蓝莓VcLon1基因的克隆、表达及抗旱性分析

Cloning,Expression and Analysis of Drought Resistance of VcLon1 Gene from Blueberry

【目的】Lon1蛋白酶具有降解叶绿体和线粒体内氧化蛋白、维持细胞正常代谢的功能。在分析蓝莓VcLon1时空表达模式的基础上,利用烟草遗传转化技术结合干旱生理分析,揭示蓝莓VcLon1的生物学功能,为运用生物技术培育蓝莓抗旱品种提供理论基础,并为其他植物抗旱机制研究提供基础信息。【方法】在从南高丛蓝莓‘夏普蓝’中克隆得到VcLon1全长(Gen Bank登录号:MF972079)的基础上,利用DNAMAN、MEGA4.0、Wo LF PSORT和Protparam等生物信息学软件及在线程序预测VcLon1序列及蛋白结构特征,分析比对氨基酸序列同源性并构建其同源蛋白序列进化树;同时,采用实时荧光定量PCR分析蓝莓VcLon1在不同组织及干旱条件下的表达模式;最后,构建表达载体,遗传转化本氏烟,以野生型、VcLon1超表达这2种基因型本氏烟为材料,进行干旱处理并分析二者在生物量生长、光合生理及氧化胁迫水平等方面的差异。【结果】1)PCR扩增得到VcLon1的ORF序列长2 982 bp,该序列编码993个氨基酸,预测蛋白的理论等电点为5.44,分子量为109.5 kDa。VcLon1编码的蛋白序列含有1个典型的AAA+结构域,属于AAA+超家族,定位分析编码蛋白在叶绿体和线粒体中表达,该蛋白与葡萄、苹果等Lon1蛋白酶亲缘关系较近。2)利用实时荧光定量PCR对VcLon1的表达量分析,发现其在‘夏普蓝’蓝莓各组织中均有表达,老叶中的表达量显著低于嫩叶,干旱胁迫显著提高蓝莓VcLon1的转录水平。3)干旱胁迫下各株系本氏烟生长均受到抑制,但VcLon1超表达植株均较野生型生长健壮,其中又以VL-6长势最佳,其叶片未发黄且根系发达,其株高、干质量分别比野生型高36.66%、114.29%。4)干旱胁迫下野生型本氏烟叶绿体肿胀明显且部分基粒片层结构模糊,分层不明显,而VcLon1超表达本氏烟仅部分叶绿体基粒片层空隙增大,其叶绿体超微结构未出现明显损伤,且叶片叶绿素含量高于野生型。同时,野生型本氏烟胞内线粒体普遍出现肿胀、变形,嵴断裂、解体并空泡化的现象,而VcLon1超表达本氏烟的线粒体仍维持正常椭球形。5)在氧化胁迫水平方面,VcLon1超表达植株干旱胁迫下的MDA含量均比野生型低34.38%~49.68%,且其叶片中H2O2的积累也较低,而野生型叶片的褐色面积明显上升。羰基化蛋白含量也呈现相似的变化趋势,VcLon1超表达本氏烟比相同条件下的野生型低36.89%,这可能由于VcLon1超表达植株各株系中SOD、GR、APX及POD等抗氧化酶的活性普遍显著高于野生型所致。【结论】干旱胁迫下,南高丛蓝莓‘夏普蓝’内VcLon1的运作机制可能是通过维护细胞膜系统及叶绿体的正常形态结构,同时通过降解线粒体内羰基化蛋白质,使线粒体结构保持完整、能量代谢等功能得以正常维护,减少活性氧自由基(ROS)的产生及维护抗氧化酶类活性,从而有效地降低细胞器内ROS的产生与积聚,并最终降低胞内的氧化胁迫水平,维持正常植物代谢,提高其抗旱能力。

【Objective 】 Lon1 protease plays a critical role in degrading oxidized proteins in chloroplast and mitochondria and maintaining the normal cellular metabolism. Up to now,its function related to abiotic stress tolerance has attracted little attention. Based on an analysis of the patterns of temporal and spatial expressions of blueberry VcLon1 gene（ Gen Bank number： MF972079）,the tobacco genetic transformation technique and drought tolerance assay were applied to elucidate the biological functions of VcLon1 involved in drought stress tolerance in plants. This study wouldprovide a theoretical basis for developing biotechnology of drought-resistant blueberry germplasms and provide basic information for understanding drought resistance mechanism of other plants.【Method】 On the basis of cloning VcLon1 full length from Vaccinium corymbosum ‘Sharpblue’,Protparam and CDD software was used to analyze the protein structure of VcLon1. Phylogenetic tree of Lon1 proteins was constructed by MEGA4. 0 software. The expression levels of VcLon1 in different tissues and in drought stress condition was analyzed by real-time fluorescence quantitative PCR（ q PCR）. After drought stress treatment,the differences of chloroplast ultrastructure,photosynthetic physiology and oxidative stress between wild type（ WT） and VcLon1 overexpressing Nicotiana benthamiana were investigated.【Result】 1） The ORF of VcLon1 was 2 982 bp,which encoding 993 amino acids. The theoretical isoelectric point was 5. 44 and the molecular weight was 109. 5 k Da. The protein sequence encoded by VcLon1 contains a typical AAA ＋ domain and belongs to the AAA ＋ superfamily. Localization analysis showed that the coding protein was expressed in chloroplast and mitochondria,and had close relationship with Lon1 protease such as grape and apple. 2） The q PCR analysis revealed that VcLon1 was expressed ubiquitously throughout‘Sharpblue’plant,with relatively lower expression levels in older tissues. The drought stress induced significantly higher transcription level of VcLon1 in blueberry. 3） The overexpression of VcLon1 in tobacco led to increased drought tolerance. The largest biomass was observed in line of VL-6,which exhibited green leaves and well-developed roots,and the height and dry weight were 36. 66% and 114. 29% higher than those of the wild type,respectively. 4） Under drought stress,the chloroplast was swollen and some of the lamellar structures were blurred of WT.However,no obvious damage was observed in chloroplast ultrastructure of VcLon1 overexpressed lines except some of the voids in the chloroplast matrix were increased,and the chlorophyll content of VcLon1 overexpressed lines was higher than that of the wild type. Meanwhile,the mitochondria of wild-type were swollen and metabolic,and the crista were fractured,disintegrated and vacuolated. The mitochondria in VcLon1 overexpressed lines still maintain the normal ellipsoid shaped structure. 5） Under drought stress,MDA content of VcLon1 overexpression plants was 34. 38%-49. 68% lower than WT,while less H2 O2 accumulation occurred in leaves of VcLon1 overexpression plants,as indicated by their brown areas.Comparatively,the brown area of the WT increased significantly. Furthermore,under drought stress,the content of carbonylated protein in the overexpressed plants was also significantly lower than that of WT,which may result from the generally higher activities of SOD, GR, APX and POD in VcLon1 overexpressed plants, as compared with WT.【Conclusion】Under drought stress,VcLon1 may play a key role in keeping the normal morphology of the chloroplasts and mitochondria and to protect the cell membrane system. Besides,the normal energy metabolism may maintain by degrading carbonylated protein in mitochondrial and in reducing productions of reactive oxygen species（ ROS）. Moreover,VcLon1 helps to maintain relatively higher activities of antioxidant enzymes to reduce the accumulations of intracellular ROS,ultimately alleviate the intracellular oxidative stress,which facilitate the maintenance of the normal cellular metabolism and improve the drought resistance capacity.