| Apple(Malus domestica Borkh.) is one of the major fruit crops in China. In recent years, as the pollution of heavy metals in soil and constantly changing, making the loss of Ca2+ in Apple increase, physiological metabolic disorders, yield and quality reduce in different degree. Ca2+/H+ antiporters (CAXs) are an important kind of membrane transport proteins, it’s an outward transporter that regulate intracellular and extracellular Ca2+ balance,which plays a very crucial role in plant nutrition and ion transport. At present, a lot of CAXs genes have been cloned, and transformed in model plants. However, which are rarely transformed in the woody plant. To improve apple’s ability to transport Ca2+ absorption and resistance, we were conduct research on the basis of previous research studies. The experiment firstly analysis the apple’s MdCAXs gene family, then according to the MdCAX1 gene sequences, cloned by restriction site analysis and overlapping PCR construction of plant binary expression vector containing sMdCAXl gene required, and construct vector, use the method of agrobacterium mediated import ’Nagafu No. 6’to obtain transgenic plants. The main results are as follows:1. Chromosome location, isoelectric point, hydrophobicity/hydrophilicity, transmembrane region, subcellular localization and conserved structure domains of MdCAXs gene family were predicted and analyzed with bioinformatics methods. Phylogenetic analysis is performed with the CAXs genes of other plants. The expression of MdCAXs genes in vegetative organs after the salt treatments was analyzed. The results showed that MdCAXs gene family contained 8 members, which are located in the A subtypes and the B subtype of the CAXs I. MdCAXs are hydrophobic proteins, containing 436 to 817 amino acids, and the isoelectric point (PI) from 4.97 to 7.12. They generally have 11 or more transmembrane regions, and contain 5 regulatory domains, i.e. N-terminal autoinhibitory domains (NRR), C-terminal domains, Ca2+ domain (CaD), C domain and D domain. MdCAXs proteins were mainly localized in plasma membrane and endoplasmic reticulum by predication of PSORT software on line. Fluorescence quantitative expression analysis showed that the expressions of MdCAXs differ in roots, shoots and leaves in response to the Na+, Ca2+,Mg2+ and Mn2+ treatments, which suggests the different functions in ion transportation of MdCAX.2. According to the NCBI MdCAX1 gene sequence design primers, we cloned MdCAX1 gene ORF from apple leaves, then to remove the N-terminal sequence of the cloned gene sMdCAX1 based on the MdCAX1 gene as a template, and get a fragment of 1272bp in size. Through the analysis of the MdCAX1 gene sequence in the ORF restriction enzyme site, two Sac Ⅰ sites were found at bases 413 bp and 65 8bp. Useing overlapping PCR method to sequence Sac Ⅰ site mutagenesis, get a fragment of 1272 bp in size, named sORFl+2Mu. While building a pYH4215-sMdl plant binary expression vector, and import the expression vector into Agrobacterium strain EHA105, get the engineering bacteria prepared for genetic transformation of ’Nagafu No.6’.3. A genetic transformtion system was studied with the ’Nagafu No.6’ tube seedling leaf as explant, mediated by Agrobacterium tumefaciens method will build good pYH4215-sMdl plant expression vector into the explant. Hygromycin (Hyg) concentration screening showed that ’Nagafu No.6’ is very sensitive to Hyg concentration.The best Hyg selection pressure is lmg·L-1 for leave,4 mg·L-1 for subculture seedlings,2 mg·L-1 for rooting. The transgenic plants were detected through X-glucuronide staining solution, then the dyeing successful plants were detected by PCR and RT-PCR. The results showed that transgenic plants containing Hyg gene and GUS gene, indicating successfully integrated pYH4215-sMd1 plasmid T-DNA region into the Apple genome. |
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