The Analysis Of Preliminary Function And Expression Pattern Of CBL Gene Family In Sweet Sorghum Under Sodium Carbonate Stress

Saline-alkali soil contains excessive sodium carbonate (Na2CO3) and sodium bicarbonate (NaHCO3), which inhibits most plant growth and development. The tradition fossil fuel is getting shortage and leads to a lot of environment problems, The development of renewable energy sources is urgently needed. Sweet sorghum (Sorghum bicolor L.) is characterized by drought and flood resistance, salt tolerance and high efficiency of biomass accumulation. It is considered to be one of the most promising bio-energy crops. In order to understand the function and the physiological expression pattern of SbCBL gene family in sweet sorghum under sodium carbonate stress and utilize this experimental results to obtain new sorghum cultivar which can tolerate sodium carbonate stress, we use sweet sorghum (Sorghum bicolor L.) cv. NengSiâ… to be our materials. In our research, we analyzed the physiological response of sweet sorghum, and identified a eligible sodium carbonate concentration. We used real-time quantitative polymerase chain reaction (RTqPCR) to analyze the expression pattern of SbCBL gene family under sodium carbonate stress and the expression pattern of SbCBL gene family in different tissues under regular condition. We cloned special gene by Gateway technology and introduced it into Arabidopsis. We analyzed the protein-protein interaction by yeast two hybrid. Meanwhile, through the bioinformatics analysis, we identified the character of SbCBL gene family. The results are as follows:(1) The results of physiological experiment showed that vitality index, radicle and the length of plumule can be the index of sorghum cultivar which can tolerate sodium carbonate stress, and we identified a eligible sodium carbonate treatment concentration: 100mM Na₂CO₃.(2) We used real-time quantitative polymerase chain reaction to analyze the expression pattern of SbCBL gene family under sodium carbonate stress and the expression pattern of SbCBL gene family in different tissues under normal condition. The results showed that SbCBL genes in sweet sorghum have different tissue-specific expression patterns under normal growth condition, the transcription of SbCBL06 and SbCBL08 were most abundant in leaves at seedling stage and others were higher expression in matured leaves. In addition, SbCBL genes showed various sodium carbonate stress responsive patterns in sweet sorghum seedlings treated with sodium carbonate, the transcriptional level of SbCBL01 and SbCBL07 showed sustained increase in roots and a similar pattern appeared in the transcription of SbCBL06 in shoots.(3) Bioinformatic analysis of SbCBLs:Firstly, Each SbCBL had its homologous ESTs in the database. Some of these ESTs were related to abiotic stresses. The results showed that SbCBL gene family might play an important role in abiotic stress.Secondly, Multiple alignments revealed that sequences of SbCBLs are highly conserved and most SbCBLs have three typical EF-hands structure. Four SbCBL proteins (SbCBL01, SbCBL04, SbCBL05, SbCBL08) have a conserved N-myristoylation domain (MGXXXS/T).Thirdly, Many stress responsive and phytohormones responsive cis-elements are found in the promoter region of SbCBLs. The conservation in intron number of the same or different species infers that the introns may have some effect on the function of CBL gene family.Phylogenetic analysis of SbCBL gene family between sweet sorghum and other plants (Arabidopsis, rice, maize, Vitis vinifera, poplar and chlorella ) suggested that sweet sorghum CBL gene family had the most closely genetic relationship with rice.(4) We cloned SbCBL01 by Gateway technology and introduced it into the expression vector pLeela. We used the floral dip method of Agrobacterium-mediated transformation to infect Arabidopsis. SbCBL01 has been inserted into the genomic DNA of Arabidopsis. We obtained six T1 transgenic lines .(5) We constructed SbCBL01 and potential target SbCIPK gene into pDEST22 and pDEST32, respectively and analyzed SbCBL and SbCIPK interaction. The result showed that SbCBL01 might interact with SbCIPK24 and SbCIPK30.