Studies Of Introduction Of Antifungal Genes Into Cotton Plants By Laser Microbeam Puncture And Clone Of Antiaphids Genes

Cotton, as the major economic crop, occupies an important place in our national economy. Verticillium wilt is one of the most serious worldwide plant diseases, which infects the cotton and decrease its yield and fiber quality. It is very difficult to obtain Verticillium wilt disease-resistant cotton varieties by traditional breeding methods. In this study, chitinase gene andβ-1,3-glucanase gene were introduced into cotton successfully by using genetic engineering methods, which were combined laser micro-beam puncture techniques and germ line transformation method, and finally transgenic plants with chitinase gene andβ-1,3-glucanase gene were obtained.The transformation experiment was carried out by using the laser micro-beam with third harmonic and 0.35μm of wavelength and 4.02μm diameter of light spot, 10 mJ export energy from a Nd:YAG laser micro-beam system under a microscopy to introduce the plasmid DNA of pBIBGC, carrying chitinase andβ-1, 3-glucanase bivalent genes into cotton target cells. The susceptive seed-germinating embryos and 40-50 days after pollination immature embryos of cotton were used as transgenic receptor. First of all the immature embryos have to pre-culturing (48 hours) and osmosis-deal (2 hours), and then treated for transformation by laser micro-beam puncture treatment when it was soaked in the exogenous plasmid DNA solution with 0.1mg/ml of concentration. The materials were screened with gradient kanamycin concentrations. The polymerase chain reaction (PCR) amplification analysis of screened cotton plants showed that one plant had specific fragment of chitinase gene andβ-1, 3-glucanase gene simultaneously, and four plants had chitinase gene and /orβ-1, 3-glucanase gene fragment. in the T1 generation, ten from 30 plants showed kanamycin-resistance. And, PCR amplification confirmed that 8 plants of them had specific fragment of chitinase, but no specific fragment ofβ-1, 3-glucanase.In this study an amaranth agglutinin gene (ARA) was also cloned from Amaranthus retroflexus for obtaining aphids resistant transgenic cotton. Aphids belong to Diptera insect pests; they damage plants directly by sucking juice of phloem, and indirectly by spreading plant disease. The plant lectins are proteins that bind reviviscence to specific mono- or oligosaccharides; therefore they have potential in plant genetic engineering. Amaranthus retroflexus grows in the field and roadside. Its adaptation is extremely strong, and it is not a poison to advanced animals. An ARA gene is consisted of 2453 base pairs, including one 1538 bp intron and two exons of 212 bp and 703 bp respectively. By using overlapping primer design and PCR amplification, we obtained coding region of the gene, thus avoided a series of problems when in extraction of mRNA and reverse transcription to get cDNA. The homology between this 915 bp fragment we obtained and the corresponding reported sequences is 98.8%.At the same time, the specific protein of phloem, bark storage protein (BSP) gene promoter fragment were cloned from plasmid pBIBGC using artificial synthetic special primer by PCR method. The length of the cloned promoter is 840 bp and it contained regions and with repeated A/T sequence and rich A/T bases. The cloned fragment showed a homology of 98.2% with the corresponding reported sequence.Consequently the ARA gene was inserted into the pET28a expression vector between T7 promoter and terminator. The recombinant protein was produced after induced by IPTG, t analyzed on SDS-PAGE. The result indicated that the molecular weight of expression protein fitted with that calculated theoretically very well.Furthermore, constructing the plant expression vector containing BSP-ARA-NOS terminator was constructed, and then integrated into Agrobacterium LBA4404. Finally the vector was introduced into leaves of tobacco through Agrobacterium tumeformation-mediated transformation and the aphid-resistance of transgenic plants was studied.