mmol·L^(-1) 3-氨基三唑)上30℃恒温培养3—5 d,观察酵母的生长情况,以确定枳NLP转录因子(PtNLP2、PtNLP4、PtNLP7及PtNLP8)与NRE的互作关系。【结果】受干旱的影响,枳叶片全氮含量表现出先升高再下降的趋势,而侧根中全氮含量则显著下降;在对照中,枳叶片和侧根全氮含量均显著上升。RT-qPCR分析表明,PtNiR的表达水平在叶片中先上调然后再显著下调,而在侧根中则随着干旱程度的增加显著下调;PtNLP2、PtNLP4及PtNLP7在枳叶片和侧根中的表达也均呈现不同程度的先上调然后再受到抑制的规律,而PtNLP8在枳叶片和侧根中的表达则在一定程度上受到干旱抑制。通过克隆测序分析,在枳NiR启动子区域-196至-154的位置发现了疑似NRE。酵母单杂交分析表明,转化pHIS2-4×NRE-HIS3载体和p GADT7-Rec-NLP载体的Y187酵母在对照培养基(-Trp/-Leu)和含有120 mmol·L^(-1) 3-氨基三唑的三缺培养基(-His/-Trp/-Leu)上均能正常生长。【结论】枳侧根中的全氮含量受干旱的影响而显著下降,以对照为参比,枳侧根和叶片中的全氮含量均随着干旱时间的延长而相对减少。氮代谢途径的枳NiR和NLP转录因子在叶片和侧根中的表达对干旱有不同程度的响应。PtNLP2、PtNLP4、PtNLP7及PtNLP8转录因子均能与PtNiR启动子中的硝酸盐响应顺式作用元件(NRE)位点绑定互作,从而激活下游基因的表达。
【Objective】The objective of this study is to investigate the changes of nitrogen content and the expression of the nitrite reductase gene（NiR） and NIN-like protein（NLP） transcription factors of Poncirus trifoliata（L.） Raf. under the drought conditions, and to analyze and confirm the binding interaction of NLP transcription factors and nitrate-responsive cis-element（NRE） of NiR promoter region. 【Method】One-year old rootstock P. trifoliata was used as the experimental material and treated with drought stress until the leaves of the plant began to wilt, and the water control was set. The total nitrogen contents in leaves and lateral roots under drought condition were measured by Kjeldahl nitrogen meter. At the same time, the gene expression of PtNiR, PtNLP2, PtNLP4, PtNLP7 and PtNLP8 under different drought conditions in leaves and lateral roots was analyzed using real-time quantitative PCR and 2^-ΔΔCT method. The NRE sequence in the promoter region of PtNiR was analyzed. The constructed vector pHIS2-4×NRE and p GADT7-Rec-NLP of Y1 H were screened using Ligation-Free Cloning Kit（abm）, and the interaction of P. trifoliata NLP transcription factors（PtNLP2, PtNLP4, PtNLP7 and PtNLP8） with NRE was analyzed by Y1 H screening using a HIS3 reporter gene under the control of NRE（4×NRE）, pHIS2-4×NRE and p GADT7-Rec-NLP plasmids were transformed into yeast Y187, and were incubated at 30℃ for 3-5 days in amino acid deficient medium SD/-Trp/-Leu（control） and SD/-His/-Trp/-Leu/＋120 mmol·L^-1 3-aminotriazole（3-AT）, and the relationship between NLPs transcription factor and NRE was determined by observing the growth of Y187 yeast. 【Result】The total nitrogen content in leaves of P. trifoliata increased first and then decreased, while the total nitrogen content in lateral roots decreased significantly under the drought conditions. In the water control, the contents of total nitrogen in leaves and lateral roots increased significantly. RT-qPCR analysis showed that the expression of PtNiR in leaves was up-regulated firstly and then down-regulated, while in lateral roots, it was significantly down-regulated with the increase of drought. The expression of PtNLP2, PtNLP4 and PtNLP7 in leaves and lateral roots was also up-regulated firstly and then inhibited. In contrast, the expression of PtNLP8 in leaves and lateral roots was inhibited to a certain extent by the drought. By cloning and sequencing, the NRE sequence was identified at the location of-196 to-154 in the promoter region of PtNiR. Y1 H assays show that the Y187 yeast transformed into pHIS2-4×NRE-HIS3 and p GADT7-Rec-NLP vector could grow normally in the control medium（-Trp/-Leu） and the nutrient deficient medium（-His/-Trp/-Leu） containing 120 mmol·L^-1 3-AT.【Conclusion】The content of total nitrogen in lateral roots of P. trifoliata decreased significantly by drought. Compared with the control, the total nitrogen contents in leaves and lateral roots reduced relatively with the extension of drought treatment time. The expression of NiR and NLP transcription factors in leaves and lateral roots of the nitrogen metabolism pathway was responsive to the drought in varying degrees. Y1 H assays show that PtNLP2, PtNLP4, PtNLP7 and PtNLP8 transcription factors can bind to the NRE of PtNiR promoter to activate the expression of the downstream genes.
作者
曹雄军
卢晓鹏
熊江
李静
谢深喜
CAO XiongJun1,2, LU XiaoPeng1, XIONG Jiang1, LI Jingl, XIE ShenXi1(1.College of Horticulture and Landscape, Hunan Agricultural University/National Center for Citrus Improvement （Changsha） Changsha 410128; 2.Grape and Wine Research Institute, Guangxi Academy of Agricultural Science, Nanning 530007)
出处
《中国农业科学》
CAS
CSCD
北大核心
2018年第17期3370-3378,共9页
Scientia Agricultura Sinica
基金
国家现代农业(柑橘)产业技术体系建设专项资金(CARS-026)
关键词
枳
NLP转录因子
干旱胁迫
硝酸盐响应顺式作用元件
绑定
Poncirus trifoliata （L.） Raf.
NIN-like protein transcription factor
drought stress
nitrate-responsive cis-element（NRE）
binding
