Identification Of Interaction Between Transcriptionfactor GmPLZ And Candidate Proteins In Vegetable Soybean And Genome-wide Analysis Of ANK Gene Family In Snap Bean

Abiotic stress such as high temperature、drought and salt seriously affect yield and quality of crops. It is important to analyze the signal transduction and responsive mechnism in wheat resistance to stresses. Including protein kinases, phospholipid binding proteins and transcription factors. In this article, through the phenotype identification of overexpress GmPLZ in Arabidopsis, GmPLZ play an important role in drought and high-salt. And based on the research of the mechanism of tolerance, Screening interactions of two candidate protein GmPAK protein kinase and GmCap phospholipid binding protein。 In order to the sdudy interaction between candidate protein and GmPLZ by yeast two-hybrid analysis. We analyze the molecular characteristic and the expression patterns of GmPAK and GmCap by constructing the recombinant and using RT-PCR technology, and expounds molecular mechanism of the GmPLZ transcription factors by improving plant resistance. Through the study of nap bean ANK family bioinformatics method、molecular characterization analysis and Expression pattern analysis, ANK protein plays an important role in resistance to abiotic stress. The results are as follows:1. Real-time PCR results indicated expression levels of GmPLZ increased under drought and salt stresses with different degrees, The expression highest when drought 2h and salt 24h. Through the phenotype identification that overexpress GmPLZ plays an important role in drought and high-salt.2. We constructed a soybean cDNA library and the bait vector GmPLZ-pGBKT7 and screened the interacting proteins of GmPLZ transcription factor by yeast two-hybrid system. The candidate positive proteins were sequenced and analyzed through using the BLAST in NCBI. The results showed that those candidate proteins were related to stress and defense, energy metabolism, growth and development in plant. Through further research selected two interacting proteins:GmPAK protein kinase and GmCap phospholipid binding protein.3. We constructed the recombinant vector GmPAK -pGADT7、GmCap-pGADT7and identified the interaction between the candidate protein and GmPLZ. Validating the interaction between GmPAK、GmCap and GmPLZ by yeast two-hybrid analysis. Protein kinase and phospholipid binding protein plays an important role in signal transduction and responsive mechanism. This result suggests that transcription factor GmPLZ play an important role in improving crops resistance to stresses.4. We constructed the recombinant vector GFP-GmPAK、GFP-GmCap and transferred it into arabidopsis protoplasts. Subcellular localization analysis revealed that GmPAK、GmCap located on cell membrane. GmPAK possesses autophosphorylation activity. RT-PCR analysis revealed that the expression of GmPAK was upregulated by drought, the high-tempreture can be detected at 12 h, but has no effect by BR and salt treatment and GmCap expression highest when drought 1h, Under the condition of high temperature, NaCl and BR stress, no obvious changes.5. A total of 30 ANK genes were systematically identified from snap bean and located on 9 chromosomes. The chromosome 5 had 13 genes, which was more than other chromosomes. Protein structure domain analysis showed that ANK25 still contained a RING domain besides an ANK domain. Ring proteins were mainly involved in gene transcriptional regulation, DNA repair and recombination. The transient expression in Arabidopsis protoplasts displayed that ANK protein mainly contributed in plasma membrane. Expression pattern analysis found that ANK25 was responsive to drought, salt, and exogenous ABA. This study provided valuable information regarding the classification and functions of ANK genes in snap bean.