刺梨CDPK基因家族的鉴定及其对供钙水平的表达响应  

Identification of CDPK family genes in Cili(Rosa roxburghii Tratt.)and its expression in response to calcium levels

作　　者：龚丽莎 向芷萱 王照 鲁敏[1] 安华明[1] GONG Lisha;XIANG Zhixuan;WANG Zhao;LU Min;AN Huaming(Engineering Research Centre of National Forestry and Grassland Administration for Rosa Roxburghii/Agricultural College of Guizhou University,Guiyang 550025,Guizhou,China)

机构地区：[1]国家林业与草原局刺梨工程技术研究中心·贵州大学农学院,贵阳550025

出　　处：《果树学报》2023年第4期639-652,共14页Journal of Fruit Science

基　　金：国家自然科学基金委员会-贵州省人民政府联合基金项目子课题(U1812401)。

摘　　要：【目的】CDPKs是植物中广泛存在的Ca2+感受器,鉴定刺梨(Rosa roxburghii Tratt.)CDPK基因家族成员,并探索其对不同供钙水平的表达响应。【方法】采用生物信息学方法鉴定并分析Rr CDPK基因家族,通过转录组测序及实时荧光定量PCR(real-time quantitative PCR,q RT-PCR)分析其组织表达特异性及在不同供钙水平下的表达响应。【结果】从刺梨基因组中共鉴定出16个具丝氨酸/苏氨酸蛋白激酶和EF-hand结构域的CDPK基因(命名为Rr CDPK1~16),结构分析显示蛋白长度在393~561 aa之间,分子质量在44.02~62.98 ku之间,平均等电点6.05;家族基因结构差别较大,外显子数量为2~10个,包括6个保守基序;亚细胞定位预测Rr CDPKs在细胞核和多种细胞器均有定位,主要定位于细胞质;进化分析可分为4个亚族,且与草莓的亲缘关系最近,其次是苹果,较远为拟南芥和水稻。启动子顺式作用元件分析表明,Rr CDPKs大多含光响应元件、多种激素响应元件及胁迫响应元件等。不同器官及果实发育时期的转录组数据显示Rr CDPKs具有时空表达特异性。Rr CDPKs对供钙水平的响应表明,相比无钙处理,0.5 mmol·L^(-1)钙水平下Rr CDPK1/2/4/8表达显著上调,2 mmol·L^(-1)钙水平下Rr CDPK2/4/9/10/12表达显著上调;Rr CDPKs在叶和根中对供钙水平及处理时间的表达响应也不尽相同。【结论】共鉴定出16个Rr CDPK基因,在刺梨中的表达具时空特异性,且在应对不同供钙水平时发挥的作用不尽相同。研究结果可为深入揭示刺梨CDPK基因家族的功能及其对外界钙水平的响应机制奠定基础。【Objective】Calcium-dependent protein kinases(CDPKs/CPKs)are a class of serine/threonine-type protein kinases,which are widespread Ca2+sensors in plants.This work was aimed at identifying and analyzing the RrCDPK gene family in Cili(Rosa roxburghii Tratt.)and exploring its expression response to different calcium levels.【Methods】The cutting nursery trees of R.roxburghii Tratt.‘Guinong 5’with basically the same growth were selected as the experimental materials in April 2021 in the R.roxburghii Tratt.Resource Garden of the Agricultural College of Guizhou University.After literature review and a series of pre-experiment screening,this work set up 3 Ca2+concentration gradients:0,0.5,2 mmol·L^(-1) and 3 sampling time points:0,1,7 d to explore the response of RrCDPKs to no calcium,low concentration and high concentration calcium treatments.Additionally,based on R.roxburghii Tratt.genome,the RrCDPK gene family was identified and analyzed by bioinformatics methods.The Arabidopsis thaliana CDPKs(AtCDPKs)protein sequences downloaded from Tair database(https://www.arabidopsis.org/)were used as queries to search against R.roxburghii Tratt.genome data.The putative genes were identified based on a local BLASTP search in TBtools software (E-value<1×10-5, bitscore>100). Combined with SMART and Pfam database for further screening, the genes with the proteinkinase domain (Pfam database ID: PF00069) and the EF-hand domain (PF13499) were recognizedas the final CDPK family members. With the help of ExPASy, WoLF PSORT, MEME, NCBI-CDD,iTOL and plantCARE online website, and MEGA7 and TBtools software, the physicochemical propertiesof the encoded proteins, the chromosome location, subcellular localization, gene structures, conservedmotifs and conserved domains, phylogeny, promoter cis-acting elements were obtained. Finally,transcriptome sequencing and qRT- PCR were used to analyze the tissue expression specificity theRrCDPKs and their expression response under the different calcium levels.【Results】A total of 16 CDPKgene

关 键 词：刺梨 钙依赖性蛋白激酶 供钙水平 

分 类 号：S661.9[农业科学—果树学]