Anther Culture And Expression Of Two Rip Genes In Jatropha Curcas

Jatropha curcas L. belongs to Euphorbia, with remarkable anticancer activity. It not only has tremendous possibilities in industrial oil using, biologic actively about disease-control and new medicines manufacture, but also is a nice tree species for afforestation among bald mountains in the xerothermic valley, so there is prospect of making good use of it widely.This experiment was mainly about the anther culture and the effect of hormone and the method of culture on the activity of PPO and POD in the anther callus of Jatropha curcas L..RIP gene curcin 2 was cloned from J. curcas., two prokaryotic expression vectors of gene Curcin2 were constructed and induced expressed in E.coli B121 and M15 respectively to compare the difference between this two prokaryotic expression systems of curcin2. On the basis of this, further study has been made on the existing form of the prokaryotic expression vectors of Curcin2 in transformant.RIP gene curcin was cloned from J. curcas., It has been determinate cloned into plasmid pBI 121, then pBI 121-curcin, a plant expression vectors,has been obtained. At last,we lead gene Curcin in tobacco by the conversion method of Agrobacterium and Leaf discs of tobacco. 1. The study on apoptosis of anther callus of Jatropha curcas L., the bud differentiation and the root differentiation by, in order to get the more suitable culture medium. Meanwhile, the cell suspension culture system has been established. The results showed that, in the inducement of anther callus, microspores at uninucleate stage were more suitable for culture; the cold pretreatment at 4℃for 3～5 days was essential to high ratio of inducement; the culture medium that MS+ NAA2.0mg/L +KT0.4mg/L + sucrose 9% is comparatively more suitable for callus inducement, the highest inducement ratio was 40.24%. In the bud differentiation, MS+KT2.0mg/L+NAA0. ling/L+ sucrose 3% was fit. And in the root differentiation, the root can be induced from the callus cultured on the culture medium 1/2MS+IAA0. lmg/L without light. But both the ratio of bud differentiation and root differentiation were low.2. The result of the effect of hormone and the method of culture on the activity of PPO and POD in the anther callus ofJatropha curcas L. showed that, the activity of PPO and POD in the anther callus culture on solid culture medium had two peaks; the first appeared at the 3rd day, and the other appeared at the 21st day; they were concerned with adversity and growth respectively. And the activity of PPO and POD in the anther callus culture on liquid culture medium just had one peak, appearing when the callus grew fastest. The hormone affects remarkably the activity of PPO and POD; 2,4-D and NAA were more powerful than IAA and IBA; KT was powerful than 6-BA. The light affected the activity of PPO remarkably, but the effect on the activity of POD was not as remarkable as on PPO. After few days, both the activity of PPO and the activity of POD descended, but the speed of PPO was faster than POD.3. Construction of two prokaryotic expression vectors of curcin2 gene: the PCR amplification was carried out by using special primers: P1: 5'-GGATCC (BamH I )ATGGCTGGTTCCACTTT-3'; P2: 5'-GAGCTC (Sacâ… ) ATACATTGGAAAGATGAG GA-3' , which was designed according to curcin2 gene order on the GenBank; and then, we reclaimed the outcome of PCR, connected it to pMD18-T, obtaining pMD-18T/curcin2 to transform E. coli JM109. It was proved that the objective segment order was correct by sequencing. Double enzyme digestion was done to pMD-18T/curcin and expression vector pET-32(c),pQE-30 by BamH I and Sac I. Then gene curcin2 segment (about 930 bp) and the linear expression vector pET-32(c),pQE-30 were obtained by reclaiming objective segment. Connecting obatained objective segment to linear expression plamid pET-32(c),pQE-30 respectively at 16℃in order to constract expression pET-C and pQE-C; then we transfered the connection to competent E. coli JM109, test it by strain-PCR, double enzyme digestion and sequencing and finally improve that objective segment curcin2 can be inserted to expression and constract prokaryotic expression pET-C and pQE-C.4. A gene fragment encoding mature peptide of curcin2 was obtained by PCR amplification from genomic DNA. The sequence was integrated into the pET-32(c) and pQE-30 vector and two recons, pET-C and pQE-C, were constructed successfully. Then, two transformants, PCB and PCM, were got when the recons were transformed into Escherichia coil strain BL21 and M 15 separately. Expression of curcin2 in recombinant bacteria was induced with different temperature, IPTG of different concentration and different time. SDS-PAGE and Western-blotting could not detect the recombinant protein of curcin2 in PCB but could in PCM. However, the curcin2 expressed in PCM existed mainly in the form of inclusion body. When inducing temperature lowered and inducing time delayed further, PCM could produce some soluble protein of curcin2. The optimized expression conditions of curcin2 in PCM were 16℃, IPTG of 1 mmol.L^-1 and 16～24 h.5. At first, ORF for curcin coding was isolated from genome of Jatropha curcas. It indicated that there was no intron existing in gene curcin. It has been determinate cloned into plasmid pBI 121, then pBI 121-curcin, a plant expression vectors, had been obtained. Leading gene curcin in tobacco by the conversion method of Agrobacterium and Leaf discs of tobacco, it was found that if the culture medium that MS₂ (MS+6-BA0.1 mg/L+Kan 100 mg/L) which contains kanamycin was used for the inducement of lusters of buds, the inducement ratio is about 40%, and 90% of them are morphologic normal; and then we induced these 90% lusters of buds to grow roots in the culture medium of MS₃(MS+Kan 100 mg/L), the ratio was about 90%;finally, we planted regenerative seedings, the success ratio was up to 95%.It was proved that 13 of 15 are integrated with gene curcin through tested by PCR and 10 of 13 can be expressed on the transcribing level. To a certain extent,transgene tobacco has antibacterial actively to Rhizoctonia solani.