Effect Of Calcium Channel Promoters And Inhibitors On Cold Resistance In Banana And Identification And Expression Analysis Of MaCNGC Family Members

Banana（Musa spp.）originated in the tropical region of Southeast Asia,is an important fruit and grain crop with high temperature requirements.It is a plant sensitive to low temperature.In the northern fringe area where bananas are planted,the damage made from the sudden drop of temperature and the cold wave invasion in winter and spring is one of the main obstacles to the development of banana industry in China.Therefore,it is of great significance for banana breeding and production to carry out scientific research related to banana cold resistance and cultivate low-temperature resistant varieties with excellent characters.In this study,‘Baxi’banana was used as the experimental material,sprayed with different concentrations of calcium channel promoter（Ca Cl₂solution）and calcium channel inhibitor（EGTA solution）to study the changes of carotenoids and several cold resistance indexes under low temperature stress.At the same time,bioinformatics and quantitative expression analysis of banana cyclic nucleotide gated channel were carried out,which laid a certain foundation for clarifying the mechanism of calcium signaling pathway in the process of low temperature stress.The main results are as follows:1 Effects of Ma CNGC Ca²⁺promoters and inhibitors on carotenoids and physiological indicators related to cold resistance in bananaTo investigate the effects of Ca²⁺promoters and Ca²⁺inhibitors on physiological and biochemical indexes related to low temperature stress of banana,taking‘Baxi’banana as material,four Ca Cl₂concentrations were applied:0（distilled water,CK）,10 m M Ca Cl₂,20 m M Ca Cl₂and40 m M Ca Cl₂,and four EGTA concentrations:0（distilled water,CK）,1m M EGTA,2 m M EGTA and 4 m M EGTA.Then,the effects of different concentrations of calcium promoter and calcium inhibitor on carotenoid,MDA,SOD,POD and CAT in banana leaves were compared at 4°C and28°C.The results showed that treated with 20 m M Ca Cl₂under 4°C,the contents of chlorophyll and carotenoid increased significantly,activated SOD and CAT,40 m M Ca Cl₂promoted the accumulation of POD and significantly reduced the content of MDA in banana leaves.At the same time,4 m M EGTA treatment under 4°C inhibited the accumulation of CAT and increased MDA content in banana leaves,while carotenoid content,SOD and POD activity showed a downward trend,and there was no significant difference under different concentrations of EGTA treatment.The results showed that after spraying calcium channel inhibitor,the activities of CAT,POD and SOD were reduced,the content of MDA was increased,the degree of membrane lipid peroxidation was intensified,and the accumulation of chlorophyll and carotenoid was inhibited.The addition of Ca Cl₂can improve the protective ability of cell plasma membrane,improve the activities of CAT,POD and SOD,reduce the content of MDA,slow down the degree of membrane lipid peroxidation,promote the accumulation of chlorophyll and carotenoid,so as to improve the ability of banana to resist low temperature stress.It indicates that Ca²⁺signal pathway plays an important role in banana cold resistance.2 The genome identification and bioinformatics analysis of cyclic nucleotide gated channelThere are 17 members in Ma CNGC family.The coding sequence of these genes ranges from 1917bp to 2247bp,with an average of 2118bp,which is distributed on all chromosomes except chromosome 2.The length of amino acid was 638 to 748aa,with an average of 705aa.The molecular weight was 72.94 to 85.41 k Da,and the isoelectric point（PI）was 8.39 to 9.58.All of them are unstable proteins,13 members are hydrophilic proteins.There is no signal peptide except Ma CNGC2-3,and all members have transmembrane structure.Subcellular localization prediction analysis shows that Ma CNGCs are located on the plasma membrane.Phylogenetic tree analysis showed that banana could be divided into four subgroups（GroupⅠ,GroupⅡ,GroupⅢand GroupⅣ）and two subgroups（GroupⅣ-A and GroupⅣ-B）,which were similar to Arabidopsis.Gene structure and protein sequence alignment analysis showed that cyclic nucleotide binding domain（c NBD）,the CNGC specific domain,included a phosphate binding domain（PBC）and a highly conserved hinge region.The results of cis-acting elements and TFBS prediction showed that there were a variety of plant hormones,stress-related response elements and 16 TFBS in the promoter region of Ma CNGC.3 Analysis of the expression pattern of Ma CNGC under low temperature stressTranscriptome data analysis showed that compared with the control,the FPKM value of 76.47%of Ma CNGC members was down-regulated at4°C for 24 hours.Compared with the control,the FPKM value of 76.47%of Ma CNGC members was down-regulated at 0°C for 24 hours.In conclusion,Ma CNGC family members show a downward trend in response to low temperature stress,which needs to be further investigated.Real-time quantification expression analysis revealed that compared with the control,82.35%of Ma CNGC members were negatively regulated at4°C/24h,which was consistent with the transcriptome analysis results.Among them,Ma CNGC1-1,Ma CNGC2-3,Ma CNGC15,Ma CNGC18-1and Ma CNGC18-2 showed a downward trend in response to cold stress with the increase of time.On this basis,after treating with Ca²⁺promoter（Ca Cl₂solution）and Ca²⁺inhibitor（EGTA solution）,the expression patterns of Ma CNGC1-1,Ma CNGC2-3,Ma CNGC15 and Ma CNGC18-2 under low temperature stress were analyzed by q RT-PCR.The results showed that relatively low concentration of calcium promoter（20 m M Ca Cl₂）significantly induced the expression of Ma CNGC under low temperature stress,while 2 m M calcium ion inhibitor（EGTA）inhibited the expression of Ma CNGC.This shows that Ma CNGC may play an important role in the response to low temperature stress.It is proposed to take Ma CNGC1-1 as the target gene to further explore the influence of Ca²⁺signaling pathway on the mechanism of banana cold resistance.