Gene Analysis And Substrate Screening Of Tobacco Calcium-dependent Protein Kinases

Calcium dependent protein kinases （CDPKs） which have been identified throughout the plants,algae and certain protests, widely distribute in plants of a variety of organizations and have significantlydifferent expression in the space. The cardamom acylation sequences in the plant CDPK N-variableregions contain the subcellular localization information. The membrane localization usually needs theN-terminal myristoylation and many CDPK N-terminals have myristoylation sites. At present, CDPKsexhibit multiple locations, including the cytosol, nucleus, plasma membrane, endoplasmic reticulum,peroxisome, mitochondrial outer membrane. Many studies showed that CDPKs have been involved in avariety of Ca²⁺-mediated signal pathways.This study mainly included two aspects. On the one hand, the CDPKs that consist of Arabidopsisthaliana, N. tabacum and other species in tobacco genus were used to search the N. tomentosiformisgenome databases; the objectives of this investigation were to further study NtoCPKs’sequencecharacteristics, evolutionary relationship and gene expression. On the other hand, tobacco yeasttwo-hybrid cDNA libraries and NtCDPK12bait proteins were constructed, and NtCDPK12substratescreening and analysis were conducted.（1）The results indicated that full-length genomic DNA sequences of25NtoCPKs were obtainedfrom N. tomentosiformis. Phylogenetic and principal coordinate analysis divided the25NtoCPKs into4subfamilies. The results of reverse transcription-PCR （RT-PCR） showed that the expression patterns ofthe25NtoCPKs were significantly different and there were four different patterns of tissue-specificexpression. This study provides an important foundation for further functional dissection of the tobaccoCDPK gene family.（2）Plasmid PCR and sequencing results showed that the yeast expression vector pGBKT7-NtCDPK12q was suceessfully constructed. The colonies of the bait expression vector which can growon SD/-Trp medium generally were more than2mm in diameter. When the colonies were inoculatedinto SD/-Trp liquid medium and the bacteria were shaken12h, the OD₆₀₀was greater than0.8. Positiveclones in the SD/-Trp-Ade-His media can not grow. So they have no self-activation and toxicity andcan be used for the yeast two-hybrid.（3）In this study, the common flue-cured variety K326was used as the experimental materials tobuild a tobacco yeast two-hybrid cDNA library. The experiments about the conversion rate, titer andrandom testing of the insert of the library showed that the conversion rate and titer were2.0x10⁷and3.0x10⁷cfu/mL, respectively, and the library insert fragments located between500and1000bp, whichmatches the requirements of the yeast two-hybrid library.（4）Combine the Y2H library with bait strain. The positive colonies were selected on theDDO/X/A and QDO/X/A media for twice. Positive clones were sent to sequence and the sequencingresults can be divided into four categories: RNase H family protein, GTBP-1, GAG-POL precursor anda kind of unknown protein. The highest proportion was the RNase H family protein, and in this studywe named it as NtPS1. （5）Quantitative analysis showed that, NtPS1and NtCDPK12can be expressed in various tissues,with the highest expression level in the stem, and no significant difference in expression level in root,leaf and flower. Induced by drought, the expression levels of NtCDPK12and NtPS1in the stemchanged significantly at6h and reached the highest. With the stress time, the expression levelsgradually returned to normal. The expression of variation in the stem was more significant than in theleaf. Their expression were increased in leaf after12h. Induced by the high salt stress, the NtCDPK12and NtPS1expression levels in the stem rose at6h. With the stress time, their expression levels weregradually restored to normal levels. These results indicated that NtCDPK12and NtPS1may be involvedin tobacco drought and salt stress response processes.