Cloning And Functional Analysis Of The CBL Family Gene In Cassava

Calcineurin B-like proteins(CBLs),which is similar to the calcineurin B proteins(CBLs),is a kind of calcium binding protein in the body of higher plants.It can interact with serine/threonine protein kinase(CBL-interacting protein kinase,CIPK)and participate in the stress response and growth process of plants.Cassava is a good material for abiotic stresses.At present,there are many studies on CBL gene and CBL-CIPK pathway in Arabidopsis thaliana,but little is known about cassava CBL gene and CBL-CIPK pathway.In this study,the CBL family gene was cloned from cassava,and the characteristics of CBL protein were analyzed by bioinformatics.The expression pattern of CBL gene under different stress was studied by fluorescence quantitative PCR,and the function of CBL protein was preliminarily studied.The main research results were as follows:1.Using the CBL protein sequence of Arabidopsis as query sequence,the sequence was found from the DNA database of cassava genome,and 8 CBL genes of cassava were cloned.According to the homologous similarity to AtCBLs,they were named MeCBL1,MeCBL2,MeCBL4,MeCBL5,MeCBL6,MeCBL8,MeCBL9,respectively.2.Using online tools such as ExPASY and CSS-Palm3.0,the molecular weight of MeCBLs protein was found to be between 24.50?28.74 kD.It was highly conservative and contained four typical EF hand domains,all of which were motif 1 and motif 4.In addition to MeCBL6,other MeCBL proteins have palmitic sites;in addition to MeCBL4,6,and 10,the N-terminal domain of other MeCBL proteins contain a conservative myrimeylation domain;according to the phylogenetic tree analysis of CBL,the MeCBL family proteins can be divided into four groups;Igroup have motif 10 and 8 introns,others contain seven introns.3.The results of MeCBLs promoter analysis,we divide 69 cis acting elements into four categories based on their biological functions:light response correlation,plant development correlation,abiotic stress correlation,and hormone response elements.Among them,8 MeCBL gene promoters contained Box 4 light response element,Skn-1 motif expressed in endosperm,and TC-rich repeats involved in defense and stress response.MeCBL6,8,9 and 10 promoters have HSE components involved in heat stress.LTR elements related to low temperature reaction were detected in MeCBL5,8 and 9 promoters.Subcellular prediction indicates that MeCBL gene is mainly located in cytoplasmic membrane.4.The expression pattern of cassava MeCBL gene in different organs was analyzed by semi quantitative analysis.The results showed that only MeCBL2 gene was expressed in different developmental stages of different tissues.Moreover,the transcription level of MeCBL5 gene is generally low,and MeCBL8 has high transcription level in mature leaves and seedling roots.In addition,the transcriptional level of MeCBL1 and MeCBL9 genes in stem and leaf tissues at maturity stage is significantly higher than that in seedling stage.5.Real-time fluorescence quantitative PCR was used to analyze the expression of MeCBL gene in roots and leaves under different stresses.The results showed that MeCBL6 and 8 were upregulated at 3 h and 9 h in root under 200 mM NaCl treatment.Under salt stress,MeCBL10 is the only gene that is up-regulated in leaves.After PEG treatment,MeCBL4 and MeCBL10 were increased at 9h in leaves,but other geneswere observed no significant difference in root or leaf.In addition,MeCBL2,4,5,9 and 10 were induced under high temperature or low temperature stress.Under high temperature stress,MeCBL4 and MeCBL5 increased at 3h and 9h in leaves,respectively.It was found that MeCIPK24 was significantly up-regulated in roots of salt streatments,and it was a salt related gene.6.In order to screen out a salt tolerance pathway similar to the Arabidopsis SOS1 in cassava,we use yeast two hybrid technique to find three MeCBL proteins interacting with MeCIPK24 protein:MeCBL2,6,and 10.Then the transgenic yeast was constructed to compare the effects of MeCIPK24,MeCIPK24+MeCBL10 protein or complex on the MeSOS1 protein.The results showed that the transgenic yeast of MeSOS1,MeCIPK24 and MeCBL10 genes was significantly enhanced salt tolerance in comparison with other single or double or three transgenic yeasts,and growthed on the salt plate of 100mM NaCl.