| The dissertation contains two parts: 1) High frequency regeneration system of kiwifruit has been established successfully. AtNHX1 gene from Arabidopsis thaliana was transformed into kiwifruit genome with Agrobacterium tumefaciens, putative transgenic plants were confirmed. 2) Through in silico cloning, a 2055bp cDNA sequence of maize Na~+/H~+ antiporter gene was assembled.1. Tissue culture and genetic transformation of kiwifruitKiwifruit is a kind of well-known fruits originate from China. Because of its distinctive flavors, high nutrition value and medical effects, kiwifruit attracts more attention in present years. Along with the improvement of living standard and changes of consumption structure, the requirement to high quality kiwifruit is increasing year after year. Abundant kiwifruit varieties distributed in China can provide resources for its breeding. However, because kiwifruit is a dioecious species with a complicated genetic background, traditional seedling propagation of kiwifruit could not meet the requirements, due to time-consuming, low survival rate and unstable properties. Modern biotechnology creates a new path for kiwifruit breeding. With the development of tissue culture system, genetic transformation has become the main method in kiwifruit breeding gradually.In our experiments, Actinidia deliciosa, with the highest yield of kiwifruit in China, was selected as the target material, and its high frequency regeneration system was established. With this system, AtNHX1 gene from Arabidopsis thaliana was transformed into kiwifruit and transgenic plantlets were regenerated. The putative transgenic kiwifruit were examined by PCR. The details are summarized as follows:(1) Establishment of high frequency regeneration system of kiwifruit. Effects of 2,4-D, 6-BA and NAA on callus induction and differentiation of stem and leaf explants of Actinidia deliciosa were studied with MS as basic medium. The results revealed that callus formation could be stimulated remarkably by 6-BA, and callus differentiation could be promoted by 6-BA in combination with NAA. In contrast, 2,4-D had an inhibition effect on callus induction. MS medium supplemented with 2mg/L 6-BA and lmg/L NAA was optimal for shoot regeneration of stem cuttings and leaf discs. On the optimal medium, the leaf discs from the regenerated sterile plants could produce secondary shoots directly and shoot regeneration rate reached 100%, each leaf disc produced 9.33 shoots on average and 23.21% of them exceeding 0.5cm in height. 1/2 MS medium supplemented with 0.4mg/L IBA was the best medium for rooting.(2) Genetic transformation of kiwifruit with Agrobacterium tumefaciens. The calli, stem segments and leaf discs were infected by LBA4404 containing pHZX1, and 15 transformants were regenerated to the differentiation medium supplemented with 20 mg/L kanamycin. The 7 putative transgenic plants confirmed by PCR.2. In silico cloning of maize Na~+/H~+ antiporter geneIn silico cloning is a new strategy of gene isolation based on the development of HGP (human genome project) and ESTs (expressed sequence tags), it has the advantages of low investment, time saving, simple operation and high pertinence. Previously, in silico cloning was mainly used in isolation of animal genes, such as human, mouse and zebra fish genes. In recent years, this method was also adopted in isolation of plant genes along with the expansion of plant EST database, and many plant genes have been cloned via this method.In this experiment, a rice Na~+/H~+ antiporter gene (GenBank Accession, AY343324) was used as the query probe in BLAST screening of the maize EST database and a 2055bp cDNA sequence was assembled. The full-length cDNA contains a complete ORF encoding a putative peptide of 539 amino acids. Compared with the Na~+/H~+ antiporters of Phragmites australis, Aeluropus littoralis and Hordeum vulgare, the similarity of the amino acid sequence excesses 90%. RT-PCR identification experiments are being carried out. |
PDF Full Text Request