摘要
【目的】鉴定对萼猕猴桃(Actinidia valvata)CDPK家族基因,并分析其在不同组织的表达模式以及对盐胁迫和淹水胁迫的响应。【方法】基于3代全长转录组测序数据,通过多种生物信息学手段,分析和鉴定对萼猕猴桃CDPK家族基因,利用实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)技术分析这些基因在不同组织中的表达,及在不同非生物胁迫条件下的表达情况。【结果】在对萼猕猴桃基因型KR5的全长转录组测序数据中共鉴定出63个CDPK基因,命名为AvCDPK1~AvCDPK63。系统发育分析将AvCDPK基因蛋白分为4个亚家族,同一亚家族具有相似的结构和基序(motif)。AvCDPK基因存在明显的组织表达特异性。AvCDPK49在盐胁迫和淹水胁迫条件下,均显著诱导表达,Av CDPK30和31在2种胁迫下均显著抑制表达。【结论】在对萼猕猴桃中共鉴定出63个CDPK基因,系统发育树显示Av CDPK基因家族与拟南芥CDPK基因家族在进化上高度保守。不同组织中AvCDPK的表达量存在明显差异,其中Av CDPK49受盐害、淹水诱导显著高表达,表明其可能在猕猴桃的耐盐和耐涝响应过程中发挥着重要作用。
【Objective】The experiment was conducted to identify the CDPK family genes in Actinidia valvata and analyze their expression patterns in different tissues and responses to salt and waterlogging stress.【Methods】Based on the full-length transcriptome sequencing data of third-generation RNA-seq,the CDPK family genes of Actinidia valvata were analyzed and identified by various bioinformatics methods.qRT-PCR was used to analyze the expression of these genes in stem,leaf,petiole,pedicel,sepal and petal,as well as their expressions under different abiotic stresses.One year old KR5 kiwifruit plantlets(16 cm×16 cm)under waterlogging stress were placed in a blue plastic turnover box(45 cm×35 cm×16 cm)filled with water.The water level was kept at 2 cm above the soil surface.Four time points of 0,3,7 and 11 d were set as sampling times.The root samples after waterlogging stress were harvested as experimental materials.KR5 potted plantlets(16 cm×16 cm)were cultured in a plastic container(39 cm×29 cm×12 cm)filled with Hoagland nutrient solution.Oxygen was supplied by an oxygenerator.The concentration of salt treatment was 0.6%NaCl.The sampling time points were set as 0,0.5,1,3,5 and 7 d.【Results】A total of 63 CDPK genes,named as AvCDPK1-AvCDPK63,were iden-tified from the full-length transcriptome sequencing data of kiwifruit genotype KR5.They all have typical characteristic domains:variable domain,catalytic domain(activator domain),junction domain(autoinhibitory domain)and regulatory domain(calmodulin-like domain/CAM-LD).Furthermore,the CDS sequence length,relative molecular weight,isoelectric point,EF-hand structure,palmitoylation and myristoylation sites of AvCDPK family genes were analyzed.The CDS sequences of 63 AvCDPK genes family members ranged from 1068 bp to 1893 bp,with amino acid length ranging from 355(AvCDPK54 and 60)to 630 aa(AvCDPK18 and 19),molecular weight ranging from 40.10 to 70.97,and isoelectric point ranging from 5.10 to 9.13.Through statistical analysis,53 AvCDPKs have four EF-hand domains,and 10 AvCDPKs have three EF-hand domains(AvCDPK3,14,23,25,28,51,59,61,62 and 63).EF-hand domain is the site of recognition and binding of Ca^(2+),which indicates that different members of AvCDPK family genes may play a different rolein change of Ca^(2+)concentrations.According to the prediction of 63 amino acid modification sites of AvCDPK proteins,37 members have palmitoylation site,11 members have myristoylation site,and 15 members have both palmitoylation and myristoylation sites.Through phylogenetic analysis,same as AtCDPK(Arabidopsis thaliana)gene family,AvCDPKs were divided into four subfamilies.The genes in the same subfamily had similar gene structure and motifs.In addition,we identified 15 conserved motifs,of which motif10 was distributed in subfamiliesⅢandⅣ,but not inⅡand some members of subfamiliesⅠ.Motif15 exists in subfamiliesⅠandⅢ,while subfamiliesⅡandⅣare absent.Motif14 only exists in subfamilyⅡ.AvCDPK genes have obvious tissue-specific expression.For example,AvCDPK11 was highly expressed in leaves,but AvCDPK29,41 and 63 were low expressed;AvCDPK43 was highly expressed in petioles,but low expressed in sepals and flowers;AvCDPK36 was highly expressed in pedicels,and AvCDPK36,38,43 and 53 were low expressed in petals.These results suggest that different AvCDPK gene may play different roles in growth and development of Actinidia valvata genotype KR5.The expression levels of AvCDPK6,11,28,44,45,49 and 61 were up-regulated under salt stress,and the expression levels of AvCD-PK36,41,44,45,46,47,48,49 and 50 were up-regulated in waterlogging test.The expression levels of AvCDPK44,45 and 49 were up-regulated in both salt stress and waterlogging stress(all up-regulated more than 2 folds).The expression levels of AvCDPK2,14,21,31 and 38 were down regulated by more than 10 folds under salt stress,and the expression levels of AvCDPK28,30 and 31 were down-regulated by more than 5 folds under waterlogging stress.The expression levels of AvCDPK31 were significantly down-regulated in both stresses.These results indicated that the different AvCDPK genes played different roles in the process of salt stress and waterlogging stress in Actinidia valvata.In addition,there were different expression patterns of the same gene under two stresses.The expression of AvCDPK28 increased significantly under waterlogging stress,but decreased under salt stress,indicating that AvCDPK28 may participate in different regulatory pathways under different stresses.【Conclusion】A total of 63 CDPK genes were identified in Actinidia valvata.Phylogenetic tree showed that these genes and AtCDPK genes were highly conserved in evolution.There were significant differences in the expression of AvCDPKs among different tissues.Three members were highly expressed,which were induced by salt and waterlogging stresses,indicating that these members may play an important role in the response to salt and waterlogging tolerance in Actinidia valvata.
作者
张永杰
白丹凤
Muhammad Abid
李志
方金豹
钟云鹏
ZHANG Yongjie;BAI Danfeng;MUHAMMAD Abid;LI Zhi;FANG Jinbao;ZHONG Yunpeng(Zhengzhou Institute of Fruit Trees/Key Laboratory of Fruit Tree Growth and Quality Control,Chinese Academy of Agricultural Sciences,Zhengzhou 450009,Henan,China)
出处
《果树学报》
CAS
CSCD
北大核心
2021年第10期1653-1667,共15页
Journal of Fruit Science
基金
国家自然科学基金青年科学基金(31801846)
中国农业科学院科技创新工程(CAAS-ASTIP-2020-ZFRI)
国家成都农业科技中心项目(NASC2020AR07)
中央级科研院所基本科研业务费专项(Y2019LM13)。
关键词
对萼猕猴桃
全长转录组
CDPK基因家族
盐胁迫
淹水胁迫
Actinidia valvata
Full-length transcriptome
CDPK gene family
Salt stress
Waterlogging stress
