英文文獻閱讀報告

1、Reading ReportAgro bacterium 農桿菌是生活在植物根的表面依靠由根組織滲透出來的營養(yǎng)物質生存的一類普遍存在于土壤中的革蘭氏陰性細菌。農桿菌是一種天然的植物遺傳轉化體系，被譽為“自然界最小的遺傳工程師”?？梢酝ㄟ^將目的基因插入到經過改造的T-DNA區(qū)，借助農桿菌的感染實現外源基因向植物細胞的轉移和整合，然后通過細胞和組織培養(yǎng)技術，得到轉基因植物。 Agro bacterium rhizogenes-mediated transformation provides a system for rapid and efcient transformation of plant

2、 tissues.The inuence of several factors plant genotype A. rhizogenes culture stage A. rhizogenes cell titre co-culture period of A. rhizogenes acetosyringone concentrationa routine, efcient and genotype-independent method of inducing hairy roots by A. rhizogenes in peanuts was established.誘導農桿菌Vir基因

3、活化，從而促進外源基因的整合Plant materialSeven peanut genotypes including Luhua11,Huayu16,Huayu28, Baisha1016, Yuanhua8, Xinhua1, and Xinhua5 were used to assess the effect of plant genotype on A.rhizogenes-mediated transformation.Agrobacterium rhizogenes strain and plasmidpCAMBIA2300The vector pGFP-GUSPlus whic

4、h contained two highly-expressed reporter genes, GFP gene and GUS gene.Induction of roots by microinjectionFig.1 Peanut hairy-root inductionA-5-day old seedling for microinjection; B-microinjection of A. rhizogenes on hypocotyls at different sides;C-Tumor(腫瘤) emerging 1 week after microinjection;D-I

5、nduced roots appearing 2 weeks after inoculation;E-Transformed root systemDetection of GFP or synthetic cry8Ea1 gene in transgenic roots by RT-PCRTotal RNA from induced roots was isolated by the TRIzol method and treated with RNase-free DNase according to the manufacturers instructions.One microlite

6、r of rst strand cDNA was used as template for PCR amplication.The two sets of primer pairs used in the assay were primers VirHF/VirHR, primer GFPF (base pairs 7693),50-CTTCTCGTTGGGGTCTTT-30, and primer GFPR (base pairs 627644), 50-ACAAGTTCAGCGTGTCCG-30. Primer jc8EF2, 50-GGCGGCAGCAT TCAAACTCAA-30, a

7、nd primer jc8ER2,50ATCTCCACCAAGATAGTGTCC-30 were used to detect the transcription of the synthetic cry8Ea1 gene（暗黑鰓金龜抗性基因）.Histochemical assay for GUS activityRoot segments (2 cm long) and hypocotyls were histochemi-cally stained to analyze GUS gene expression according to Jefferson.Fig.2 GUS staini

8、ng assayGUS expression in vertical sectionVertical sectiontransverse sectiontransverse sectionhypocotyls injected with K599and induced roots induced roots Effect of plant genotype on hairy roots inductionLeaves, cotyledons or embryonic axes were inoculated with a suspension culture of A. rhizogenes.

9、 Only embryonic axes developed crown gall tumors，and early forms of roots 2 weeks after injection and hairy roots were harvested 3 weeks later.It was about 11 days of induced roots emerging after co-cultivation(DIRC)for most gen-otypes, although the DI-RCof Huayu28 was signicantly lon-ger than that

10、of the other 6 enotypes.These results showed that A. rhizog-enesmediated transformation was not genotypes-dependent. Therefore Baisha1016 was randomly chosen forthe following experiments.Table1 DIRC and number of induced roots of different peanuts genotypesEffects of A. rhizogenes growth phase and c

11、ell titre on transformation efciencyTable2 Effect of A. rhizogenes growth stage on DIRC and number of induced roots of peanuts.Table3 Effect of A. rhizogenes vitality on transformation efciencyexponential growth phasetransition phase and stationary phaseIt took more time for embryonic axes to develo

12、p hairy roots when injected with A. rhizogenes atstationary phase (OD600= 5.5) while it only took 910 days for the other three treatmentsBecause the overgrowth of A. rhizogenes(OD600of 3.0 and 5.5)inhibited root emergence, only sa-mples from the OD600= 0.2 and 0.8 treatments were used for RT-PCR and

13、 GUS staining detection;The transformation efciency were all above 20%Table4 Effect of A. rhizogenes cell titre on hairy-root inductionThe frequency of RT-PCR positivity for GFP reached a plateau when each plantlet was inoculated with 5x107 cells, and did not continue to rise as the infecting cell n

14、umber increased.Effects of co-culture period of A. rhizogenes with seedlings on transformation efciencyTable5 Effect of coculture time on DIRC and number of induced roots of peanutsTable6 Effect of coculture time on transformation efciencyA 2-day period of co-cultivation was optimal since hairy root

15、s emerged approximately 5 daysafter inj-ection, and 11 roots were obtained per embryonic axeCompared with the remaining treatments, the explants with 2-day co-cultivation developedmore roots in a shorter time period and exhi-bited a higher transformation efciencyEffects of acetosyringone concentrati

16、on in co-culture medium on transformation efciencyTable7 Effect of acetosyringone concentration on root inductionThe average number of induced roots and positive frequency of molecular detection peaked at 16 and 47%, respectively when explants were placed onto MS medium with 50 umol l-1 acetosyringo

17、ne.An efcient and fast system for A. rhizogenes-mediated transformation of peanuts5-day old seedlings without roots were microinjected with 5x107 cells from exponential growth phase and placed on MS medium supplemented with 50 umol l-1 acetosyringone for 2 days. Then explantswere transferred to MS m

18、edium supplemented with hygromycin 500 mg l-1 carbenicillin and 50 mg l-1hygromycin.Under optimal conditions, 61% of 89 explants were positive for GFP transcription based on RT-PCR assay Covalent integration of the GFP gene into the peanut genome was conrmed.Assessment of insecticidal activity of pe

19、anut roots transformed with the synthetic cry8Ea1 geneFig.4 RT-PCR detection of peanut roots transformed with A. rhizogenes RT-PCR assay showed that 59% of 27 composite plants were positive and the positive composite plants carrying the synthetic cry8Ea1 gene were transferred to pots.Fig5 Bioassay o

20、f peanuts root lines against Holotrichia parallela Motschulsky larvaeA-Composite plants with induced roots transformed with synthetic cry8Ea1 geneB-Non-transgenic plantlets as negative controlC-Live larvaeThe results elucidated that the synthetic cry8Ea1 gene had certain insecticidal activity to Hol

21、otrichia parallela larvae, but further study should be Conducted.Seeds of J.curcas(L.) population IP-2P from Indonesia were used as the plant material.Plant regeneration from cotyledon and hypocotyl explants. the explants were placed on 33 different kinds of callus induction medium(CIM) consisting o

22、f MS medium supplemented with various combinations of phytohormones. (BA、TDZ、IBA、GA3).the binary vectors pIG121-Hm, pBIN30,and A2 were introduced into A.tumefaciens strain LBA4404 and used for stable transformation.the cell density was adjusted to an OD600 of 0.250.35 before co-cultivation in transf

23、ormation experiments. Stable transformation using cotyledon explants Cotyledons from 12-day-old plants were cut into55mm pieces and incubated with A.tumefaciens LBA4404 cells harboring the A2 vector in liquid hormone-free MS medium containing 20mg l1 AS for 10 min at room temperatureHairy root induc

24、tion from explants Hypocotyls, cotyledons, and roots from 4-day-old seedlings were cut into 5-mm segments and incubated with A. rhizogenes ATCC15834 cells harboring the A2-IG vector in liquid hormone-free MS medium for 10 min at room temperature.MaterialsOptimization of shoot and root regeneration p

25、rotocolsTable1 Effects of concentrations of IBA and MS salts on induction of roots from Jatropha shootsFigure1 Effciency of shoot generation from cotyledon explantsIn total, we examined 132 different combinations of phytohormones in the CIM and SRM for both hypocotyl and cotyledon explantsTable2 Com

26、parison of selection markers for screening of transgenic Jatropha shootsThe most effcient shoot regeneration (shoot/explant) was obtained after callus induction on the CIM containing 1mg l1TDZ and 0.05mg l1 IBA (hereafter denoted as CIM-1) and shoot induction on the SRM containing 1.5 mg l1 BA and 0

27、.05 mg l1 IBA (denoted as SRM-1) .The highest rooting effciency was observed in the RIM containing half-strength MS salts and 0.25 mg l1 IBA,Most of the root-induced plants were able to be acclimated in soil.Figure2 Regeneration of Jatropha from hypocotyl (AD) and cotyledon (EH) explants.Figure3 Tra

28、nsformation of Jatropha with A.tumefaciens carrying mutated ALS gene Cotyledon explants were co-incu- bated with A.t harboring the A2 vector and placed on selective callus induction medium containing 10 mM bispyribac sodium salt Cotyledon explants were co-incubated with A.tumefaciens harboring the A

29、2 vector and placed on selective callus induction medium containing 10 nM bispyribac sodium salt.Resistant shoots grown for 2 mont-hs were cut from calli and transpl-anted onto fresh selective shootregeneration mediumRoot-induced transgenic shootsScreening of transgenic hairy roots expressing GUS geneTo determine which tissues are best suited for use in this system, we used A.rhizogenes to infect hypocotyl, cotyle