葡萄APX基因家族的鉴定与表达分析

Identification and expression analysis of APX gene family in grape

【目的】当植物遭受土壤盐碱、干旱和低温等非生物逆境胁迫时,其体内产生大量活性氧(Reactive Oxygen Species,ROS),影响植物正常生长发育甚至死亡。抗坏血酸过氧化物酶(Ascorbate Peroxidase,APX)能够有效清除逆境胁迫下产生的ROS,从而保护植物免受损害。本研究旨在葡萄全基因组中鉴定出APX基因家族成员,并探究其在非生物胁迫下的表达情况,为后续葡萄抗逆基因的挖掘和功能研究提供理论依据。【方法】以生长30 d的葡萄‘黑比诺’试管苗为材料,通过生物信息学方法分析该基因家族的理化性质、系统进化、启动子顺式作用元件分布、基因芯片表达情况,利用qRT-PCR分析该基因家族成员在不同逆境胁迫下表达情况。【结果】葡萄APX基因家族具有7个成员,其氨基酸数目分布在104~421,等电点(Isoelectric Point,pI)介于5.75~8.77。系统进化分析发现,葡萄VvAPX与水稻OsAPX同源性较高,拟南芥AtAPX次之。蛋白质二级结构以α-螺旋和不规则卷曲为主。亚细胞定位预测发现,VvAPX基因在叶绿体、线粒体和细胞质中表达较高。VvAPXs基因均含有A活性位点、H结合位点以及motif1保守基序。顺式作用元件分析结果发现,VvAPXs基因家族所有成员中均含有干旱胁迫应答元件(MYB)和抗旱元件识别位点(MYC)。基因芯片表达图谱显示,在高盐、PEG、低温处理条件下,与CK3相比7个基因均上调表达,其中VvAPX04较明显。qRT-PCR结果表明,VvAPX04在200 mmol·L-1NaCl处理下与对照相比显著性上调,是对照的14.5倍,在10%PEG处理下的表达量是对照的1.5倍。VvAPX05在10%PEG处理下与对照相比显著性上调,是对照的1.5倍。【结论】初步鉴定并提供了葡萄APX基因家族成员信息,预测了所具有的部分功能,为进一步研究葡萄APX基因参与非生物逆境胁迫的机制提供参考。

【Objective】Grape is one of the oldest fruit trees in the world,native to Europe and Asia.Although the cultivation area of grape is very large and wide,grape cultivation is now facing many abiotic stresses,such as drought,low temperature,salt damage and so on.APX gene plays an important role in removing ROS(reaction oxygen species)producing under stress in plants.This experiment aims to identify APX gene family members in grape,and predict the characteristics of this gene family,so as to provide a basis for the analysis of VvAPX genes’function and the mining and utilization of resources of resistant genes to the stresses in grape.【Methods】Plantlets of’Pinot noir’(Vitis vinifera)cultured in vitro,were stored in the laboratory and stem-segments with single bud were subcultured on the medium(GS+0.2 mg·L-1 IAA)in a growth chamber under 16 h of light/8 h of dark conditions at 25℃/20℃for30 d.The subcultures,which had consistent growth state and were healthy,were treated with abiotic stresses,including 10%PEG,500μmol·L-1 ABA,200 mmol·L-1 NaCl and 4℃low temperature and pure water as a CK.Each treatment was repeated three times.APX gene family members were searched from the grape genome in the website.The physicochemical property,phylogenetic relationship,cis-acting element distribution in the 2 kb upstream region of the promoter,gene chip expression were analyzed.The expression of genes under different abiotic stresses was analyzed using qRT-PCR.【Results】Seven APX gene members in the grape had been homogenously searched,and the gene family could be divided into two sub families(Ⅰ,Ⅱ).The physicochemical property analysis showed that VvAPX02 had the longest protein sequence,CDS sequence and the largest molecular weight in all genes.In addition,VvAPX07 had the shortest protein sequence and the largest full length of genome.We find that all genes were located on 5 chromosomes of the grape,namely the 3 th,the 4 th,the 6 th,the 8 th and the 18 th chromosomes,respectively.Among them,both the 4 th and the 18 th chromosomes had two genes,the former had VvAPX06 and VvAPX07,and the latter had VvAPX01 and VvAPX02.Analysis of gene structure showed that VvAPX07 had the longest intron region beyond 14 kb.Genes from the same subgroup had similar structures,such as VvAPX03 and VvAPX04 in groupⅠ,both of them had 8 exons.VvAPX02 had 12 exons,VvAPX01 had 11 exons,VvAPX06 had 10 exons,VvAPX05 had 9 exons,and VvAPX07 had 3 exons.Besides,only VvAPX01,VvAPX02,VvAPX03 and VvAPX05 had integrated structure.VvAPX04 had no downstream(3’UTR),and VvAPX06 and VvAPX07 had no upstream(5’UTR)and downstream.Multiple sequence alignment revealed that the seven APX genes had high homology because all of them had active site A and binding site H.The conservative motif analysis showed that all APX genes in the grape had motif1,among them VvAPX01 and VvAPX07 only had motif1.Secondary structure analysis showed that the 7 proteins encoded by VvAPX gene family were mainly composed of alpha helix and random coil.The subcellular localization indicated that APX gene family was mainly expressed in cytoplasm,chloroplast,nucleus,mitochondria,vacuole and plasma membrane.VvAPX03,VvAPX04,VvAPX05,VvAPX06 and VvAPX07 were expressed in cytoplasm.VvAPX01,VvAPX02,VvAPX05,VvAPX06 and VvAPX07 were expressed in chloroplast.VvAPX03 and VvAPX06 were expressed in nucleus.VvAPX02,VvAPX03,VvAPX04,VvAPX05 and VvAPX07 were expressed in mitochondria.VvAPX05 was the only one expressed in the vacuole.Both VvAPX02 and VvAPX06 were expressed in plasma membrane.The cis-acting element analysis showed that all VvAPX genes contained MYB and MYC,which were concerned with drought resistance.Gene chip expression analysis showed that the expression level of VvAPX01,VvAPX03 and VvAPX07 decreased significantly under ABA stress.Compared with the control group 3(CK3),all of genes were up-regulated under the treatments of salt,PEG and 4℃,among which VvAPX01,VvAPX04 and VvAPX05 were more obvious than the others.Besides,the genes family expressed differently under different abiotic stresses at different time,such as VvAPX03 was up-regulated significantly higher than those of the control group 3(CK3),salt and PEG treatment for 24 h was up-regulated significantly higher than them under other time period.Real-time fluorescence quantitative analysis showed that the VvAPX genes family expressed distinguishingly under different abiotic stresses,including 500μmol·L-1 ABA,200 mmol·L-1 NaCl,10%PEG and 4℃low temperature,and there were significant differences in the expression levels between the treatments.It was found that the expression pattern of VvAPX02,VvAPX03,VvAPX06 and VvAPX07 were analogous,the expression of all of them was lower than those of the control group(CK),500μmol·L-1 ABA,200 mmol·L-1 NaCl,10%PEG and 4℃stresses.The expression pattern of VvAPX01 and VvAPX05 were analogous,the expression of both of them was higher in the leaves under10%PEG than those of the control group(CK).VvAPX04 gene was distinctly up expressed,which was14.5 times higher than that of the control group(CK).【Conclusion】The experiment preliminarily identified the APX gene family members in the grape and predicted the functions of this gene family,which would provide references for further studies on APX gene functions in the grape.