Molecular Identification Of Borer Resistance Of Yunnan-BtRice Lines

The occurrence of pests reduced the yield of crop during rice production. How to reduce the pests has become a big problem? Using pesticides to control pests will lead to pesticide residues that are not only cause environmental pollution, but also harm to human health. The Bt gene is an insect-resistant gene that was cloned from Bacillus thuringiensis(Bt) against the pests, which are Lepidoptera, Dipteral, Coleoptera,respectively. So far, the insect-resistant genes, such as Cry1Ab/Ac, RCry1 C and Cry1 C, which have more effective to insect-resistance were obtained through genetic engineering. The development and utilization of the Bt gene provides a new way for rice insect-resistant breeding.In this study, the insect-resistant variety of rice, Hua-Hui-1, RJ-5 and T1C-19, carrying Bt genes, which possessed Cry1Ab/Ac、RCry1C and Cry1 C, respectively, were employed as donor to transfer Bt genes into the cultivar of Chu-Geng-28, which is a main variety in Yunnan rice production. To screen offspring of Bt gene by marker-assisted selection(MAS), we put Chu-Geng-28 as the recurrent parent, backcrossing 3-4 generations and intercrossing by themselves with 4 generations. Receptor of backcrossing generations and plant of intercrossing generations were screened by MAS for Bt gene. Final, the varieties possessed the Bt gene was bred and named “Yunnan-BtRice”. Based on this, the thesis was focused on both phenotypic evaluation of insect-resistance under naturally induced pest and molecular identified of borer resistance of Yunnan-BtRcie lines using DNA, RNA and protein molecular level, respectively. The results are as follows:1. The phenotypic evaluation of borer resistance of Yunnan-BtRice. The Yunnan-Bt Rice, which are Yunnan-BtRice 1(Chu-Gen 28/Hua Hui 1, Cry1Ab/Ac), Yunnan BtRice 2(Chu-Gen 28/RJ-5, RCry1C), Yunnan-BtRice 3(Chu-Gen 28/T1C19, Cry1C) and Chu-Gen 28 as receptor control, and the completely randomized design with three replications were employed to evaluate the phenotypic of borer resistance with two rice seasons in Jinghong in 2015. The evaluation results shown that: 1) In the first rice season, occurrence of borer is lighter, and four materials nearly have no borer damage. 2) In the second rice season, occurrence of borer is serious. Dead heart rates(striped stem borer, yellow rice borer) of Yunnan-BtRice 1, Yunnan-BtRice 2 and Yunnan-Bt Rice 3 and Chu-Gen 28 are 4.37%、 4.25%、1.32%、15.31%, respectively. Curled leaves rate(rice leaf folder) of Yunnan-BtRice 1, Yunnan-BtRice 2 and Yunnan-BtRice 3 and Chu-Gen 28 are 18.10%、8.02%�'� 6.14%、42.21%, respectively. It indicated that the borer resistance of the Yunnan-BtRice are different with very significant(P<0.01) compared with Chu-Gen 28. The results had shown that the Yunnan-BtRice was bred by MAS strategy.2. DNA molecular identification of Bt insecticidal genes of Yunnan-Bt Rice. We extracted DNA of rice leaves by CTAB method, then identification the Bt genes by PCR amplification reactions with Bt gene specific primers. Electrophoresis’ results showed that Yunnan-BtRices were all positive but the control was negative. The molecular identification of Yunnan-BtRice lines based on DNA shown that Bt genes(Cry1Ab/Ac, RCry1 C and Cry1C) was transferred into Chu-Geng-28 cultivar, respectively and bred the Yunnan-BtRice lines.3. RNA molecular identification of Bt insecticidal genes of Yunnan-BtRice. We extracted RNA of both Yunnan-BtRices and the control. Then we tested the expression level of real time fluorescence quantitative PCR(qRT-PCR) with the high-quality RNA. The result of qRT-PCR of Yunnan-BtRice shown that the Bt genes, which is Cry1Ab/Ac, RCry1 C and Cry1 C gene, was expressed at the RNA level.4. Protein level identification of Bt insecticidal genes of Yunnan-BtRices. First, Yunnan-BtRice and the control were tested by rapid diagnosis strip of BtCry1Ab/Ac and BtCry1 C. The results shown that plant samples of Yunnan-Bt Rice 1, Yunnan-BtRice 2 and Yunnan-BtRice 3 had red stripe appeared at T line but the control had not; To validate results rapid diagnosis strip of BtCry1Ab/Ac and BtCry1 C, we carried out western blot of Yunnan-BtRice to test again. Western bolt results shown that Cry1Ab/Ac protein, RCry1 protein and Cry1 C protein were all expressed in Yunnan-Bt Rices; Finally, we determined the Bt protein content in Yunnan-BtRice by ELISA. Bt protein content of nontransgenic control is-0.002±0.02 μg/g. Bt protein content of Yunnan-BtRice 1, Yunnan-BtRcie 2 and Yunnan-BtRcie 3 are 0.22 ± 0.16 μg/g, 0.25 ± 0.18 μg/g and 0.26 ± 0.18 μg/g, respectively. Bt protein content is much same with their donator. Protein level identification testified that Bt gene of Yunnan-BtRice was expressed in the protein level.Above results shown that agronomic characteristic of Yunnan-BtRice 1(Cry1Ab/Ac),Yunnan-BtRice 2(RCry1C) and Yunnan-BtRice 3(Cry1C) are behave well, and have ability of borer resistance and also identificated that the borer resistance of Yunnan-BtRices are mediated by Bt genes based on the molecular levels of DNA, RNA and protein, respectively. These insect-resistant lines would be provided genetic resources and insect-resistance rice variety for Yunnan rice industry in future.