外源硼元素对喀斯特地貌先锋植物黄褐毛忍冬LfMYB基因表达的影响

Influences of Exogenous Boron Stress on Expression of LfMYB in Pioneer Plant Lonicera fulvotomentosa Hsu et S.C.Cheng under Karst Landforms

为了明确黄褐毛忍冬对矿质元素胁迫的响应机制,该研究采用转录组测序筛选和RACE克隆的方法分离并鉴定了喀斯特地貌地区的先锋植物黄褐毛忍冬MYB转录因子基因LfMYB,进行LfMYB原核表达活性检测和转录活性验证,并测定不同浓度(0、0.25、1.0、2.5 mg/L)硼(B)元素处理下黄褐毛忍冬不同部位B元素含量的变化,采用qRT-PCR分析LfMYB基因表达量的变化。结果表明:(1)LfMYB是转录激活因子,属于R2R3 MYB转录因子家族。(2)原核表达结果显示,pCOLD-LfMYB重组蛋白经0.2、0.4和0.8 mmol/L IPTG诱导后都能表达,蛋白大小为75 kD左右;转录活性实验表明,LfMYB在酵母中能够正确转录和翻译,且能够激活报告基因的转录和翻译,表明LfMYB有自激活活性。(3)qRT-PCR分析显示,黄褐毛忍冬LfMYB基因在老叶、白花和黄花中表达量明显高于其他部位,幼茎和幼叶表达量相对较低。(4)在喀斯特土壤基质中,B元素主要积累在黄褐毛忍冬的老叶和茎中,且随着喷施的B浓度升高,各部位B含量随之升高,其中以幼叶、老叶和幼茎中B含量升高最为明显(P<0.05);黄褐毛忍冬各部位的LfMYB基因表达量也随着B元素浓度增加而提高,但当外源B浓度为2.5 mg/L时,黄褐毛忍冬各部位的LfMYB表达量反而减少,且以幼叶、幼茎和绿蕾中的降低最明显(P<0.05)。该研究结果为探索黄褐毛忍冬耐受B胁迫的分子机制奠定了实验基础。

To investigate the expression and regulation mechanism of MYB in Lonicera fulvotomentosa under mineral element stress.The MYB transcription factor LfMYB of pioneer plant L.fulvotomentosa under karst landforms was isolated and identified by a combination of transcriptome sequencing and RACE cloning.Prokaryotic expression activity detection and transcription activity verification of LfMYB were carried out.The changes on the content of B element in different organs of L.fulvotomentosa were detected under different concentrations(0,0.25,1.0,2.5 mg/L)of boron(B).The expression characteristics of LfMYB were analyzed by qRT-PCR.The expression pattern of LfMYB was analyzed by the treatment of different concentrations(0,0.25,1.0 and 2.5 mg/L)of B element and qPCR analysis.The results showed:(1)LfMYB is a transcriptional activator belongs to the R2R3 MYB transcription factor family.(2)The prokaryotic expression results showed that the pCOLD-LfMYB recombinant protein could be expressed after different concentrations(0.2,0.4,0.8 mg/L)of IPTG induction and the size of the recombinant protein is about 75 KD.The transcription experiments showed that the LfMYB protein can be transcribed and translated correctly in yeast,and LfMYB can activate transcription reporter gene.All these proved that LfMYB has self-activating activity.(3)The results of qPCR analysis showed the expression of LfMYB in old leaves,white flowers,and yellow flowers was significantly higher than that in other parts,while the expression in young stems and young leaves was relatively low.(4)In karst soil,the B accumulated in the old leaves and stems of L.fulvotomentosa.When a certain concentration of B was applied to the karst soil,the B content in all organs of L.fulvotomentosa increased as the concentration of applied B increased,and the content of B in young stems and green buds increased most obviously,which reached a significant level(P<0.05).The expression of LfMYB increased in all organs of L.fulvotomentosa as the concentration of applied B increased.When the exogenous B concentration is 2.5 mg/L,the expression of LfMYB in all organs of L.fulvotomentosa decreased,among which the expression of LfMYB in young leaves,young stems and green buds decreased most obviously,which reached a significant level(P<0.05).This study provides an experimental basis for revealing the molecular mechanism of B tolerance in L.fulvotomentosa.