| Chemical pesticide was the most convenient,effective and commonly used method for pest control.However,besides the improvement of economic efficiency in agricultural production,irrational use of the pesticide would cause many health and environmental problems.Various medicinal plant contain a large number of antifungal protein,identify the genes that encoded those proteins and use plant transgenic technology to generate novel antifungal germplasm is the other efficient way to prevent the damages of disease in agricultural production.In this study,the genes of chitinase family from Eucommia ulmoides Oliver(E.ulmoides),a traditional medicinal plant in China,had been cloned;the gene expression pattern and function of the cloned gene had been analyzed.The results shown in this thesis could provide the basic documents of E.ulmoides utilization in antifungal activity.1.Cloning and bioinformatic analysis of chitinase genes in E.ulmoidesBased on the transcriptome analysis of E.ulmoides,total of 15 fragments had been discoved as the possible chitinase gene.The experimental material were collected from the E.ulmoides tree,which were grown in the laboratory’s farm for over 10 years.The samples,which include the shoot buds,young leaves,young stems,barks and young fruits of both male and female E.ulmoides tree,were harvested during the flowering period.The total RNA were extracted from the samples,reversed transcript into c DNA,and used to amplify the 5’ and 3’end of the target gene via RACE(Rapid Amplification of c DNA Ends).The predicted full c DNA sequence was splicing according to the overlap fragement from transcriptome.According to the predicted full c DNA sequence,two primers were designed to amplificationthe full c DNA sequence.After sequencing verification,three full ORF(Open Reading Frame)of the chitinase gene with the length at 1402 bp,1218 bp and 1269 bp were identified from E.ulmoides,which were named as Eu CHIT1,Eu CHIT2 and Eu CHIT3 respectively.Conservation district and motif analysis indicated that Eu CHIT1 and Eu CHIT2 are belong to chitinase glycolhydrolase 19 family,while Eu CHIT3 is belong to chitinase glycolhydrolase 18 family.Phylogentic tree had been drew among the target chitinases and the chitinases origin from other organisms(including insects,bacteria,fungi,and other plants),the results shown that all three chitinase from E.ulmoides were cluster together with other plant chitinase,which indicated that those chitinases may share a close relationship and have a common evolutionary ancestors.2.Analysis the Eu CHITs gene expression pattern in E.ulmoidesThe relative expression levels of Eu CHIT1,Eu CHIT2 and Eu CHIT3 in different plant tissues(including lateral roots,young stems and leaves,old stem)were measured via q RTPCR.The result shown that all three genes had the highest expression level in young stems.Eu CHIT1 and Eu CHIT2 also had higher expression level in young leaves compare to the expression in other tissues.Whereas there no tissue specific difference among roots,leaves and old stems in Eu CHIT3 expression.The expression pattern were measured and analyzed on E.ulmoides seeding with F.oxysporum(Fusarium oxysporum)inoculation to the root after 24,48,72 and 96 h.The summit of Eu CHIT1 and Eu CHIT2 expression level were shown at 72 h after the inoculations,which were 13.6 and 5.77 times elevation comparing to the expression level before inoculation.While the expression level of Eu CHIT3 were only increased in 48 h,and only had 2.75 times increment.The protein components assay were carried on the same samples and the results shown that the number of differential protein reaches the maximum in72 h after inoculation,which including 18 up-regulated proteins and 69 down-regulated proteins.It is noticeable that β-glycosidase and phospholipase-D were always remain downregulated after inoculation.In the protein components analysis,Eu CHIT2 is the only target chitinase that can be detected,which were down-regulated 24 h after inoculation.Additionally,GO analysis shown that the membrane protein,DNA transcription activation factor and Ca2+ions response receptors were seriously affected with the inoculation,and the Serine/threonine kinase,the kinase that related to plant resistance,was activated.KEGG analysis shown that in all the F.oxysporum stress related pathways,the Bip(binding immunoglobulin protein)was always up-regulation.3.The construction of Eu CHIT1 and Eu CHIT2 plant overexpression vector and the generation of transgenic tobacco plantThe plant overexpression vector p SH-35S-Eu CHIT1 and p SH-35S-Eu CHIT2,which contained the full sequence of Eu CHIT1 and Eu CHIT2 respectively,were constructed.The vector were design to generate the Eu CHITs overexpressed transgenic tobacco plant.The transgenic Nicotiana tabacum CV.Xanthin were generated by A.tumefaciens strain LBA4404 and identified via the histochemical Gus assay and PCR.Total of 53 lines of Eu CHIT1 and 36 lines of Eu CHIT2 overexpressed transgenic plant were obtained.4.The effects of Eu CHITs on chitinase activity in transgenic tobaccoChitinase activities were measured among the wild-type and the T1 generation of Eu CHIT1 or Eu CHIT2 overexpression transgenic tobacco.The result shown that compare to the enzyme activities in the wild-type,the Eu CHIT1 and Eu CHIT2 transgenic tobacco plant were 2.4 times and 2.11 times significantly higher respectively.With powdery mildew inoculation,the enzymatic activity in Eu CHIT2 overexpressed transgenic tobacco were only0.32 times increasing,and there is no significant difference in increment between the wildtype and the transgenic plant.However,the enzymatic elevation of transgenic tobacco was noticeably less than the wild-type after Botrytis cinerea(B.cinerea)inoculation,the wild type tobacco was increased 1.866 times 6 h after inoculation,while the elevation in Eu CHIT1 overexpressed transgenic plant were only 1.19 times compare to the before.This result suggested that overexpression chitinase gene in tobacco could increase the chitinase activity before fungus inoculation,however there was no obvious increment of chitinase activity in Eu CHITs overexpressed transgenic after the fungus inoculation.5.The effects of fungal diseases resistance in transgenic tobacco with Eu CHIT1 and Eu CHIT2 overexpressionB.cinerea(HM2)were inoculated to tobacco leaf pieces to evaluate fungal resistance,the leaf pieces were collected from plant with similar condition in both wild-type and Eu CHIT1 overexpressed transgenic plant.Small pinpoint lesions appeared 2 days after the inoculation in wild-type and transgenic plant,and were enlarged by time.The difference of the lesion diameters between wild-type and transgenic plant were reach the maximum in 4 days,thus,lesion diameters after 4 days were measured as an indicator to evaluate the severity of the disease.The average of the lesion diameters in wild-type were 2.6 cm,while the average lesion diameter were only 1.2 cm,1.8 cm and 1.7 cm respectively in Eu CHIT1-31,Eu CHIT1-37 and EuCHIT-151 three transgenic lines.It is obvious that the average diameters were significantly smaller on transgenic plants compared to wild-type plants(T test P<0.01).The result indicated that overexpressed Eu CHIT could increase resistance against B.cinerea in transgenic tobacco plants.The fungus that cause powdery mildew disease in tobacco,Erysiphe cichoracearum DC(E.cichoracearum),were inoculated to the wild-type and Eu CHIT2 overexpression transgenic plant.Disease spot was not appeared in the first 2 days after the inoculation,but it appears in the wild-type and one of the transgenic line Eu CHIT2-2 on the 3rd day.The disease spots were visibly bigger in the wild-type compare to the Eu CHIT2 overexpression transgenic plant.The spots were keep grew,which covered the whole leaf with mycelium and spore after12 days in the wild-type.On the contrast,this syndrome was severely relived in the transgenic tobacco plants.The spores per the unit area of disease leaf was 69444 / cm2,186111 / cm2 and33333 / cm2 on Eu CHIT2-1,Eu CHIT2-2 and Eu CHIT2-15 respectively,whereas on the wildtype,the spore number reaches 327778 / cm2 for each unit area.The results indicated that Eu CHIT2 overexpression could strength the resistance of E.cichoracearum in tobacco.6.The effects of Eu CHITs on pathogen related genes in tobacco The PR-1a(pathogenesis related protein 1a)and COI1(Coronatine insensitive 1)gene expression were tested in wild-type and transgenic tobacco plants by q RT-PCR.The result shown that before the inoculation the relative expression levels of the COI1 in three Eu CHIT1 overexpression transgenic tobacco lines were 1.75,2.04 and 2.28 times respectively higher than the expression level of the wild-type.However,the relative expression level of COI1 gene were significantly decreased in transgenic tobacco plant after B.cinerea inoculation.It is demonstrated that overexpression of Eu CHIT1 gene in plant could increase the relative expression of COI1 gene,on the contrary,inhibited the COI1 gene expression with B.cinerea infection.The relative expression levels of PR-1a were 4.75,3.84 and 2.74 times greater in three individual Eu CHIT1 overexpression transgenic line than of wild-type before B.cinerea inoculation,and the gene expression reaches the apex 72 h after the inoculation in transgenic tobacco,which was 33.8 times higher than the highest expression point of the wild-type.The relative expression level of PR-1a were keep increasing in the first 6 h to reach the highest expression level,then start to decrease.These results demonstrated that Eu CHIT1 overexpressed in transgenic tobacoo plant could increase the PR-1a expression with or without the B.cinerea inoculation.After induced the E.cichoracearum infection,the relative expression level of the Eu CHIT2 overexpression transgenic tobacco and the wild-type were analyzed.Similar with the influence of overexpressed Eu CHIT1 in transgenic tobacco plant,the expression level of COI1 gene were 1.31,2.07,and 1.2 times higher than in the wild-type respectively.With E.cichoracearum inoculation,the expression level COI1 gene of the wild-type had the highest expression level in 24 h after the inoculation,which was 3.6 times higher than the expression level before the infection.Interestingly,COI1 gene expression were decreased in transgenic plant with Eu CHIT2 overexpression after inoculate E.cichoracearum.The results indicated that overexpressed Eu CHIT2 gene could increase the relative expression in the transgenic plant,but inhibited it after E.cichoracearum infection.The relative gene expression level of PR-1a is already 2.67,1.23 and 2.29 times higher in three individual trangenic lines compare to the wild-type before the E.cichoracearum infection and this difference were enlarged to307.22,112.22,and 146.67 times respectively higher 72 h after the infection.The result indicated that PR-1a may involves in increase of E.cichoracearum resistance in Eu CHIT2 overexpressed transgenic tobacco.7.The effects of Eu CHIT1 and Eu CHIT2 overexpression on protective enzyme activity in transgenic tobaccoThe activity of peroxidase(POD),catalase(CAT)and the content of malondialdehyde(MDA)were measured in both wild-type and transgenic plant.When compare between the wild-type and Eu CHIT1 overexpressed transgenic plant,the result shown that CAT activity was 122.40 U/g in the transgenic lines 72 h after inoculation with B.cinerea,which was significantly higher than in the wild-type(50 U/g).Overexpressed Eu CHIT1 gene dramatically increased the activity of CAT to protect the plant cell membrane of infected plants.The content of MDA was 5.7 mmol/g in transgenic tobacco before inoculation,which is significantly lower than that of wild-type(9.4 mmol/g)(P < 0.05).The content of MDA was changed to 8.7 mmol/g 72 h after B.cinerea inoculation,which was again,significantly lower than that of the wild-type(10.5 mmol/g).For comparison of Eu CHIT2 overexpressed plant with the wild-type,the POD activity was higher in the transgenic tobacco plant(5158 U/g)with Eu CHIT2 overexpression compare to the wild-type(3425 U/g)before E.cichoracearum inoculation,and this advantage of the transgenic plants remains in 12 h(8625 U/g compare to4716 U/g)and 24 h(10816 U/g compare to 3450 U/g)after the inoculation.Similar with Eu CHIT1 overexpression plant,the MDA concentration were lower in Eu CHIT2 overexpressed transgenic plant compare to the wild-type before and after E.cichoracearuminoculaiton.The results of protective enzyme activity assay indicated that in Eu CHITs induced antifungal activity,the POD or CAT activity were increase to avoid the cell damage caused by hypersensitive response. |
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