| Banana belongs to the genus Musa,a member of the family Musaceae,which is the most popular fruit in the world.Banana plants are thought to be thermophilic crops that originated in the tropics.Banana cultivation has a long history in China,but Fujian,Guangxi,and other provinces in China,belong to the northern margins of banana cultivation,where the banana often suffers from cold stress,resulting in severe damage to the production.The wild banana germplasm resources are rich in Fujian province.For further utilizing the useful wild banana genetic resources,the wild banana from Sanming City of Fujian Province(Musa itinerans)was used as materials,conducted for multiple omics analyses of response under low temperature stress in the wild banana in this study,including:physiological and biochemical analysis under the cold stress;the miRNAs,mRNA-lncRNA omics analysis of responses to low temperature using high-throughput sequencing technologies and validation of the relationship between miRNAs,IncRNAs and their target genes using qPCR;cloning of some cold-resistant related genes(CKII family and others)and miRNAs and qPCR validation under the low temperature stress in the wild banana.In addition,the gene structure and qPCR expression of 4 overlapping genes were conducted.This study was aimed at exploring the wild banana response to low temperature stress and cold resistance mechanism,and providing the scientific evidences for mining cold-resistant genetic resource in the wild banana and the cold resistance breeding of banana.The main results were as follows:1.The analysis of physiological and biochemical changes under the low temperature stress in the wild bananaThe wild banana after different low-temperature treatments,i.e.,at the normal growing temperature of 28? as the control,at critical growth temperature of 13?,at the chilling temperature of 4?,and the water freezing temperature of 0? as the test materials,the changes of the activities of SOD,POD and CAT and the contents of H2O2,sucrose were determined by spectrophotometer in the wild banana.The results showed that the activities of SOD,POD and CAT were highest at 4?,and slightly lower at 0? but higher than in control(28?),which suggested that the low temperature activated the enzymatic antioxidant system in the wild banana.At the same time,the contents of H2O2 were highest at 4?,and slightly lower at 0? but higher than that of control,which was similar to the changes of the activities of antioxidant system.Morever,sucrose content rose markedly as the temperature dropped.Notably,the sucrose content in wild banana at 4? was only about 1.16-fold higher than control.At 0?,however,the sucrose content was even 1.5-fold higher than control.The results were showed that the low temperature stress increased the contents of H2O2 and sucrose,and further improved the cold resistance ability in the wild banana.2.The analysis of miRNAs of low-temperature responses in the wild bananaThe miRNAs of the wild banana at different low temperatures(13?,4? and 0?,28?for control)were sequenced by using the method of high throughput sequencing,and the sequencing analysis and validation were conducted.The mature of 265 known miRNAs and 41 novel miRNAs were obtained.Cluster analysis of differentially expressed(DE)miRNAs indicated that some miRNAs were specific for chilling or 0? treated responses,e.g.,miR395,miR408,miR172,suggesting that they maybe play key roles in response to cold stress.The GO and KEGG pathway enrichment analysis of DE miRNAs targets indicated that there existed diversified cold-responsive pathways,and miR172 was found likely to play a central coordinating role in response to cold stress,especially in the regulation of CK? and the circadian rhythm.Finally,qPCR assays indicated the related targets were negatively regulated by the tested DE miRNAs during cold stress in the wild banana.3.The analysis of mRNAs-lncRNAs of low-temperature responses in the wild bananaThe wild banana from Sanming City was used to profile the cold-responsive mRNAs and lncRNAs by RNA-seq at different low temperatures(13?,4? and 0?,28? for control),and the sequencing results were analyzed and validated.The transcriptome different expressed genes KEGG analysis indicated that some essential pathways concerning secondary metabolism,the circadian rhythm,etc.,were highly enriched during cold stress,and some pathways were specifically enriched in one to six groups,which implied that the wild banana might have some specific mechanism for cold acclimation.The total number of alternative splicing events(especially the intron retention)at low temperatures were higher than those of the control,implying that the AS events might be closely related to the cold responses.The IncRNA identification indicated that 12,462 IncRNAs were totally identified during cold stress in the wild banana.The lncRNA targets predict indicated that the 4 TF genes(Zinc finger,MYB,WD40 and bHLH,all related to regulating flavonoid biosynthesis)ranked ahead in frequency,and the top 3 enriched terms of non-TF targets were the genes for hydrolases,protein kinases and domain of unknown function proteins.The KEGG analysis of differential expression IncRNA targets indicated that the glycolysis/gluconeogenesis,spliceosome,carbon metabolism,biotin metabolism pathways occurred in the most groups,but some other pathways occurred in specific temperature points.Furthermore,comparing the differential expression mRNAs and the differential expression lncRNAs,it is inferred that majority of differential expression mRNAs were regulated by lncRNAs,suggesting that the IncRNAs play a vital role in regulating the differential expression genes during cold stress.The qPCR validation results showed that there was complicated regulation relationship between the IncRNAs and their target genes the wild banana,with positive or negative regulation.Some lncRNAs might regulate their target genes during all the low temperature stress,but others might regulate their target genes only at one or two specific temperature points.4.The several precursors of miRNAs cloning,molecular characteristics and expression analysis of some miRNAs related to cold resistance in the wild bananaBased on the analysis results of miRNA omics data in the wild banana,the differentially expressed miR172,miR408,miR395 family members were chose for further analyses.Firstly,the precursor and mature of miRNAs registered in miRbase(21.0 version)and bioinformatics analysis method were used to reveal evolution and molecular characteristics of miR172,miR408 and miR395 family members.The results showed that the specificity of species was an important factor affecting the precursor evolution of MIR408 family,while the conservation of sequences was one of the main factors affecting the evolution of the mature miR408 family.The members of miR172,miR408 and miR395 from 5p arm were quite specific,on the contrary,conserved for the members of miR172,miR408 and miR395 from 3p arm.The stem sequence was more conserved than loop sequence,and the miRNAs from 3p arm sequence was more conserved than the 5p arm.Secondly,based on the analysis of evolution and molecular characteristics of plant miR 172,miR408 and miR395 family members,and referring to the result of wild banana miRNAs omics,the precursor sequences of miR172c,miR172a,miR408b and miR395a in the wild banana were obtained by PCR method,and the lengths were 497 bp,126 bp,85 bp and 81 bp,respectively.Meanwhile,the promoter sequence analysis of miR172c,miR172a,miR408b and miR395a indicated that this 4 miRNAs promoters included not only the core components,but also many cis-elements such as light,hormone(ABA,auxin,GI,SA,MeJA and ethylene),stress responses(low temperature,defense and stress response,drought induced,drought-inducibility,wound responsive)and circadian control,etc.The 4 miRNAs promoters included light responsive elements,as many as 12-14.The most type of hormone cis-elements were included in miR395a promoter,and the numbers was 5,miR172c,miR408b and miR172a followed.The most number of hormone cis-elements was miR172c,among which the MeJA cis-elements number was up to 14.The circadian control cis-elements were included in miR172a and miR395a promoter.The low temperature cis-elements were included in miR172c,the defense and stress response cis-elements were included in miR172a and miR408b.The results of cis-elements analysis showed that these miRNAs might play an important role in response to low temperature stress in the wild banana.At last,the expression levels of the members of miR172c,miR172a,miR408b and miR395a were detected by qPCR at different temperatures(at the growth temperature of 28? as the control,and the low temperatures of 13?,4? and 0?)and different tissues(leaves,pseudo-stems and roots)in the wild banana.The results showed that the expression levels of miR172c,miR172a,miR408b and miR395a were specific in different tissues,and responded to low temperature stress to some extents in the wild banana.5.The genome-wide identification and qPCR expression of CK? family in the wild bananaThe analysis result of miRNA omics during wild banana in response to low temperature stress showed that CK? was the target gene of differentially expressed miR172,and CK? was also differentially expressed in the transcriptome results of the wild banana in response to low temperature stress.Therefore,the further identification and the expression analysis of CKII gene family members were performed from the wild banana in response to the low temperature stress.Based on the data of the banana genome and combined with bioinformatics analysis method,using the term 'casein kinase ?' as the key word for search in the Banana Genome A from'DH-Pahang'(Musa accuminata,AA group)and Banana Genome B from 'PKW'(Musa balbisiana,BB group)wild banana,13 CKII family members from Banana Genome A and the 11 CKII family members from Genome B were identified.The analysis of conservative structure domains and clustering showed that the member difference of CKII family sequences was quite big between the wild bananas of 'DH-Pahang' and 'PKW',and the analyses of the functional domains for the 13 CKII of Banana Genome A showed that all the members of the 13 CKII family members contained STKc_CK2_alpha or CK_?_beta functional domains,but only 3 members among the 11 CK? members of Banana Genome B contained the complete functional domains(STKc_CK2_alpha or CK_?_beta).Therefore,the integrity and accuracy of CK? from Genome A were high,which was suggested as the reference for identification of the CK? gene family in Musa plants.Based on the Banana Genome,using RT-PCR combined with RACE techniques,13 sequences of the CK? family members were obtained both in the Musa spp.cv.Tianbaojiao and in the wild banana(Musa itinerans),respectively.Among them,the AS event of exon deletion occurred in the 2 transcripts of MiCKII/?-like-2 of the wild banana(Musa itinerans).The analysis of the functional domains indicated that all the members of the CK? family contained the whole STKc_CK2 alpha or CK_?_beta conserved domains in the wild banana and the Tianbaojiao banana.The sequences comparisons of the members of CK? family among the wild banana,Tianbaojiao and the Banana Genome A were conducted,most of the members were the same or similar among them.The sequences of the CKII family members of the wild banana and the Tianbaojiao banana were more close to those of the Banana Genome A than to those of the Banana Genome B,and furthermore,the sequences of the CK? family members of the Tianbaojiao banana were more close to those of the Banana Genome A than to those of the wild Banana.Apart from the CKII ?-like-1 from Tianbaojiao and the Banana Genome A,all the other CK? ? subunits members were located in nucleus,and the subcellular locations of the CKII a subunits members were different,including cytoplasmic,mitochondrial,extracellular(including cell wall),etc.The gene structure of the CK? family among the wild banana,Tianbaojiao and the Banana Genome A was analyzed,and the results showed that except for MiCK??-like-2b from the wild banana and MaCK??-like-2 from Tianbaojiao banana,all the other CKII a members contained 10 exons and 9 introns,and all the other CK?? members contained 5 exons and 4 introns.Both MiCK??-like-2b from the wild banana and Tianbaojiao banana contained 4 exons and 3 introns.The structures define the functions,therefore,the CK? a members and CK?(3 members subunits functioned differently.The occurrence of the CK? phosphorylation sites were predicted among the wild banana,Tianbaojiao and the Banana Genome A.The results showed that CK?? members had more CK? phosphorylation sites than CK? a members.The conserved motifs of CK? family were analyzed among the wild banana,Tianbaojiao and the Banana Genome A.The results showed that the motifs among CK? a or CK?? family members were highly conserved.The motifs among CK? a or CK?? family members were highly conserved,which confirmed that CK? existed among the eukaryotes,which were very highly conserved in structure.The expression levels of all the members of the CK? family members were detected by qPCR under different temperatures(at the growth temperature of 28? as the control,and the low temperatures of 13? for the critical growth,4? for chilling and 0? for freezing,using the leaves as the materials)and different tissues(leaves,pseudo-stems and roots)in the wild banana.The results showed that the expression levels of CK?-14a,CK?-15 and CK?-4 were the highest at 4?,especially the expression levels of CK?-14a and CK?-15 at 4? were significantly higher than those at the control,and the expression levels of CK?-14a,CK?-15 and CK?-4 at the leaves were also higher than those at pseudo-stems and roots.The expression levels of CK?-9 and CK?-10 were both the highest at 0?,furthermore,the expression level of CK?-9 in the root was remarkably higher than that in the leaf,and the expression levels of CK?-10 in the root and pseudo-stem were both lower than that in the leaf.The expression levels of CK?-1,CK?-2 and CK?-7 were all high but not significant at 13?,and the expression levels of the 3 members in the leaves were significantly the highest among the 3 tissues.The expression levels of the 5 members of CK?-3,CK?-5,CK?-6,CK?-13 and CK?-16 changed unremarkably at low temperature points.The member of CK?-14b did not display in our results,which was the alternatively spliced transcript of the normal CK?-14a transcript,and expressed at very low levels even could not be detected under different temperatures and different tissues,which was quite different from the expression patterns of CK?-14a.Very interestingly,the wild banana of Musa itinerans from Sanming City and the Banana Genome A from 'DH-Pahang'(Musa accuminata,AA group)of the Malaysian wild banana had the normal CK?-14 transcript CK?-14a,but the cultivated cultivar'Tianbaojiao'(AAA group)had only the alternatively spliced transcript CKII-14b(low-expressed in the wild banana)and had not the normal transcript CK?-14a(the wild banana had the both),which might result from the differences of the species and evolution,or the sensibility to the cold stress between the wild and the cultivated bananas.6.The analysis of 4 cold overlapping genes related to cold resistance based on the banana genome in wild bananaBased on the analysis results of the wild banana miRNNAs omics in response to low temperature stress,both of CK? and ADA1 being target genes of differentially expressed miRNA172,might play important roles in response to low temperature stress.Further analysis found that CK? and ADA1 had a special arrangement in the chromosome 6 of banana genome A:the unknown function protein gene(GSMUA_Achr6G 34700,for short 700)(only 171 bp)encoding a small peptide,and the whole ORF existed in 3' UTR of CK?(GSMUA_Achr6G 34710,for short 710).There was also only 1200 bp between CKll and formamidase(GSMUA_Achr6G 34720,for short 720),and the three genes(700,710 and 720)existed reversely in chromosome 6.There was also only 2111 bp sequence between 720 and ADA1(GSMUA_Achr6G 34730,for short 730),and the two genes were opposite direction,so the two genes shared one promoter,namely the bidirectional promoter.Due to the particularity location of the 4 genes in the chromosome,700 and 710 were the synthetic array overlapping genes,720 and 730 were the reversed arrangement overlapping genes.To learn whether 4 overlapping genes exist in the cold resistance of wild banana and their special arrangement structure is related to response to low temperature stress,the identification and gene expression analysis of 4 overlapping genes were performed in wild banana and cultivation banana 'Tianbaojiao'.Referring to the Banana Genome A,using the method of TA cloning combined with RACE,the complete cDNA,gDNA and promoters sequences of 4 overlapping genes from Tianbaojiao were obtained.The analyses of promoter sequence of 710 and bidirectional promoter of 720-730 indicated that both contained the cis-elements response to low temperatures such as hormone,low temperature response and circadian control,etc.Referring to the Banana Genome-Musa itinerans,combined with the 4 overlapping gene sequences from the Tianbaojiao banana,the sequences and structure of the corresponding 4 overlapping genes from the wild banana(Musa itinerans)were analysed by local blast.The results indicated that there were also 4 overlapping gene sequences in the scaffold1345 of the genome.The alignment of overlapping genes gDNA sequence analysis between Musa itinerans and Tianbaojiao showed that the sequence conservation was higher,and the conservation of the gDNA of 730 sequence was the highest,with the sequence consensus of 98.96%,the sequence consensus of the 700,710 and 720 was also more than 90%.The conservation of the intergenic sequence was relatively lower,with sequence consensus below 90%,the conservation of 710 promoter was the lowest,sequence consensus at 81.05%,and the sequence consensus of the bidirectional promoter was 87.59%.The expression levels of the 4 overlapping genes were detected by qPCR under different temperatures(28? as the control,and the low temperatures of 13?,4? and 0?)and different tissues(leaves,pseudo-stems and roots)in the wild banana.The results showed that the expression patterns of 700 and 730,710 and 720 were similar.The 4 overlapping genes played an important positive or negative regulatory role in response to different low temperature stress in the wild banana.In addition,the expression trends of the 710 and 730 genes in different tissue parts were similar,both were pseudo-stems>root>leaf;however,the expression trends of the 700 and 720 genes were opposite,the expression trends of the 710 were root>pseudo-stems>leaf,and the expression trends of the 720 were leaf>pseudo-stems>root.The highest expression level was 720 in leaf,the highest expression levels were 710 and 730 in pseudo-stems,the highest expression level was 700 in root,which showed that the expression levels of 4 overlapping genes were specific in different tissues in wild banana.Interestingly,the gene 730,encoding adal,is one of the components of acetylation complex,which was newly discovered in plants,and the gene 710 encoded CKII protein,both of which were closely related to the gene expression in response to stress response,and deserved further exploration.In conclusion,based on the analysis of the physiological and biochemical during on the low temperature stress in wild banana,the miRNAs,transcriptome and lncRNAs were sequenced using high-throughput sequencing technologies,a large amount of data were obtained,which was beneficial to digging the resistant genetic resources of the wild banana,and in the meanwhile,the results also found that the mechanism of stress responses to the different low temperatures was different,and there existed several pathways in response to low temperature stress in the wild banana,which suggested that the wild banana can be probably used as the model species in the studies on the responses to low temperature stress mechanism for tropical plants.At the same time,based on the results of transcription of RNA-seq analyses,the identification and expression analyses of several miRNAs,CK? family and 4 overlapping genes related to cold resistance were performed,which provided new clues for further exploring the mechanism of cold resistance in the wild banana or cultivated bananas,and also provided important scientific basis for cold resistant of banana breeding. |
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