| Weeds compete with crops for moisture, nourishment and light, moreover, they are hosts of disease or the place where disease survives in the winter, which influences the output and quality of crops. It is indispensable for modernized agriculture to control weeds by using herbicide. But berbicide also injures crops while killing weeds. At present, cultivating berbicide resistant crops by genetic engineering has solved this contradiction.PPT(Phosphinothricin) is the active composition of broad-spectrum, non-selective hercicide Basta. It inhibits the activity of glutamine synthetase(GS) which is necessary for the production of glutamine and for ammonia detoxification. Inhibition of GS by PPT causes a rapid buildup of intracellular ammonia levels. Bar gene codes phosphinothricin acetyl- trasferase(PAT) which acetylizes the freedom amino of PPT and makes PPT lose activity. Bar gene also is one of widely used selective marker gene. The cloning of bar gene is important to plant transgenic engineering, gene expression and studying physiology.In the experiment, the full code sequence of bar gene was cloned by PCR from transgenic herbicide resistant Bobwhite wheat and checked. It was expressed in E.coli and its protein was determined. After having been properly modified, the bar gene which correctly codes PAT was cloned into binary vector pBI121 and then transferred into LBA4404 by triparantal crossing, which is the prerequisite work for genetic transformation. The system of wheat transformation mediated by Agrobacterium tumefaciens was preliminarily studied on, which make foundation for cultivating high yield, high-quality and herbicide resistant wheat in Anhui. The research suggested:1. PCR is a simple and effective method for cloning gene whose sequence is known. But a lot of factors influence PCR specificity, among which anneal temperature play a key role. In our experiment, the specific fragment was amplified from transgenic Bobwhite genome DNA at annealing temperature 61 by using high-fidelity pfu DNA polymerase and cloned into clone vector pGEM-7fz(+), then sequenced. The cloned sequence was completely identical to the sequencewhich was issued in GenBank.2. The sequence was cloned into express vector pET32a and expressed in BL21trxB( DE3). The molecular weight of fusion protein induced by IPTG was 39.6kDa, and the molecular weight of target protein was 20.1kDa, which accorded with the estimated molecular weight 20.0kDa. Then its enzyme activity was determined by DTNB spectra chromatography.3. In order that bar gene efficiently transcript and translate in eukaryote, the sequence next to ATG was modified by PCR. The modified sequence was cloned into binary vector pBI121. The constructed expression vector was named pBI121-bar, then transferred into Agrobacterium tumefaciens LBA4404 by triparental crossing. Thus the project bacterium has been successfully constructed.4.In the cultivation, wheat genotype, concentration of hormone in the medium and the kind of explant are the three key factors that influence frequency of induction embryogenic calli and plant regeneration. Genotype mainly influences the frequency of embryogenic calli formation, 81.35% immature embryo of Yangmai 87158 can form embryogenic calli, while only 16.81% immature embryo of Annong 92484 can form embryogenic calli. When mature embryo was used as explant, as for above three lines of wheat, the optimize concentration of 2,4-D for inducting calli and embryonic calli is 4.0 mg/L. However, when immature embryo was used as explant, the optimize concentration of 2,4-D is different, as for yangmai 87158 and Ann 98005,it is 2.0 mg/L,but as for Ann92484, it is 4.0mg/L. Research also indicate that explant influence the frequency of callus induction, especially influence embryogenic calli formation.5.The proper concentration of PPT is 3.0mg/L in screening resistant callus after transformation. Effective concentration of Cef restrain Agrobacterium tumefaciens is 200|xg/ml. The efficient density of AS is 100 umol/L in the co-cultivatio...
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