Molecular Identification And Insect Resistance Evaluation Of Transgenic Barley - Resistant Rice

Rice (Oryza sativa) is a single cotyledon germanous plant; nearly half the world’s population consumes rice as the main food crop; it has a vital role for the survival of mankind. However, insect pest is one of the most important factors that hinder the development of rice production; it will have a big impact on rice quality on serious time; at the same time, it also can reduce rice production. Nowadays, it is the most economic and effective method to restrain the harm of pests through plant gene engineering technology to cultivating insect-resistant rice. In plant insect-resistant genetic engineering research, insecticidal crystal protein gene form Bacillus thuringiensis is the most popular in the research and application of insect-resistant genes. The protein products of Bt gene expression can be specific poison to lepidoptera, coleoptera, diptera, and other harmful organisms, but harmless to humans and the environment in comparison with chemical control. Thus, the use of transgenic technology to solve the pests especially borer damage has extremely far-reaching significance.this study of genetically modified (gm) rice HD2 harboring cry2A* which is a resistance genes, in which Conducted experiments including Basta resistance screening, PCR test, survey of insect resistance, Southern blot detection and RT-PCR detection; and the experiments quantitative test of Bt protein, rice quality and yield traits examination was following. This study, such as identification, the transgenic rice materials resistant to borers rice lines was obtained by PCR positive of the purpose gene, exogenous gene copy low consolidation, higher transcription and translation level of Bt portion, the stronger borer resistance; and yield and rice quality compared with no significant differences between the non-transgenic rice.The main results were as follows:1. Transformation of rice varieties (pBar-cry2A*) empty) 131 was based on agrobacterium EHA105. This study begins from transforming 101 different independent rice plants of resistant callus which harboring the cry2A *Basta resistance genes. The principle is based on the positive PCR testing of bar gene, normal growth and development, by selecting T0, T1 and T2 generation, and nurtured several transgenic rice plant named the HD2 strain.2. The expression of genetically modified (gm) rice strains HD2 insect-resistant gene RNA was studied; in the results for different strain amount of mRNA, the level of the expression of RNA in leaf of HD2-14 is the highest, and the lowest is the HD2-2; the level of stem are highest in HD2-14, and the lowest is HD2-2; the level of young ear are highest in HD2-14, and the lowest is the HD2-2. The exogenous gene expression quantity of RNA in leaves; stems and young ears have a trend of synchronization, which means that high or low at the same time.3. The expression of genetically modified (gm) rice strains HD2 insect-resistant gene protein was studied; in the results for different strain quantity of protein expression, the level of the expression of protein in leaf of HD2-14 is the highest, the lowest is the HD2-2; the level of stem are highest in HD2-14, HD2-2 are the lowest,the level of young ear are highest in HD2-14. The exogenous gene expression quantity of protein in leaves; stems and young ears have a trend of synchronization, which means that high or low at the same time.4. The expression of genetically modified (gm) rice strains HD2 exogenous gene copy number was studied; the copy number of different lines is different. As analysis of cry2A* gene, Four genetically modified (gm) rice lines including HD2-2, HD2-6, HD2-12, HD2-14 and HD2-19, which strains single copy integration. HD2-4 and HD2-5 have tow copies integration, and HD2-16 and HD2-18 harboring 3 copies. Except the HD2-16, bar gene copy number of other strains the same as the cry2A gene copy number. The bar gene copy number of HD2-16 is 4, which is more than the cry2A* gene copy number which is 3. Both gene expression and the Bt protein expression of HD2-14 was the highest in single copy strains. Although HD2-16 and HD2-18 are integration of three copies, but the purpose gene expression and the Bt protein content are in the medium of high level, and the copy number and insecticidal crystal protein expression level have no obvious correlation or no dose effect.5. The expression of genetically modified (gm) rice strains HD2 insect resistance was studied; the results according to the resistance from high to low sequence is HD2-14, HD2-5, HD2-10, HD2-18, HD2-19, HD2-6, HD2-4, HD2-16, HD2-12 and HD2-2. Comprehensive mRNA expression and protein gene results analysis, the purpose gene transcription level and the quantity of protein expression more high, the more resistance is strongest.6. The expression of genetically modified (gm) rice strains HD2 agronomic was studied; Four genetically modified (gm) rice lines including HD2-14, HD2-5, HD2-4, HD2-18 were compared with the reference material at agronomic traits, the difference is not big. But only the HD2-14 is single copy; both HD2-5 and HD2-4 are two copies. The purpose gene expression and protein content of HD2-5 in medium to high level; in addition, the gene copy number of HD2-18 is three copies, but the purpose gene expression and protein content in the medium to high level, that is means that visible multi-copy strain breeding again also can completed as selection criteria. This study selected the HD2-14, the HD2-5, and the HD2-18 in commercial varieties.