Fertility Gene And Protein Expression Regulating In Genetic Engineering

Hybrid varieties of crop plants are grown when the increased productivity gained from heterosis offsets the extra cost of their development and seed production. One important factor in economically visble hybrid seed production is the availability of a practical and effective pollination control system, which is employed to prevent sib- or self-pollination of the female parent. Such a system with the development of recombinant DNA technologies has opened new possibilities for creating and manipulating male sterility for pollination control. Fertility factors in nucleus and cytoplasm interact complicatedly, their inherit basis is very important to create pollination control system. A number of studies have focused on the cloning and using of specific genes throughout the development of plants. There is the most successful pollination control system 棗 the SeedLink?system of PGS NV(its components include the TA29-barnase male-sterile gene, the TA29-barstar RF gene, and the 35S-bar selectable marker gene).Arabidopsis 5'-UTR of anther-specific genes A6 and A9 were obtained from genomic DNA of Arabidopsis thaliana WS ecotype . Tobacco (Nicotiana tabacum L.) "NC89" and "K326" plants were transformed with LBA4404 including each promoter fused with BN aimed gene and Bxn marked geneWA6N, pWA9N, pWATN and pW3PN expression vector respectively. The transformed plants were analyzed with polymerase chain reaction, Southern and bromoxynil resistance experiment. The results showed BN and Bxn gene linked with these promoters had been integrated into plants genome. TI generation of the transgenic plants tested under field conditions showed characteristics of male sterile obviously, and these promoter showed anther-specific promoter activity in detached tobacco anther, but still observed obvious temperature sensitivity.In order to analyze the function of G9 gene regulation and control section which was expressed in cotton pollen specifically, we separated this section fromupland cottOn "Anddle resistal No. l7 l' and conStnJCted glucuronidase gene (GUband cytotOAn gene (bro) fusion gene. Wth the first geneic tIansformation'plant exPression veCtor, gUs enzyme was exPressed at cottOn pollen by particlebombardment transndssion. GUs gene trallient exPression procldried 4-MUreachon produCt's fund quantity was five times of untransformed pollens, seventiInes of transfOrmed leaves. An evidence to upStIeam -l ~-567bp regions of G9transcribe haal point possessed highly exPressive biological activity andpromoter function for the first time, and can st8rt the exPression of extraneoussources gene at pollen. Moreover, G9-Bamase fusion gene was transmitted into thegenome of tobacco by using Apobacterium. The result of bo gene eapressedin poliens showed we had obtained tranSgenic tobacco plants of recessivenucleus-sterile. It is the first basis of ho1ding the male-Sterile characters and makinggood use in practice.On the other hans, we has c1oned and erpressed the inhibitor of Bamase -Barstar protein, It can be exPressed in the way of soluble protain by our imPovedexPression and purification exPeriment, and the quanities of prOtein comes70-l00mg /L. We Inake the antibody production and deteCt the Bamase andBarstu protein exPressed in the transgenic rnale-sterile and fertile plants in thelevel of molecular immunoassays. The results of immune preciPitation are ourBarstar anibody has the high biological immunoreachvity with the tWo antigenproteins. Thus the antigen-anibody reaction has already develOped into practicaltools for screening transfOrmed crop plans which have special difficulties in theDNA molecular 1evel testing.