Studies On Construction Of Plant Expression Vectors Containing CAPD Gene Driven By A Phloem Specific Promoter And Genetic Transformation In Fruit Trees

China is a leading country in fruit industry, ranking the top of the world both in cultivation area and in total output. The development of fruit industry, however, is facing the threat from various devastating quarantine diseases such as Citrus huanglongbing, Banana fusarium wilt and Kiwifruit bacterial canker etc. The breeding of disease-resistant cultivars, therefore, is the essential approach to those problems. The conventional method, e.g. hybrid breeding, seems not working well because of the lack of suitable germplasmic resources, the complexity in genomes and ploidy and the long period for breeding. The rapid development of the modern biotechnology has provided new ways for breeding of disease-resistant cultivars, as it can produce new genotypes and germplasms that could hardly be attained by conventional techniques. To date, most of the disease-resistance genetic engineering has adopted CaMV 35S promoter for constitutive expression and the target genes come from microorganisms or animals. But in fact these genes, if not optimized in codons, have the problems of security, specificity and efficiency. This study focused on the devastating diseases conducted via phloem, e.g Citrus huanglongbing and Banana fusarium wilt. First, a phloem-specific promoter was cloned from Cucurbita maxima and, based on the codon usage analysis, a new version of Antibacterial peptide D (APD) gene that kills the pathogens of these diseases were designed and synthesized. Then the different plant expression vectors that contained marker genes or target gene driven by various promoters were constructed. Finally the newly constructed vectors were studied by transforming into dicotylous woody fruit tree -citrus, lianoid fruit tree - kiwifruit and herbaceous fruit tree - strawberry, respectively. The results were summarized below:(1). A pair of primers was designed according to the sequence of phloem protein 2 (PP2), which shows phloem-specificity from C. maxima, and a 966bp fragment of PP2 gene in the promoter region was amplified by polymerase chain reaction (PCR) using the genomic DNA of C. maxima cv. 'Shanxi Bendizhong' as the template. The fragment was cloned into pUCm-T vector, and a new recombined vector named pUCm-PSP was obtained after confirmed by Blue/White screening, PCR and restriction digestion. Sequence analysis indicated that this fragment had 95% and 98% homology compared with two reported promoters respectively. This result suggested that it might have the same function as other PSPs.(2). Coding DNA sequences (CDS) of four different citrus species, including sweet orange (Citrus sinensis), satsuma mandarin (C. unshiu), grapefruit (C. paradisi) and lemon (C limon) were used to analyze the relative frequency of synonymous codon (RFSC), and high-frequency codons were selected by high-frequency codon analysis. The results indicate that there was little difference in codon preference among the four citrus species. With similar method, 177 CDS were used to analyze the relative frequency of synonymous codon (RFSC), and 7 high-frequency codons including TAA, GCT, GAT, CTT, AGG, AGA and GTT were revealed by high-frequency codon analysis. When the frequency of codon usage of citrus was compared to those of Homo sapiens, Drosophila melanogaster, Saccharomyces cerevisiae and Escherichia coli, we found that the codon preference was considerably different between citrus and non-plant species. While comparing with Arabidopsis thaliana, Lycopersicon esculentum, Oryza sativa and Musa acuminata, we found that the codon preference of citrus is identical to those of the dicotyledons (A. thaliana and L, esculentum), but different from those of the monocotyledons (O. sativa and M. acuminate). The findings are helpful to researches involving introducing genes from animals or microbials or vice versa and designing primers for functional gene cloning.(3). Based on the codon usage for the amino acids of 177 citrus functional proteins deposited in GenBank, a new Antibacterial Peptide D (APD) gene with citrus preference codons was designed and chemcially synthesized. The peptide has an ability to kill the phloem conducted pathogens such as Huanglongbing, a destructive and widespread disease in citrus. The new gene encoding the same peptide, referred to as CAPD, was cloned into pUC19 to generate a recombinant plasmid pUC19-CAPD, and was verified by sequencing.(4). Four new plant expression vectors were constructed by recombinant DNA technology. pHZ01 with reporter gene GUS droved by PSP was constructed after cutting two vectors pUCm-PSP and pBI121 plant expression vector; pHZ02 whose reporter gene GUSA was droved by PSP was constructed after cutting two vectors pUCm-PSP and pCAMBIA1301 plant expression vector; pHZ03, the reporter gene GFP droved by PSP was constructed after cutting two vectors pUCm-PSP and pCAMBIA1302 plant expression vector; and pHZ04 in which the fusion reporter genes GFP and GUSA were droved by PSP was constructed after cutting two vectors pUCm-PSP and pCAMBIA1303 plant expression vector. The recombined plant expression vector was transferred into Agrobacterium tumefaciens strains of LBA4404, GV3101 and EHA105 and A.rhizogenes strain of 15834 by using cell competent method, and a good foundation had been laid for further research work in expression and function of this promoter.(5). Two new plant expression vectors, pHZ05, harboring CAPD target gene driven by the constitutive 35S promoter of CaMV (35SP), and pHZ06, harboring CAPD gene driven by PSP were constructed using recombinant DNA technology. The two new vectors, which harbor the same target gene CAPD but driven by different promoters, were introduced into competent cells of A. tumefaciens strains LBA4404, GV3101 and EHA105 and A rhizogenes strains Ri15834 respectively. The study is helpful for finding a new way to develop new germplasms resistant to some diseases in fruit trees.(6). Since the transformation system requires highly efficient regeneration system, we studied the in vitro regeneration of different explants of C. sinensis (L.) Osbeck cv 'Anliu' orange in order to get suitable medium and explants. On the 15 media with different combinations of 6-BA, IBA and NAA, redifferentiation was observed for different parts of epicotyl and cotyledons from the sterilized etiolated seedlings of 'Anliu' orange, and a medium prescription suitable for genetic transformation of 'Anliu' orange were obtained, i.e, MT+IBA 0.5mg/L + 6-BA 2.5mg/L +sucrose 30g/L + agar 8g/L. It was found that the epicotyl parts were better than other explants for genetic transformation. We used the cultures of Agrobacteria EHA105 containing the newly constructed expression vector pHZ05 or pHZ06 to transform the epicotyl from sterile etiolated seedlings of 'Anliu' orange. After pre-culture, infection and co-culture, we used a medium to which only cef were added to inhibit the growth of agro-bacteria through different selection modes for differentiation. Upon the onset of differentiation of the explants, the media containing antibiotics at different concentrations were used for selecting. This method of selecting was not only good for the growth of the transformed buds but also helpful for preventing the inhibitive effects of antibiotics on buds. In addition, it could prevent the untransformed buds from escaping. After 4 months of selecting culture, a batch of transgenic resistant buds was obtained. DNA was extracted from leaves of some of the big buds for PCR detection with several primers and the products of the target gene was sequenced and compared. 3 transgenic plants were obtained from the 10 buds transformed with pHZ05 and 11 from 60 buds transformed with pHZ06.(7). Using the leaf discs from the sterile seedlings of 'Bruno' kiwifruit as explants, we studied the effects of the placing method and the dark culture time on the adventitious buds regeneration. A highly efficient regeneration system was established. Then using leaf discs and Agrobacteria-mediated transformation method, we conducted transformation of CAPD gene droved by different promoters. After pre-culture, infection, co-culture and selective culture, some transgenic resistant buds were obtained. DNA was extracted from the leaves of some of the big buds for PCR detection with several primers and the products of the target gene was sequenced and compared. The results showed that 7 transgenic plants were obtained from the 60 buds transformed with pHZ05 and 12 from 40 buds transformed with pHZ06.(8). Using Agrobacteria-mediated transformation method and leaf discs from tissue cultured plants of 'Toyonoka' strawberry as explants, we transformed CAPD gene droved by 35SP and PSP respectively. After pre-culture, infection and co-culture, the explants were cultured for 2 weeks in bud inducing medium without any antibiotic, then selected by using kan antibiotic. Some transgenic resistant plantlets were obtained. DNA was extracted from leaves of some plantlets for PCR detection with several primers and the products of the target gene was sequenced and compared. The results showed that 1 transgenic plants were obtained from the 5 plantlets transformed with pHZ05 and 14 from 65 buds transformed with pHZ06.This study has laid a solid foundation for researches in the gene expressions with different promoters in fruit trees and for the breeding of new fruit cultivars resistant to diseases through genetic engineering.