| Rice is an important food crop all over the world. Rice blast and sheath blight are important diseases influencing rice yield and quality. It has been approved that breeding and planting resistant cultivars is the most economical and effective way to control rice blast and sheath blight.To date, a series of rice varieties with resistance to disease were bred by various conventional methods, which exit enormous social and economic function in agricultural production. However, as the result of genetic diversity of nosophyte, absence of resistance genes and long breeding circle in traditional methods, the breeding effect for disease resistance was lower to a great degree. To improve breeding effect in rice, conventional breeding methods connected with tissue culture and molecular marker assisted selection technique were applied to breeding for disease resistance. Though overcoming large amount of defects in traditional breeding, some issues were still existed, i.e. insufficient genes for disease resistance. Just the transgenic technique can remedy the deficiency, and has become an effective approach in breeding for disease resistance.In our laboratory, disease resistance genes McCHIT from balsam pear (Momordica charantia L.), alfAFP from clover (Medicago sativa L.) and their bivalent gene (McCHIT1-alfAFP) were transferred to rice restorer line Jinhui35 by Agrobacterium-mediated transformation method. By detection of disease resistance and GUS as well as PCR, we obtained a set of transgenic rice plants harboring McCHITl, alfAFP and McCHIT1-alfAFP gene whose descendants obviously strong resistance to blast and sheath blight. For further understanding the inherited character of the genes in transgenic plant descendant and screening stable plant descendant with blast and sheath blight resistance, estimating and screening resistance to blast and sheath blight were done from T3 in the study. Once the stable and outstanding transgenic plants with blast and sheath blight resistance were obtained, on the one hand, their resistance spectrum and main agronomic traits were valued, on the other hand, hybrid rice F1 crossed by sterile lines with them were appraised by the same targets involved above. We expect screening excellent transgenic rice restorer and exploring a novel route for broad-spectrum disease resistance. The main results were as follows:1. Genetic variation of disease resistance for rice transgenic plants harboring McCHITl,alfAFP and McCHIT1-alfAFP in various generationsTogether with the prophase study results, there were same trends basically in disease resistance of different generations for transgenic plants harboring McCHITl, alfAFP and McCHIT1-alfAFP gene. At the low generation, especially before T2 generation, resistance inheritance for blast and sheath blight were unstable, exhibiting larger difference among various lines even plants within the same line. At T3 generation, disease resistance was stable basically for most lines, only unstable for a few heterozygosis plants in which resistance still exist polarization among plants. With the increasing of generation after T3, transgenic lines to resistance were gradually stable. In T5 generation, different types of stable transgenic plants with excellent disease resistance were screened out by successive detection of disease resistance and GUS test.2. Stable and noticeable M. grisea-resistance transgenic rice lines harboring McCHITl, alfAFP and McCHITl-alfAFP gene and its major agronomic characteristicsBased on the analysis of resistance to rice blast in T3, T4 and T5 generations and on identification of resistance spectrum incubated by M. grisea in T6 generation. transgenic plant lines increasing resistance and extended resistance-spectrum to rice blast were screened as follows:seven McCHITl-transgenic rice lines i.e. C36-2-1, C35-6-2, C10-7-1, C24-4-1, C24-4-2, C21-6-2 and C21-3-1, eight alfAFP-transforming rice lines i.e. A3-6-1, A3-6-2, A7-3-1, A7-6-1, A7-6-2, A7-8-1, A4-8-1 and A10-4-1, and four McCHIT1-alfAFP-transforming rice lines i.e. AC2-10-1, AC4-2-2, AC7-3-1 and AC8-5-1. These lines demonstrated the mid-level resistance to leaf and neck blast as well as resistance in seedling stage to leaf blast or at least closely. In detail, when the wild-type control was highly sensitive to M. grisea was of a disease, transgenic plants were slightly destroyed no matter leaf and neck blast or leaf blast at seedling stage. The value of disease-resisted frequency increased more than 15% in transgenic plant lines compared with the control of wild type 36.50%, and the majority of transgenic rice acquired the resistance to ZE groups and enhanced the resisted-level to ZA, ZB, ZG and ZF groups by identification of broad-spectrum resistance. Consistently, excellent transgenic rice lines could be found based on the method of selection pressure increased from generation to generation and mid-resistance lines kept.After self-pollination of generations, the key agronomic characteristics have inherited inherited stably in despite of the distinct differences between transgenic lines. In the case of McCHITl gene over-expression, compared with the wild type, the transgenic lines mainly showed the shorten life cycle, dwarfism, decreased effective panicle number per plant, reduced panicle length, increased grain number and seed density per panicle, poorly seed setting, lower weight of 1000-seeds and filled seeds per plant, interestingly, four of which demonstrated universal and significant difference with these of the wild type, that is life cycle span, plant height, seed setting and 1000-seeds weight. As for transgenic plant alfAFP gene over-expression, effective panicle number per plant showed no significant difference between wild type and transgenic lines, life cycle showed significant longer or shorter and plant height increased or decreased significantly in the transgenic rice compared with those of wild type control, and other agronomic traits changed consistently with the plants of McCHIT1 gene over-expression, of which, life cycle, seed setting ratio and plant height were severely reconstructed. When McCHITl-alfAFP genes were transformed to rice, the progeny of transgenic lines displayed no significantly difference in grain number and seed density per panicle, other agronomic characteristics such as life cycle, effective panicle number per plant and seed weight per plant were significantly negatively or dominantly affected in some off-springs of transgenic plant, and the changing of seed setting ratio and 1000-seeds weight was consistent with rice plant harboring McCHITl and alfAFP respectively. For all agronomic traits,1000-seeds weight, life cycle, plant height, panicle length and seed setting ratio were wildly and severely influenced at the McCHIT1-alfAFP-transforming rice. Given the breeding of rice restorer lines, seven lines C36-2-1, C21-6-2, C21-3-1, A-7-3-1, A7-8-1, AC2-10-1 and AC4-2-2 were excellent transgenic plant lines against to rice blast.3. M. grisea resistance and agronomic traits analysis of hybrid rice varieties crossed with screened transgenic lines as male, harboring McCHITl, alfAFP and McCHITl-alfAFP gene respectively.Hybrid combinations were acquired by crossing predominant transgenic lines as males, rice blast resistances were performed and agronomic characteristics were identified subsequently. The results showed as follows:M. grisea resistances were remarkably increased in hybrid combinations with the male of M. grisea-resisted transgenic lines than in those with wild type control or M. grisea-unresisted transgenic lines as male, particularly for alfAFP over-expression lines, the combinations were nearly coincided with transgenic line in neck-blast resistance, revealed mid-resistance level at least closely, which suggested the resistance could be inherited to F1 generations dominantly in the transgenic rice. In essence, the main agronomic traits were inherited stably and equivalent to the wild-type control in F1 generations, significant discrepancies were only detected in single or several traits of the combinations crossed with exceptive transgenic lines by comparison with these of wild type, this result disaccord with the multiple mutations of major agronomic characteristics in transgenic lines. Taken together, the hybrid combinations with excellent productive traits and higher resistance to M. grisea were bred by crossing CMS line II-32A with transgenic lines of C21-6-2, C21-3-1, C36-2-1, A7-3-1, A7-8-1, AC2-10-1, AC4-2-2 and AC7-3-1 respectively.4. Identification of rice sheath blight resistance transgenic lines harboring McCHIT1, alfAFP and McCHIT1-alfAFP gene respectivelyEight plant lines were screened against to rice sheath blight (the value of disease index less than 45%) by T3, T4 and T5 generation identification of McCHITl-transforming rice, which were C36-2-1, C35-6-2, C10-7-1, C24-4-1, C10-2-1, C24-2-1, C24-2-2 and C21-13-2, of which, four lines i.e. C36-2-1, C10-7-1, C21-6-2, C21-3-1 showed predominant resistance to both rice blast and rice sheath blight. By over-expression of alfAFP gene, seven transgenic lines acquired rice sheath blight resistance (the value of disease index less than 45%), they were A3-6-1, A3-6-2, A7-6-1, A4-8-1, A10-4-1, A3-2-2 and A2-9-3, comparably, five of which showed double resistance including AC2-10-1, AC4-2-2, AC7-3-1, AC3-2-1 and AC6-2-2. As for McCHIT1-alfAFP genes, only five transgenic lines i.e. AC2-10-1, AC4-2-2, AC7-3-1, AC3-2-1 and AC6-2-2 exhibited the value of disease index less than 45% by identification of rice sheath blight resistance, of which, AC2-10-1, AC4-2-2 and AC7-3-1 revealed double resistance to both rice blast and rice sheath blight.5. Analysis of the differentiation of resistance strength to M. grisea and R.solani. among different stable transgenic lines harboring McCHITl,alfAFP and McCHIT1-alfAFP gene respectively.The analysis of grisea resistance spectrum showed that 7 McCHITl-transforming rice lines have excellent blast resistance, the blast resistance of 8 alfAFP-transforming lines and 4 McCHIT1-alfAFP-transforming lines represent a highly significant compared with control and other transgenic lines. The mainly reason is that the anti-spectrum of the total population of magnaporthe grisea are significantly wider, especially the physiological race of dominant group B and important group A have higher frequency of resistance. The analysis of chitinase activity showed that the chitinase activity of good disease resistance in McCHITl-transforming lines before and after inoculation Rhizoctonia solani were more significant than those of control control and other transgenic lines. The total chitinase activity were 3.4 to 4.2 times as much as the receptor at 1 d after inoculation and 2.1 to 2.7 times as much as susceptible transgenic plants. With the rapidly increase of the total chitinase activity of receptor at 2d after inoculation. The total chitinase activity of good disease resistance of transgenic decreased compared with the times of control were 1.8 to 2.9. But the total chitinase activity increased more than 2 d before, which showed that the McCHIT-transforming plants with excellent disease resistance have strong constitutive expression. Meanwhile, endogenous chitinase gene can be induced to express. Susceptible McCHIT1-transforming lines have not been a high level of constitutive expression, which is the reason why the disease resistance of susceptible transgenic plants was significantly weaker than excellent disease resistance of transgenic rice lines. Otherwise, the disease resistance of excellent disease resistance of transgenic rice was significantly higher than those of control or the other transgenic lines. The reason of physiological reasons is that McCHITl has a higher constitutive expression in McCHIT-transforming rice with excellent disease resistance than in the susceptible transgenic lines. |
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