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

【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 CDPKgenes with serine/threonine protein kinase and EF-hand domains were identified from the R. roxburghiigenome. They were randomly distributed on the other 6 chromosomes except for chromosome 4and were named as the RrCDPK1-16 according to their chromosome location from the top to bottom.The structural analysis displayed that the gene length ranged from 1496 bp (RrCDPK6) to 5524 bp(RrCDPK14), the lengths of the proteins from 393 aa (RrCDPK6) to 561 aa (RrCDPK16), the molecularweights form 44.02 (RrCDPK6) ku-62.98 ku (RrCDPK16);RrCDPKs were hydrophilic proteins;exceptfor RrCDPK5/8, other members were acidic proteins;unlike RrCDPK1/4/11/12/14/15, others werestable proteins. The gene structure was quite different, the number of exons was 2-10, and all the RrCDPKsincluded 6 conserved motifs. The subcellular localization predicted that the RrCDPKs were localizedin the nucleus and various organelles, mainly in the cytoplasm. The RrCDPKs were divided intofour subfamilies by evolutionary analysis and most closely related to those of Fragaria × ananassaDuch., followed by Malus pumila Mill., and farther than A. thaliana (L.) Heynh. and Oryza sativa L..The analysis of promoter cis- acting elements in the upstream 2000 bp sequence showed that most ofthem contained light response elements, various hormone response elements and stress response elements.Transcriptome data from different organs and fruit developmental stages showed that the RrCDPKshad spatiotemporal expression specificity. Among them, the high expression of the RrCDPK3/5/10/13 in the stems and the RrCDPK4/8/9/15 in the leaves indicated that they might be related to vegetativegrowth. The RrCDPK2/8/11/15 was continuously up-regulated with fruit development, so that it reachedthe highest level at the later stage, which was consistent with the accumulation trend of flavonoids. Theexpression of the RrCDPK1/10/12/14, RrCDPK7 and RrCDPK3/4/5/13/16 was the highest in the earlystage, the fruit expansion stage, and the early and middle stages of fruit development, respectively.Therefore, it was speculated that these RrCDPKs were involved in the regulation of various biologicalactivities synthesis of substances. The response of the RrCDPKs to different calcium levels showed thatafter calcium supply, a few RrCDPKs showed obvious up-regulation effect: the expression levels of theRrCDPK4/8 in the leaves 1 day after the treatment and the RrCDPK1/2 in the roots 7 days after thetreatment were significantly increased (p<0.05) under 0.5 mmol · L- 1 Ca2 +;the RrCDPK4/10/12 in theleaves and the RrCDPK9 in the roots 1 day after the treatment, and the RrCDPK9 in the leaves and theRrCDPK2 in the roots 7 days after the treatment were significantly up-regulated under 2 mmol·L^(-1) Ca2+.It was suggested that the RrCDPKs in the leaves and roots could be expressed differently in response tothe different calcium levels and treatment times.【Conclusion】Totally 16 RrCDPK genes were identifiedin the whole genome of R. roxburghii Tratt., and all of them contained the typical serine/threonineprotein kinase and EF-hand domains. The expression of the RrCDPK genes in R. roxburghii were spatiotemporallyspecific, indicating that they might play an important role in the different organs and developmentalstages of fruits. The RrCDPK family members might play different roles in response to the different calcium levels, especially the RrCDPK1/2/4/8/9/10/12. The results could provide relevant informationfor further revealing the function of R. roxburghii Tratt. CDPK gene family and its responsemechanism to the external calcium environment.