摘要
【目的】磷是植物生长发育所必需的大量营养元素,研究无机磷酸盐(inorganic phosphate,Pi)胁迫下羊草的响应,筛选不同浓度Pi胁迫下的内参基因,并分析Pi响应相关基因的表达,为羊草Pi胁迫响应的分子机制研究提供基础数据。【方法】以羊草幼苗为材料,进行不同浓度Pi处理培养,对羊草的株高和根长进行检测,利用钒钼黄比色法检测羊草植株中Pi的含量。根据羊草转录组数据选取7个候选内参基因,从NCBI核酸序列数据库选取1个候选内参基因,对羊草不同处理材料进行总RNA提取和cDNA合成,利用qRT-PCR对候选内参基因的表达进行检测,并通过geNorm、NormFinder和Bestkeeper软件对其稳定性进行评估。根据筛选获得的较稳定内参基因,对Pi响应基因的qRT-PCR检测数据进行相对定量分析。【结果】表型观测结果显示,无论是低Pi还是高Pi胁迫都使得羊草的生长减缓;株高对低Pi或缺Pi胁迫较为敏感,根长对高Pi胁迫较为敏感;并随着处理浓度的增加羊草中Pi的含量也增加。qRT-PCR溶解曲线分析显示8个候选内参基因均具有单一的溶解峰,基因表达谱分析表明8个候选内参基因的CT值范围是17.16-26.61,其中,LcGAPDH的表达丰度最高为17.16-20.22,Lc18SrRNA的表达丰度最低为23.28-26.61,CT值变异系数最小的为LcARPT(2.09%),最大的为LcTUA(6.8%)。综合geNorm、NormFinder和Bestkeeper稳定性排名,通过计算几何平均数,获得8个候选内参基因综合稳定性排名,其中排名靠前的3个基因分别为Lc18SrRNA、LcCAP和LcEF1α,排名最后的2个基因为LcTUA和LcTUB。分别以Lc18SrRNA、LcCAP和LcEF1α作为内参基因,qRT-PCR相对定量表达分析结果显示,与对照相比,LcPHO1-2的表达受低Pi或者缺Pi诱导;LcPAP2的表达受高Pi诱导;而LcPAP27的表达同时受低Pi和高Pi下调。【结论】低Pi和高Pi胁迫均使羊草生长受阻,羊草地上组织与地下组织对Pi胁迫响应的模式不同。筛选出3个�
【Objective】Phosphorus is the nutrient elements that is essential for plant growth.It would provide the basic data for molecular mechanism investigation of Leymus chinensis responding to inorganic phosphate(Pi)treatment,via analyzing the response under Pi stress in L.chinensis,and selection of the reference genes for qRT-PCR analysis,along with relative expression analysis of Pi-responsive genes.【Method】Using L.chinensis seedlings as materials,the length of shoots and roots were measured,and the contents of Pi were examined via vanadium molybdenum yellow colorimetric method following different concentration of Pi treatments.Based on transcriptome data of L.chinensis,as well as NCBI nucleic acid sequences database,7 and 1 candidate reference genes were selected,respectively.Total RNA was isolated from each sample after different concentration of Pi treatment.Expression level of candidate reference genes was examined through qRT-PCR,and the stability was calculated by ge Norm,Norm Finder and Bestkeeper.According to the most stable reference genes selected,the relative expression of Pi-responsive genes was analyzed according to the qRT-PCR results.【Result】The phenotype observation showed that both lower and higher concentration of Pi stresses reduced the growth of L.chinensis,and the shoot growth was more sensitive to low Pi(or Pi deficiency)stress,whereas root length was more sensitive to high Pi stress.In addition,the accumulation of Pi was enhanced along with the increased concentration of Pi treatments.The melting curve analysis showed that single peak of every candidate reference gene was observed,and gene expression profiles indicated that their CT values ranged from 17.16 to 26.61,among which Lc GAPDH exhibited the highest expression abundance with CT values ranged form 17.16 to 20.22,and Lc18 Sr RNA showed the lowest expression level with CT values ranged form 23.28 to 26.61.Lc ARPT(2.09%)showed the smallest expression variation,and Lc TUA(6.8%)had the largest expression variation.Finally,accordin
作者
万东莉
侯向阳
丁勇
任卫波
王凯
李西良
万永青
WAN DongLi;HOU XiangYang;DING Yong;REN WeiBo;WANG Kai;LI XiLiang;WAN YongQing(Institute of Grassland Research,Chinese Academy of Agricultural Sciences/Key Laboratory of Grassland Ecology and Restoration,Ministry of Agriculture,Hohhot 010010;College of Life Sciences,Inner Mongolia Agricultural University/Inner Mongolia Key Laboratory of Plant Stress Physiology and Molecular Biology,Hohhot 010011)
出处
《中国农业科学》
CAS
CSCD
北大核心
2019年第23期4215-4227,共13页
Scientia Agricultura Sinica
基金
内蒙古自治区自然科学基金(2019MS03002)
中央级公益性科研院所基本科研业务费项目(1610332016018,1610332018001)
内蒙古农业大学高层次人才引进科研启动项目(NDYB2018-61)
