Cloning Study Of AP2/ERF And CaM On Bruguiera Gymnorhiza And Kandelia Candel Of Mangrove

Mangroves are woody plant community which grow in tropical and subtropical coastal intertidal zone and has a natural salt balance system. AP2/ERF transcription factor can regulate a number of functional genes downstream in the biological and abiotic stress signal transduction process, combination with the cis-acting element of promoter. Calmodulin (CaM) could interact with the target proteins through its own unique CaM-binding domain, thereby regulating cell's normal growth and development as well as the important physiological functions. Bruguiera gymnorrhiza and Kandelia candel were as materials in this study which collected from the Shenzhen mangrove nature reserves. Clone new genes using the methods of genetic engineering principles and technology. Provide new genetic sources for plant genetic engineering research and application. It has laid a foundation to make further using of these genes into plants. The main results are as follows:1. Conserved domain analysis according to AP2/ERF transcription factor family, design of degenerate primers and get 2 conserved sequences of ERF transcription factor.2. Design gene 3'end and 5'end-specific primers according to the middle of the sequence has been obtained, and rapid amplification of cDNA ends, get gene 3'end and 5'end sequence of Bruguiera gymnorrhiza and Kandelia candel. Design gene full-length primers, get full-length ERF genes of Bruguiera gymnorrhiza and Kandelia candel, full-length cDNA are 1870bp and 1209bp respectively, We named MlERF and QqERF.3. This study referenced other CaM genes have been cloned in several crops (maize, sugar cane, rice and other crops) from GenBank. Design gene primers based on the highly conserved nucleotide sequence. We cloned the full length CaM cDNA sequence which is 735bp from Bruguiera gymnorrhiza and named MlCaM.4. Sequence analysis showed that the MlERF and QqERF have a conservative AP2/EREBP domain, there are oneα-spiral and threeβ-folding in protein spatial structure. The results of homology compared and the phylogenetic tree analysis showed that it has a high homology on conservative region of amino acids when compared with the majority of crops ERF transcription factor. MlCaM has high homology on conservative region of amino acids when compared with the arabidopsis thaliana, peanuts, barley, beech, sweet potato and other plants by sequence alignment and phylogenetic tree analysis. The complete open reading frame of MlERF gene is 1428bp, encoding 475 amino acids. Predicted protein isoelectric point is 8.77, molecular weight is 50.6kDa.The complete open reading frame of QqERF gene is 870bp, encoding 290 amino acids. Predicted protein isoelectric point is 8.09, molecular weight is 31.5 kDa. The complete open reading frame of MlCaM is 450bp, encoding 149 amino acids. Predicted protein isoelectric point is 4.11, molecular weight is 16.8kDa.5. We construct plant expression vector pC13rd29A-MlERF, pC13rd29A-QqERF and pC13rd29A-MlCaM.