Screening And Bioinformatics Analysis Of Transcription Factor Interacting With MeCWINV6 Promoter In Manihot Esculenta Crantz

Cell wall acid invertase (CWI) is a key enzyme involved in the sucrose metabolism pathway in plants. MeCWINV6 gene is one of six cell wall invertase genes in cassava. Promoter plays a vital role in regulating gene transcription,and transcription factors play an important role in the regulation of gene expression by interaction with the promoter. Therefore, the study of the CWI gene promoter and its interacting transcription factor can help to elucidate the regulation mechanism of gene expression of CWI gene,and provides a basis for further study on the signal pathway and the regulation mode of cassava CWI gene during abiotic stress responsive processes.In our research,we got potential upstream genes by yeast one-hybrid technique,interacting with the promoter region of MeCWINV6 gene, which provide a basis for further study of its expression and regulation. A bait vector, which was constructed by the promoter region of MeCWINV6, was transformed yeast cells to build bait yeast strain. The one-Hybrid cDNA library of cassava was built via SMART technology.Then the cDNA and pGADT7-Rec vector were used to co-transform into the bait yeast strain. We screened upstream transcription factors of the promoter region of MeCWINV6 gene via homologous recombination. The estimated cDNA library storage capacity is almost 7.08×106 and inserted PCR fragments sizes are 250-2000 bp.There are 35 positive yeast colonies greater than 1000 bp. The AT-hook nuclear localization protein transcription factor was screened out by sequencing analysis and BLAST homology analysis,named MeAHL31. AT-hook is a DNA-binding protein that plays an important role in plant growth, organogenesis, and response to hormonal and adversity stress.The promoter of MeCWINV6 and MeAHL31 were used to construct Pcw6 reporter vector and AHL-62-SK effect vector with pGreen ? 0800 and pGreen II 62-SK respectively. The vectors of effector and reporters were separately co-transformed into A.tumefaciens GV3010 cells with the Psoup vector, and the transformed GV3010 cells were used to infiltrate young tobacco leaves for transient expression, which was evaluated 3 days after infiltration. The results showed that the co-transformed Pcw6 reporter vector and the AHL-62-SK effect pGreen II 62-SK vector increased in the double fluorescence ratio of Pcw6 and co-transformed tobacco leaves, indicating that MeAHL31 protein could bind to the promoter of MeCWINV6 and could enhance its starting activity.The expression pattern of MeAHL31 was analyzed in the roots of the cassava by treating under 4?, ABA, GA3, H2O2 and SA. The results showed that the expression level of MeAHL31 was significantly increased. The MeAHL31 was induced by these factors and it might be involved in plant stress. We also found that the expression pattern of MeAHL31 was similar to that of MeCWINV6, which suggested that MeAHL31 protein could regulate the expression of MeCWINV6 and increase its expression level.The 42 genes of the AT-hook gene family were obtained from the cassava genome by bioinformatics method, their phylogenetic, chromosome distribution and the structure and function of their encoding protein were analyzed systematically. It was found that these proteins had different physicochemical properties,and the subcellular localization analysis showed that the AT-hook gene family members had the highest position in the nucleus, reaching 13. Cassava AT-hook gene is located in the nucleus, plasma membrane, lysosome and endoplasmic reticulum, and also there are few in microbody, cytoplasm and mitochondria. In addition to chromosomes 12,13 and 16, these AT-hook genes were distributed over the other 15 chromosomes unevenly. Cassava AT-hook family can be divided into three types, Type I contains 18,Type II contains 11 and Type III contains 11, the other two AT-hook genes can not be attributed into these 3 types.The results of the transcriptome analysis of AT-hook family genes in cassava roots showed that, the expression of AT-hook family gene in cassava cultivar RYG-1 was significantly lower than that of conventional cultivar SC8 with the prolongation of storage time and the decay of cassava roots. And the expression of 7 genes among 8 AT-hook family genes that differentially expressed were significantly increased in the roots rotting process of conventional variety of SC8. These phenomena suggest that some of the genes in the AT-hook family may be involved in the post-harvest physiological deterioration (PPD) of cassava, and may be promote the decay of cassava roots through regulating the genes on certain signaling pathways,which also indicates that the AT-hook family may have important research value in the breeding of anti-physiological deterioration of cassava.