Identification And Expression Characteristics Of Genes Involved In Calcium Absorption In Rosa Roxburghii Tratt.

Calcium is an essential nutrient element for plants,and as the second messenger in cells,it has complex physiological functions.It is widely involved in the biological processes of fruit tree growth and development,stress response,secondary metabolite accumulation and quality formation.Rosa roxburghii Tratt.belongs to Rosaceae Rosa shrub,which is widely distributed in Guizhou karst area with high content of soil exchangeable calcium and rich in secondary metabolites in fruits.Previously,our research group also found that Ca²⁺can participate in the regulation of L-ascorbic acid（As A）synthesis in fruits at the biochemical and molecular levels,but so far,the absorption and transport process of Ca²⁺by R.roxburghii and its main genes are not clear.In this study,Rosa roxburghii Tratt.cv.guinong 5 was used as material to analyze the gene expression differences in the process of calcium absorption by RNA-Seq and other techniques,and screen the candidate genes related to calcium absorption;Combined with the expression characteristics of these genes under different Ca²⁺levels,further identify the vector or channel protein members closely related to calcium absorption,and explore the possible relationship between them and the accumulation of main active substances（As A,flavonoids,triterpenes,etc.）in fruit,so as to lay a foundation for revealing the calcium absorption mechanism of R.roxburghii and its regulation process on fruit quality.The main results are as follows:1.Analysis of transcriptome response and differentially expressed genes in R.roxburghii during calcium absorptionThe Clonal Seedlings starved for 24 hours were used as materials for Ca²⁺absorption test.Through the reduction range of Ca content in nutrient solution and repeated for many times,we first determined that 0min,5 min and 6 h were the sampling time nodes of starvation period,rapid calcium absorption period and stable calcium absorption period respectively.Then,the roots and leaves of the three periods were collected for RNA-Seq analysis,and a total of 12314 differentially expressed genes in the process of calcium absorption were screened under the conditions of fold change≥1.2 and FDR<0.01.GO annotation results show that DEG is mainly annotated into 53 functions.The enrichment results of KEGG pathway showed that the differentially expressed genes were mainly enriched in three pathways involved in Plant-pathogen interaction,MAPK signal pathway and Plant hormone signal transduction.Transcription factor analysis showed that the number of DEG in WRKY,MYB,C2H2 and b HLH families was the largest among the differentially expressed genes,all of which were more than 40,indicating that a variety of transcription factors were involved in the process of calcium absorption or affected by calcium content.2.Identification and expression verification of genes involved in calcium absorption and transport in R.roxburghiiThrough gene annotation and bioinformatics comparison,combined with the correlation analysis between gene expression and calcium absorption rate,taking the correlation coefficient>0.7 and FPKM value>10 as the conditions,focus on screening ion channels and carrier proteins related to ion absorption and transport as candidate genes,and analyze their expression patterns under different Ca²⁺levels such as 0mmol·L^-1,0.5 mmol·L^-1and 2 mmol·L^-1.The results showed that a total of 25 candidate genes were directly related to calcium absorption and transport,including 11 in roots and 15 in leaves.According to the gene annotation information,it is found that 25 genes belong to 7 gene families,including cyclic nucleotide-gated ion channel（CNGC）,Zinc transporter（ZTP）,Metal tolerance protein（MTP）,ER-type calcium ATPase（ECA）,Autoinhibited calcium ATPase（ACA）,Glutamate receptor（GLR）and Natural resistance-associated macrophage protein（NRAMP）,of which the number of members of CNGC family is the largest（12）.In addition to the above seven families,the genes positively related to calcium content in roots and leaves also include the following four families:Ca²⁺/H⁺antiporter（CAX）,mitochondrial calcium uniporter（MCU）,hyperosmolality-gated calcium-permeable channel（OSCA）and plasma Membrane H⁺-ATPase（PMA）.The expression of Rr CAX was positively correlated with the calcium content in roots and leaves,indicating that it played an important role in the calcium accumulation of R.roxburghii.The expression of 10 gene family related members such as cation/Ca²⁺exchanger（CCX）,heavy metal transporting ATPase（HMA）,two pore Ca²⁺channel（TPC）,ACA,CNGC,ECA,GLR,MTP,OSCA and PMA was up-regulated during starvation,which may be involved in the steady-state regulation of Ca²⁺in plants during starvation.Under different calcium concentrations,Rr CNGC7 was significantly positively correlated with calcium content in roots,and Rr CNGC1/10/12 and Rr MTP2 were significantly positively correlated with calcium content in leaves,respectively,indicating that CNGC family may play a key role in responding to different calcium supply levels.3.Spatial and temporal expression characteristics of genes involved in calcium absorption and transport in R.roxburghii and their possible relationship with the contents of major active substances in fruitThe temporal and spatial expression patterns of 25 related genes were analyzed with different organs and fruits at different development stages of R.roxburghii as materials;The effects of in vitro feeding and foliar calcium spraying on R.roxburghii on the main active substances（As A,flavonoids,triterpenes,etc.）were observed.The results showed that the expression of these genes was tissue-specific and time-specific.The transcription level of most calcium absorption related genes in fruits and leaves was higher than that in petals,and the expression level in the middle and late stage of fruit development was higher than that in young fruit.In vitro feeding or field spraying of exogenous calcium can increase the calcium content of fruit and promote the accumulation of As A,total flavonoids and total triterpenes,while the addition of calcium channel inhibitor verapamil（Vp）,calcium chelator EGTA or intracellular calcium channel inhibitor Li Cl can inhibit this effect.On this basis,we deeply analyzed the correlation between gene expression and the main active substances of R.roxburghii fruit.The results showed that the expression of multiple genes was significantly correlated with the accumulation of As A,total flavonoids or total triterpenes in R.roxburghii fruit,including Rr CNGC2/3/12,Rr GLR1/2,Rr ECA1,etc.among them,Rr CNGC12 was the only gene with significant positive correlation with the content of these three substances in the fruit,indicating that it may promote Ca²⁺absorption Transport and response to calcium play a key role in the regulation of R.roxburghii fruit quality formation.