Research On Improving Resistance To Stem Borer In Three Japonica Rice Via Co-expressing Cry1Ac And CpTI Or Tissue-specific Expressing Cry1Ac

With the development of plant bio-technology, the technologies of crop transformation has been greatly developed. Since the first report of transgenic tobacco for insect resistance, over 40 insecticidal genes with different insect-resistant mechanisms have been used in crop genetic engineering and some transgenic insect-resistant crops have been realized commercial production. In recent years, the insect-resistance duration and the bio-safety of transgenic insect-resistant crops has become a hot issue for publics, and many breeders are trying to seek molecular ways efficient and safe enough to avoid the risks of GM crops. In this study, we established an efficient and quick Agrobacterium-mediated transgenic system of rice (oryza saliva L.) and has applied it successfully to three rice varieties. In order to prolonging the insect-resistance duration of transgenic rice, we constructed a plant expression vector harboring two insecticidal genes and used it in Agrobacterium-mediated rice transformation. The segregation laws of Cry 1 Ac in TI lines of transgenic rice has also been analyzed. The RSuSl (rice sucrose synthase gene 1) is an important gene in plant amylase synthesis pathway and the phloem-specific expression pattern of its promoter in transgenic tobacco plant has been reported. In order to ensure the insecticidal genes do not express in edible parts of transgenic rice, we cloned the promoter sequences from rice and characterized its expression pattern in transgenic rice plant. The insecticidal gene Cry 1 Ac driven by the promoter has also been used in rice transformation. The major results can been summarized as below:1. An efficient and quick rice transgenic system of Agrobacterium-mediated transformation has been established and the transformation ratio ofJaponica rice Zheda 19, Xiushui 11 and Xiushui 63 can all reach to 80%.2. A plant expression vector, PCAM-Bt-CpTI, was constructed, which harboring two insecticidal genes CrylA(c) and CpTI under the control of two 35S promoters, and was used in transformation of an early Japonica rice Zheda 19 via Agrobacterium turner-faciens. About 1500 transgenic plantlets have been regenerated from 1300- x-f>1-7hygromycin-resistant calli and 70 independent transgenic plantlets (absolutely from different calli) of them have been assayed by PCR and PCR-Southern. The results showed that 62 plantlets contained both of the two transgenes and the positive ratio is 88.6%. The resistance assay in lab and field of 9 independent transgenic TI lines against striped stem borer (Chilo suppresalis) indicated that 3 of them are highly resistant to the insect, the larvae mortalities of these three lines are all 100% and the white head ratioes are all less than 4%.3. The segregation laws of the insecticidal genes are also studied and the results revealed that the segregation rules of most TI lines tested fit to the ratio 3:1 except the other two lines. One of the two lines fit to the segregation ratio of 15:1 and the southern assaying revealed that the Bt gene had inserted in the genome of the plant with two copies, then deduced that the two copies of the transgene has integrated in different and unlocked loci in genome; The segregation ratio of the other line is 3:2 and the cross test results indicated that the unmoral segregation of transgene may due to the low transmission ability of the transgene through pollen.4. Two pairs of specific primers was designed according to the published 5' upstream sequence of RSuSl (rice sucrose synthase genel). The sequence before the expression initiation site (including the first intron denoted by RSP1) and the sequence before the first intron (denoted by RSP2) were amplified from genomic DMA template of an Indica rice variety 'Minghui 63' by PCR. Sequence analysis of the two sequences showed that no differences were found between the cloned and the published sequences in important functional regions. The cloned promoter sequences RSP1 and RSP2 were fused to Gus gene respectively and...