Identification Of Cassava TCP Transcription Factor Family And Study On MeTCP4 Anti-stress Function

Low temperature and drought seriously affect plant growth and reproduction.In order to adapt to adverse environmental changes,plants have developed a variety of regulatory mechanisms at physiological and biochemical levels.It has been proved for a long time that plants are often accompanied by the expression of stress-related genes during adaptation to stress.The TCP transcription factor family genes are involved in multiple regulation of plant growth and environmental stress.At present,this family of genes is rarely studied in cassava.Therefore,the phylogenetic and expression patterns of cassava TCP family genes under low temperature and drought stress were studied,which has guiding significance for cassava breeding.In this experiment,the cassava TCP family members were studied by bioinformatics and gene expression methods.At the same time,MeTCP4 gene was cloned and its function was studied by overexpression of this gene in Arabidopsis thaliana.Below are key research findings1.The whole genome of cassava encodes 36 MeTCP genes family member genes and the naming numbers are MeTCP2 to MeTCP23.The cassava TCP family gene has a typical TCP domain.The number of amino acids in 36 cassava TCP gene-encoded proteins ranged from 73 to 562;the relevant molecular mass ranged from 8.3 to 58.1 KDa;the isoelectric point ranged from 5.53 to 10.08.The phylogenetic tree analysis showed that the cassava TCP family genes can be divided into 8 different subfamilies(A-H).The cassava TCP family gene and the Arabidopsis TCP family gene are the closest.2.The gene structure and conserved domain analysis of the cassava TCP family showed that the TCP gene of cassava in the same subfamily showed exon-intron distributions with similar exon lengths and intron numbers,which is consistent with the results of phylogenetic analysis.16 of 36 MeTCP genes had no intron,while the other MeTCP genes possess 1-3 introns,with the exception of MeTCP16 containing four introns.The proteins encoded by the cassava TCP family member genes have a total of 20 conserved domains and are named motifl to motif20.The cassava TCP genes of the same subfamily have similar domain composition and are highly separated among different subfamilies.3.Quantitative real-time PCR analysis showed that the expression of MeTCP family members was different in different tissues.The expression of most genes in stems,leaves,cusps was higher than that in roots.The expression of cassava TCP family members in different tissues varies greatly and the expression of a single gene in different tissues also differs.Among the differentially expressed genes in the TCP family of cassava,there are some genes that express high levels in specific tissues.For example:MeTCP2a?MeTCP3a?MeTCP5b?MeTCP8a?MeTCPl3a and MeTCP20d are highly expressed in leaves,indicating that these genes play an important role in leaf organs.4.Real-time quantitative PCR was used to detect the expression of TCP genes in 36 cassava TCP family members that are treated with ABA,GA3,IAA,JA,ZT and 6-BA.The results showed that 6 kinds of hormones can cause differential expression of cassava TCP family gene.At the same time,22 TCP genes in the TCP family of cassava were found to be particularly sensitive to ABA,which indicates that these genes may mediate the ABA signaling pathway involved in the regulation of plant abiotic stress.5.Under low temperature and drought stress,RNA-seq analysis of cassava TCP family genes expression patterns showed that 18 and 24 MeTCP genes showed significantly change under cold and drought treatment,respectively and the fold change was greater than 2 times,the expression levels of MeTCP genes in response to cold and drought stresses were dramatically changed,implying their putative roles in stress tolerance.The analysis of cis-acting regulatory elements in the cassava MeTCPs family gene promoter region revealed that the altered expression of 12 MeTCPs that were co-induced/repressed by cold and drought stresses,which indicates that they may be regulated by key stress regulatory genes.6.MeTCP4 contained an open reading frame(ORF)of 1269bp,which encoded a protein with 422 amino acids with a predicted molecular weight of 45.75KDand PI 6.17.The full length cDNA sequence of MeTCP4 was cloned from the cassava genome,Quantitative real time RT-PCR PCR results showed that the expression of MeTCP4 can be found in all the tested tissues,with the highest expression in leaves but very lower in stems and roots.MeTCP4 gene was repressed by drought and low temperature stress.7.Arabidopsis thaliana are transformed to obtain 35s::rMeTCP4 over-expressed Arabidopsis plants.Phenotypic observation revealed that the rosette leaves of the over-expressed MeTCP4 transgenic Arabidopsis were smaller,more round,and often darker green than the petiole of the wild type leaf,with a large proportion of leaves.Leaf margins are more rounded than wild-type Arabidopsis thaliana.Under low temperature stress,the over-expression of MeTCP4 in transgenic Arabidopsis thaliana increased the content of proline and soluble sugar and decreased the content of MDA.This indicates that the MeTCP4 gene can enhance the adaptation mechanism of transgenic Arabidopsis to low temperature stress.qRT-PCR analysis showed that the expression of the key genes(CBF transcription factor?RD29a?COR15a and COR47)in 35s::rMeTCP4 overexpressed Arabidopsis plants were significantly up-regulated compared to the wild type,which indicates that the key genes to low temperature stress were directly affected by MeTCP4.Expression the key genes enhances the cold Resistance of 35s::rMeTCP4 overexpressing Transgenic Arabidopsis thaliana Plants.8.Under low temperature stress,the GO enrichment analysis of the differentially expressed genes in transgenic and wild Arabidopsis thaliana revealed that the GO enrichment of the differential genes was mainly concentrated on oxidoreductase activity,peroxidase activity,antioxidant activity and catalyst activity.RNA-seq results showed that many genes that responded to low temperature and ABA stress were significantly differently expressed and most of them were significantly upregulated in transgenic Arabidopsis;qPCR validation was consistent with RNA-seq results.