| Sugar beet is a important sugar crops in China, but it’s always infested bymany insect pests, especially Barathra brassicae L. The chemical insecticidescann’t exterminate the insects effectively and also have bad effect on theentironment. With the emphasis of ecological and environmental protection,ecological agriculture has become a significant demand in the future and insect-resistant genetic engineering has been an effective method for breeding insect-resistant sugar beet. In this research, insect-resistant gene was transformed andexpressed into the chloroplast genome of sugar beet though the sequence ofchloroplast genome are unknown. We constructed sugar beet chloroplastmulticistron site integration expression vector. The atpB and rbcL genes clonedfrom sugar beet chloroplast genome is homologous fragments and Bt genecry1Ac acted on lepidopteran insects is target gene, and aadA gene is selectivemarker. After transformation with particle-bombardment, screening of putativeplants was antibiotic, molecular detection and bioassay, finally transgenic sugarbeet plants and the T1seeds were obtained which have resistance to pests. Thegenetic stability of transgenic plants were studied and the mechanism ofchloroplast transformation was researched. The sugar beet chloroplasttransformation system was established.The culture medium component were confirmed after the experiment whichwere designed based on the different combination of genotype, explant type andplant growth regulators. Optimize sugar beet regeneration system was established.It provided a good foundation for regeneration of transgenic sugar beet.Afterselected with5antibiotics, kanamycin and spectinomycin were chosen as theselective agent, and their respective concentration were50mg/L and30mg/L. TheaadA gene was chosen as selectable marker in chloroplast transformation system.Because the feature of sugar beet which contain much sugar, the existingmethods of chloroplast DNA extraction methods are not applicable for sugar beet.Therefore, according to the advantages of existing methods and the feature ofsugar beet,a new method of sugar beet chloroplast DNA extraction was used in the research. The chloroplast DNA prepared with such method had not only high-yield more three times, but also high-quality, which could be directly used forgenome sequencing.The sequence of sugar beet chloroplast genome is unknown, so it is difficultto clone homologous fragments. In this research, we analyzed the completesequences chloroplast genome of higher plants, such as tobacco, rice, spinach andmaize, by referring to the conservation status of plant chloroplast genome formedthrough evolution. With the primers that designed according to the nucleotidesequences of atpB/rbcL and rps7/ndhB genes, we cloned the four significantfunctional genes from sugar beet chloroplast genome successfully by PCRreaction. The sequencing results showed no difference with expectancies and theatpB/rbcL genes had been submissioned to Genbank, the Accession No. areDQ067451and DQ067450. The atpB and rbcL genes cloning would alsocontribute to studying the photosynthesis mechanisms of sugar beet.A sugar beet chloroplast transformation vector was constructed, whichcontained homologous recombinant fragments atpB and rbcL, insect-resistant Btgene cry1Ac expression cassette（Prrn-cry1Ac-psbA3’）and selective markergene aadA expression cassette（Prrn-aadA-TpsbA）. After transformation withparticle-bombardment and screening several times, some transgenic sugar beetwere obtained. The chloroplast transformation system of sugar beet wasestablished, and it would be a good foundation for further research.After PCR identification, Southern blot, Northern blot and Western blot, theresults indicated that insect-resistant Bt gene cry1Ac had integrated andexpressed in sugar beet chloroplast genome. We detected the insecticidal activityof cloning recombinant strain and transgenic plants with the second instar larvaesto Barathra brassicae L. The results indicated that the Bt toxin protein of cloningrecombinant strain had high insecticidal activity and the mortality of larvaes was90%; the mortality was33.3%~66.7%when the larvaes weve bred with leavesfrom transgenic plants. The difference of mortality is relevant to the differenthomoplasmic level. As we know, there are hundreds of chloroplasts in onemesophyll cell. When the numbers of chloroplasts integrated with target gene islarger, the homoplasmic level of transgenic plants and the expression level oftarget gene will be higher, and the insecticidal activity will be higher too. Secondary regeneration and many times of subculture on medium with antibioticswere carried out to filtrate homoplasmic plants. The results indicated that twoadditional rounds of regeneration and were subcultured for many times on MSmedium containing spectinomycin in the stem sections, increased thehomoplasmic level of transgenic plants markedly. This study of increasinghomoplasmic level of transgenic plants will extremely contribute to the obtainingof homopladmic pure line. The genetic stability of transgenic plants were studiedand it showed that the Bt gene could express steadily and expressed insecticidalactivity in the T1generation. The mortality of larvaes was33.3%-55.6%.The research also used the bar gene and aadA gene as a selective marker forcontrast, and the result showed that different selective marker had different effecton screening of transgenic plants. Meanwhile, the research used rps7rps12/ndhBwhich were also cloned from sugar beet chloroplast genome as homologousfragments, and the result showed that the position of the homologous fragmentshad no effect on tansformation. Those study may contribute to chloroplasttransformation on other crops. |
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