荔枝CDPK基因家族鉴定及其在霜疫病胁迫下的表达分析

Identification of LcCDPKs and analysis of their expression patterns in response to downy mildew stresses in lychee

【目的】鉴定荔枝(Litchi chinensis Sonn.)钙依赖蛋白激酶(CDPK)基因家族,并分析其在不同组织和荔枝霜疫病胁迫下的表达模式。【方法】基于荔枝基因组数据,利用生物信息学方法鉴定CDPK家族基因,并对其序列特征、基因结构、启动子顺式作用元件、染色体定位和进化关系等进行分析。依赖276份荔枝种质材料,筛选高抗霜疫病的优良荔枝品种。另外,通过RNA-Seq和qRT-PCR方法检测高抗病荔枝品种中LcCDPK基因家族成员在荔枝霜疫病胁迫处理下的表达模式。【结果】从276份荔枝自然群体材料中鉴定到荔枝高抗霜疫病品种裕荣1号(YR1)。从荔枝基因组中共鉴定出19个LcCDPK基因家族成员,分布于11条染色体上。根据保守结构域和系统发育分析将其分为4个亚家族。基因结构分析表明,LcCDPKs外显子数量为7~19。蛋白结构分析发现,所有LcCDPK蛋白均具有1~4个EF-hand结构域。顺式作用元件分析表明,LcCDPKs成员拥有大量的生物和非生物胁迫响应元件。组织表达分析发现,LcCDPK基因在荔枝不同组织存在组织特异性。另外,表达分析发现在高抗霜疫病荔枝裕荣1号(YR1)中,LcCDPK5、LcCDPK17和LcCDPK19在霜疫病胁迫后急剧上调表达,LcCDPK3和LcCDPK8急剧下调表达。【结论】裕荣1号(YR1)是一种高抗霜疫病的荔枝品种。荔枝基因组中共鉴定出19个CDPK基因家族成员,具有明显的组织特异性,LcCDPK5、LcCDPK17、LcCDPK19、LcCDPK3和LcCDPK8可能在荔枝抗霜疫病过程中发挥重要作用。研究结果为进一步探究荔枝CDPK基因家族提供了参考。

【Objective】Lychee(Litchi chinensis Sonn.)is a delicious and nutritional fruit widely accepted by consumers.However,the fruits are highly susceptible to various diseases.Lychee downy blight is one of the major diseases in lychee.The calcium dependent protein kinases(CDPKs)play vital roles in regulating plant growth,development and response to abiotic or biotic stresses.However,the potential role of CDPK gene family in lychee(LcCDPK)have not been reported.Here,we aimed to identify and analyze the CDPK gene family from the lychee genome with bioinformatic technology,and investigate the phylogeny,protein structure,expression patterns and response of CDPK gene in lychee under lychee downy mildew stress.This study would provide a basis foundation for further functional characterization of the lychee CDPK genes.【Methods】The lychee genome was used to identify and analyze the LcCDPK genes by bioinformatics using a local BLASTP search of TBtool software.All putative candidates were manually verified with the InterProScan program to confirm the presence of the protein kinase domain and the CaM domain.The length of sequences,isoelectric point(pI),molecular weights(MW)and predication subcellular location of the LcCDPKs were estimated by ExPASy Compute pI/Mw tool.The multiple alignments of amino acid sequences were performed using MEGA-X program.The neighbor-joining phylogenetic tree was constructed via applying the MEGA6 program,with bootstrapping set at 1000 replications.The gene structure of the LcCDPKs were analyzed using the Gene Structure Display Server(GSDS)program with default settings.The LcCDPK genes were mapped to lychee chromosomes based on physical location from the database of the lychee genome using MapChart.The cis-acting regulatory elements located in a region upstream 2 kb to the start codon of LcCDPK genes were identified using the PlantCARE database,and visualized with TBtools.The germplasm used in this study contained 276 lychee landraces from different geographic areas.This population was selected for the present study because the collection was representative of the diverse genetic variation in lychee.These 276 germplasm materials were provided by Institute of Fruit Trees,Guangdong Academy of Agricultural Sciences and were planted in National Fruit Tree Germplasm,Guangzhou Lychee Nursery.The pathogen of P.litchii was provided by South China Agricultural University.P.litchi were cultured in juice agar(CJA)medium at 27℃.The spore suspension was filtrated using double sterile layer and adjusted to 104 spores mL^(-1) for inoculation.For transcription analysis,the leaves of Yurong 1(YR1)lychee were inoculated with 5μL sporangia suspension of P.litchii(104 spores·mL^(-1))or a mock suspension(sterile water),and then the leaves were collected at the indicated time points.All tissues were immediately placed in liquid nitrogen and stored at-80℃.The sequencing data of lychee were available from NCBI Sequence Read Archive database under the accession number PRJNA747875.The expression levels of the LcCDPK genes were analyzed in various tissues,including female flowers ovary,root,pericarp,aril,embryo,epicarp,male flowers anther,leaf and seed.The heat map with hierarchical clustering of the LcCDPK genes were constructed using MeV4.9 software by average linkage with Euclidean distance method,to visualize the expression levels in nine tissues based on the log10(FPKM+1)values of the LcCDPK genes.Based on the transcriptome data,the expression of the CDPK was analyzed and verified by qRT-PCR.【Results】This study identified and comprehensively analyzed the CDPK family genes based on the whole genome data of lychee.The sequence characteristics,gene structure,promoter cis-acting elements,chromosome localization,evolutionary relationships and expression patterns in different tissues under the stress of lychee downy mildew were analyzed.A total of 19 CDPK family members were identified from lychee genome,which were distributed on 11 chromosomes.According to conserved domain and phylogenetic analysis,the 19 CDPK family members were divided into four subfamilies.The gene structure analysis indicated that the number of exon-intron ranged in the LcCDPKs from 7 to 19.The protein structure domain analysis showed that all LcCDPK proteins had 1-4 EF-hand domains.The promoter cis-acting element analysis exhibited that LcCDPKs had a large number of biological and abiotic stress response elements.Tissue-specific expression analysis showed that many LcCDPKs genes could be detected in all tissues,while a few of LcCDPKs genes were tissue specific.The differential gene expression analysis of the LcCDPK genes in response to lychee downy mildew stresses helped us identify the LcCDPK5,LcCDPK17 and LcCDPK19 as the candidate genes for disease resistance in lychee,whose relative transcript abundance rapidly increased after lychee downy mildew infection.【Conclusion】A total of 19 CDPK family genes were identified in lychee genome.The expression of the LcCDPK genes in different tissues and their expression patterns in the response to lychee downy mildew stresses elucidated that the LcCDPK5,LcCDPK17,LcCDPK19,LcCDPK3 and LcCDPK8 might play an important role in the resistances of lychee to lychee downy mildew.Our study would provide critical foundation for further functional characterization of the lychee CDPK gene family.