Transgenic japonica rice expressing the cry1C gene is resistant to striped stem borers in Northeast China

Rice production and quality are seriously affected by the lepidopteran pest,striped stem borer(SSB),in Northeast China.In this study,a synthetic cry1 C gene encoding Bacillus thuringiensis(Bt)δ-endotoxin,which is toxic to lepidopteran pest,was transformed into a japonica rice variety(Jigeng 88)in Northeast China by Agrobacterium-mediated transformation.Through molecular detection and the Basta resistance germination assay,a total of 16 single-copy homozygous transgenic lines were obtained from 126 independent transformants expressing cry1 C.Finally,four cry1 C-transgenic lines(JL16,JL23,JL41,and JL42)were selected by evaluation of the Cry1 C protein level,insect-resistance and agronomic traits.The cry1 C-transgenic lines had higher resistance to SSB and higher yield compared with non-transgenic(NT)control plants.T-DNA flanking sequence analysis of the transgenic line JL42 showed that the cry1 C gene was inserted into the intergenic region of chromosome 11,indicating that its insertion may not interfere with the genes near insertion site.In summary,this study developed four cry1 C-transgenic japonica rice lines with high insect resistance and high yield.They can be used as insect-resistant germplasm materials to overcome the problem of rice yield reduction caused by SSB and reduce the use of pesticides in Northeast China.

Identification of Bt-transgenic Rice Plants for Resistance to Stripe Stem Borer (Chilo suppressalis) and Genetic Analysis of the Transgenes

Resistance to herbicide Basta was used to identify Bt-transgenic rice plants and the progenies of crosses between the Bt-transgenic rice and the rice varieties cultivated in the Huang Huai area of China. The results demonstrated that the Basta-positive rice plants were highly resistant to stripe stem borer (Chilo sup-pressalis) both in the laboratory and field tests. Both cryIA (b) and bar genes were expressed and co-inherited in both self ing and crossing progenies. Mendelian segregation of the marker gene bar was observed in F2 and BC1 progenies. The results implicates that it is possible to transfer cryIA (b) gene into other cultivated varieties through crosses and back crosses.

Rice gene expression profiles responding to larval feeding of the striped stem borer at the 1st to 2nd instar stage

The objective of this study was to identify rice genes that are in response to the striped stem borer （SSB） （Chilo suppressalis Walker） feeding at the first to second larval stage. Using combined suppression subtractive hybridization （SSH） and dot blot approaches, we analyzed the induced defense genes that took place during the first 72 h of infesting intact rice （Oryza sativa L.） plants in sheath tissues with SSB larvae. By sequencing the whole SSH library, 39 expressed sequence tags involved in disease stress, insect stress or other stress responses were identified to be up-regulated by SSB larvae feeding. Among these genes, rice allene oxide cyclase （AOC）, terpene synthase （TPS） and four proteinase inhibitor （PI） genes were up-regulated by SSB larvae feeding. Real-time quantitative reverse transcription polymerase chain reaction analysis showed that four rice PI genes were already up-regulated at 6 h, and reached peaks between 6 h to 12 h. In addition, the transcription ofgene involving in jasmonate signaling pathway such as allene oxide cyclase （AOC） concerning rice early defense response to SSB feeding was activated after rice feeding by SSB for 2 h. Although the expression office terpene synthase （TPS） gene, involved in the biosynthesis ofmonoterpenes or diterpenes, was already up-regulated at 7 h, a significant increase in the expression was delayed until 12 h and reached its peak at 24 h. The present study identified six SSB-response genes and their expression patterns, which provides evidence and information to understand insect stress-response in plants.

Development of lepidopteran pest-resistant transgenic japonica rice harboring a synthetic cry2A* gene

A synthetic cry2A^＊ gene enco ding Bacillus thuringiensis（Bt） δ-endotoxi n that resi st ance to lepidopteran pest was transformed into japonica rice variety Jijing 88, which is the most widely cultivated variety in Jilin Province, Northeast China, by Agrobacterium-mediated transformation. A total of 106 independent transformants overexpressing cry2A^＊ gene driven by ubiquitin（Ubi） promoter was produced. Three single-copy homozygous transgenic lines were finally selected based on the results of PCR analysis, se gregation ratio of Bast a resistance, and Southern hybridiza tion analyse s. RT-PCR and enzyme linke dimmune sorbent assay（ELISA） revealed that cry2A^＊ transcripts and protein were highly expressed in these lines. The high level of Cry2A^＊ protein expression resulted in high resistance to rice striped stem borer as evidence d by insect feeding bioassays. Our results demonst rate that cry2A^＊ transgenic japonica rice confers resistance to the rice striped stem borer in the laboratory conditions.

Cloning of a Potato Proteinase Inhibitor Gene PINII-2x from Diploid Potato(Solanum phurejia L.) and Transgenic Investigation of Its Potential to Confer Insect Resistance in Rice

Both cDNA and a genomic DNA fragment encoding a new potato proteinase inhibitor Ⅱ were isolated from a diploid potato IVP101 （Solanum phurejia L.） and named PINII-2x. Nucleotlde sequencing confirmed that the DNA fragment of PINll-2xwas 580 bp, including a 115-bp intron and two exons. The deduced PINII-2x proteln contained an Intact signal peptide and two active sites. The PINII-2x gene and its deduced PINII-2x protein had 88% and 93% homology with another tetraploid potato proteinase inhibitor Ⅱ, respectively. Northern blotting analysis Indicated that the mRNA of PINII-2x gene was wound induced in potato leaves. Binary vector pNAR301 and pNAR302 were constructed for rice transformation, in which the PINII-2x cDNA was driven, respectively, by rice actin I promoter （Actl） and maize ubiquitin promoter （Ubll）. Via an Agrobacteriummediated method, these two constructs were transferred into japonica rice cv. Xiushui 63. PCR and Southern blotUng analysis for transgenic rice revealed the integration of the PINII-2x gene. Northern blotting analysis also confirmed transcripts of the PINII.2x gene in transgenic rice plants. Insect bloassays using stripe stem borer （Chilo auppressalis Walker） demonstrated that the average weight and body length of larvae In transgenic plants were only nearly 50% and 61% of those of larvae in control plants, respectively. These results Indicate that the PINII-2x gene should be an effective insect-resistance gene and could be valuable for application in crop breeding for insect resistance.