Breeding And Identification Of Insect Resistant Rice By Transferring Two Insecticidal Genes

Rice is one of the most important food crops in the world. With the increasing population and decreasing arable land, to further promote rice output is a very critical problem for rice industry. As we know, insect damage is one of the main factors that constraints rice yield, and the transgene technique could be an effective way to solve the problem. Although the number of transgenic insect resistance rice plants generated through genetic engineering is increasing, plantation of those transgenic plants are facing several problems, the first is it’s narrow insect resistant spectrum, the second is that insects showed easy to become more tolerance to Bt transgenic plants. More recently, new-generation transgenic insect-resistant plant which contains two or more insect-resistance genes is available.The results are shown as follows:1. Two insecticidal genes (sbk and sck) in the plasmid of pCDMARUBA-Hyg have been transferred into a rice variety, Dular, which itself has the wide compatible gene, S5-n gene and three stripe virus resistant gene loci, mediated by Agrobacterium tumerfaciens. By conventional PCR,18marker-free transgenic plants were obtained. Southern blot analysis showed that15of them were single copy insertion, and these15single-copy transgenic plants could normally express the insect-resistant gene by detecting of RT-PCR analysis. By detecting markers for wide compatible gene (S5-n) and anti-stripe genes (qSTV-11b and qSTV-11c), results indicated that those15single-copy transgenic plants had belts for S5-n, qSTV-11b and qSTV-11c. Field test results about seed setting rate of the hybrids between such single-copy transgenic plants and some indica or japonica rice varieties, and the cases of rice stripe virus and damage of insects showed that these single-copy transgenic plants not only retained themselves wide compatible and stripe virus resistant property, resistant ability of rice stalk borer had also been significantly increased. It can be helpful for the wider application of hybrid rice by pyramiding disease resistance genes, insect resistance genes and wide compatible gene.2. Plasmid of pCDMARUBA-Hyg which contained two insect resistant genes (sbk and sck) was transformed into Agrobacterium EHA105, and infected the callus of Nanjing45. Through PCR analysis with the primers of sbk and sck genes,42positive individuals which showed marker-free and contained the target genes were selected from56regeneration plants. Results of Southern-blotting indicated that38plants showed single copy, three plants showed two copies and one plant was three copies of exogenous genes. Analysis with RT-PCR and Bt gene testing paper of such single copy insert plants indicated that the two insect resistant genes could be normally expressed. Results of feeding rice stem borer by manual also indicated that insect resistant ability of those transgenic plants was greatly increased, and the insecticidal effect on rice stem borer showed97%to100%. It greatly increased the insect resistant ability of Nanjing45after being transferred into the two insect resistant genes. Among those generation plants, T45-2, T45-11and T45-14were elite lines for rice breeding and production since their suitable agronomic traits.