Improvement Of Rice Resistance And Grain Quality Via Transgenic Approach

Rice is one of the most important crops in the world, providing staple food for more than half of the world's population. It have got great achievement for improvement of rice yielding, resistance and quality through conventional breeding approach, but it becomes much difficult to further increase rice yielding and quality as the limition of conventional breeding and genetic resource. Recent advancements in molecular genetics and biotechnology offer new opportunities for rice breeders to increase rice resistance to diseases, insects and herbicide or enhance rice starch quality through transformation.The objective of this research was to study improving rice resistance and grain quality of several elite rice varieties through genetical engineering. To increase the resistance to diseases, insects and herbicide in rice, four target genes were used, which include the Bt cryIA(c) gene from Bacillus thuringiensis, GNA (Galanthus nivalis agglutinin) gene from snowdrop, AP1(Amphipathic protein 1)gene from sweet pepper (Capsicum annuum L.) and the herbicide resistance Bar gene from Streptomyces hygroscopicus. The another work was to regulate Wx gene expression in rice grains to improve starch quality via transformation of different Wx constructs, such as the antisense RNA structure, full length of Wx genome sequences and Wx-cDNA. The main results obtained were showed as follows: 1. Bt gene transgenic rice. To produce selectable marker-free transgenic rice lines with increased resistance to pests, calli derived from immature seeds of two elite japonica rice varieties, Guanglingxiangjing and Wuxiangjing 9, were used for Agrobacterium-mediated co-transformation of the Bt cryIA(c) and hygromycin phosphotransferase (HPT) genes. Two different transformation approaches, a single twin T-DNA binary vector in one agrobacterial strain(EHA105/pSBBt) or two separate binary vectors in two separate agrobacterial culture(sEHA105/p03Bt:EHA105/pCAMBIA1300) were used. The twin T-DNA binary vector was composed of two separate T-DNA regions, one carrying the Bt cryIA(c) while the other the HPT gene. The two separate binary vectors either contained the Bt cryIA(c) gene or the HPT gene in individual plasmid. Many transgenic rice lines co-transformed with both the target and HPT genes were obtained, and, subsequenctly, the selectable marker-free Bt transgenic rice lines were selected from the offspring of the co-transgenic plants. The resistance evaluation indicated that: (1) The affected area of the Bt transgenic rice lines leaves in vitro caused by rice leaf folder was smaller than that of the wild types, and all the larvas were dead after 3 dayes feeded on the Bt transgenic rice lines leaves. Affected area of the Bt transgenic rice lines culms in vitro caused by five instar striped stem borers was also smaller than that of the wild types, and the excretion of striped stem borers was little on the Bt transgenic rice lines culms. (2) All the striped stem borer larvas were dead 3 dayes after feeded on the Bt transgenic rice seedlings. The dead hart rate of the Bt transgenic rice lines caused by striped stem borers during maturing stage was lower than that of the wild types, and the resistance grade of some Bt transgenic rice lines reached to middle resistance or resistance level. In natural condition, no Bt transgenic rice lines was disserved by rice leaf folder, and all the wild types were disserved.2. AP1 gene transgenic rice. To produce selectable marker-free transgenic rice lines with increased resistance to diseases, calli derived from immature seeds of Guanglingxiangjing and Wuxiangjing 9 were used for co-transformation of the 14 AP1 and HPT genes mediated with the twin T-DNA binary vector pSBAP1. Many transgenic rice lines co-transformed with the AP1 and HPT genes were obtained and HPT gene free AP1 transgenic rice lines were selected from the offering of the co-transgenic plants. The resistance evaluation after inoculation indicated that the AP1 gene confered the transgenic rice lines improved resistance both to bacterial leaf blight and sheath blight. (1)The bacterial leaf blight lesion length of AP1 transgenic rice lines was shorter than that of the wild types. And the resistance grade of the transgenic rice lines reached to resistance level; (2) Despite the disease resistance grade to sheath blight of the transgenic lines and the wild type was at the same level, middle sensitive, the ratio of the lesion length divided by plant height was reduced significantly compared to the wild type, i.e. the distance the lesions moved up the stem was decreased in the transgenic plants. The resistance of most AP1 transgenic rice lines to false smut was also improved in natural condition.3. GNA and Bar genes transgenic rice. To produce transgenic rice lines with increased resistance to brown planthopper and herbicide, calli derived from immature seeds of Guanglingxiangjing, Wuxiang9915 and Yangfujing 8 were used for transformation mediated with the binary vector pCUGNA-BAR. The resistance evaluation indicated that the GNA and Bar genes confered the transgenic rice lines improvemed resistance both to BPH and herbicide Basta. Honeydew production of BPH feeding on transgenic lines was signifcant lower than that of the wild types and the sensitive control TN1. The propagating frequecy of BPH feeding on the transgenic lines was also lower than that of the wild type. The resistance of one transgenic line, 4901-1, to BPH at seedling stage reached to middle resistance grade.4. Multi-resistance transgenic rice. To produce marker-free transgenic rice lines with improved resistance to pests, diseases and herbicide, calli derived from immature seeds of Guanglingxiangjing were used for co-transformation of the Bt cryIA(c), AP1 and HPT genes mediated with EHA105/pSBAP1 and EHA105/p03Bt. Some of the transgenic rice lines co-transformed with the Bt cryIA(c), AP1 and HPT genes were obtained. These plants were crossed with transgenic rice lines containing both GNA and Bar genes, and, therefore, the HPT gene free transgenic plants with different combination of target genes were selected from their offspring. The resistance evaluation showed that: (1) The AP1 gene confered the multi-gene transgenic rice lines improved resistance both to bacterial leaf blight and sheath blight. The leasion length of multi-gene transgenic plants caused by bacterial leaf blight(KS-1-20) was shorter than that of only AP1 gene transgenic rice. And the leasion length of AP1 gene, GNA gene and Bar gene transgenic rice lines was shorter than that of AP1 and Bt genes transgenic rice; After inoculated with the mixture of four bacterial leaf blight strains, the resistance to bacterial leaf blight was enhanced in multi-transgenic rice lines containing AP1, Bt, GNA and Bar genes when comparing with that of the transgenic lines containing the AP1 and Bt genes; (2) The sheath blight leasion length of multi-gene transgenic plants was shorter than that of the wild type and only AP1 gene transgenic rice. The resistance grades of multi-gene transgenic plants to sheath blight reached to resistance levels. The resistance to sheath blight was enhanced in multi-transgenic rice lines containing AP1, Bt, GNA and Bar genes when comparing with that of the transgenic lines containing the AP1 and Bt genes; (3) The resistance evaluation indicated that the Bt gene confered the multi-gene transgenic rice lines improved resistance to rice leaf folder and striped stem borer. Affected area of the Bt transgenic rice lines leaves in vitro caused by rice leaf folder was smaller than that of the wild type, and all the larvas were dead after 3 dayes feeded on the multi-gene transgenic rice lines leaves. The resistance grade of multi-gene transgenic rice lines to striped stem borers reached to resistance or middle resistance level. In natural condition, no multi-gene transgenic rice lines was affected by rice leaf folder, and all the wild types were affected. (4) The GNA and Bar genes confered the multi-gene transgenic rice lines improved resistance both to BPH and Basta. Honeydew production of BPH feeding on the multi-gene transgenic lines was signifcant lower than that of the wild type and the sensitive control TN1.5. Antisense Wx transgenic rice lines and their quality performance. In order to obtain marker-free and quality improved rice, HPT and anti-Wx genes were co-transformed to Xieqingzao, Longtefu and Wuxiangjing 9 mediated with EHA105/p13W0 and EHA105/pCAMBIA1300 or EHA105/pYH592. Several transgenic lines without the HPT gene were selected from the offspring of the co-transgenic plants. The analyses of Wx gene expression and starch quality indicated that: (1) The expression of Wx gene was suppressed to different level in different transgenic lines, and the amount of mature 2.3kb Wx mRNA and un-spliced 3.3kb Wx pre-mRNA in developing seeds of transgenic rice lines was lower than that of the wild types. The amount of Wx protein and amylose content in mature seeds of transgenic rice lines was also reduced in different transgenic lines, and some of which was reduced to the level of waxy rice, both in japonica rice and indica rice. (2) The other quality was also changed after the amylose content reduced, such as gel consistency (GC) changed to softer. The gelatination temperature (GT) of the transgenic rice lines which derived from Longtefu was increased as their amylose content reduced to that of the waxy rice. But the variance was not notable between the GT of the transgenic rice lines which derived from Wuxiangjing 9 and Xieqingzao and that of their wild types. There was also some variance in RVA profile in the anti-Wx transgenic rice. The PKV, HPV and CPV of the transgenic rice lines were reduced significantly as their amylose content was reduced to that of waxy rice. (4) The starch granules of the transgenic rice lines were not filled as good as that of the wild type when their amylose content was reduced notably. (5)The grain weight was reduced obviously in the transgenic rice lines as their amylose content was reduced to that of waxy rice and the decrease reached to 17.05% compared to that of the wild type. 6. Sense Wx gene transgenic rice lines and their quality performance. The homozygous Wx gene transgenic rice lines were selected from the offspring of the transgenic plants mediated with EHA105/p13W2 derived from Xieqingzao, Longtefu,Wuxiangjing 9, Wuxiang 9915 and Guanglingxiangnuo. Wx gene expression and starch quality analysis indicated that: (1)The expression of Wx gene, as well as the amylose content, was increased to different level in different transgenic rice. The quantity of mature 2.3kb Wx mRNA in transgenic rice lines immature seeds was enhanced while the amylose content was increased. The Wx protein amount and amylose content in transgenic rice lines mature seeds were also enhanced and the enhancement was more obvious in the transgenic rice lines which derived from japonica rice and waxy rice than that from indica rice. For instance, the highest amylose content of transgenic rice lines derived from waxy rice, Japonica rice and indica rice reached to 24.92%, 24.14% and 27.52%,and the enhancement reached to 22.91%, 9.72% and 2.99%,respectively; (2) The other quality was also changed when the amylose content increased, and there were different exhibition in the different transgenic lines which derived from indica rice or japonica rice, such as gel consistency (GC) changed to harder in the transgenic rice lines which derived from japonica rice and waxy rice, but the variance was not obvious in the transgenic rice lines which derived from indica rice. The GT of the transgenic rice lines only which derived from Guanglingxiangnuo was reduced as their amylose content was increased. There were also some variances in RVA profile in different transgenic lines; (3) The starch granules of the transgenic rice lines were filled better than that of the wild types as their amylose content was increased obviously; (4)The 1000-grain brown rice weight was increased in the transgenic rice lines which derived from japonica rice and waxy rice when their amylose content was increased notably, and the enhancement reached to 16.89%.7. Wx cDNA transgenic rice lines and their quality performance. In order to study the effect of Wx cDNA on rice quality, Wx cDNA and HPT gene were co-transformed to Guanglingxiangnuo and Suyunuo mediated with EHA105/pCAMBIA1300 and EHA105/p585. The analyses of Wx gene expression and starch quality indicated that: (1)The Wx protein quantity in the transgenic rice lines mature seeds was enhanced as their amylose content was increased; (2)The amylose content in most of the transgenic rice lines was increased differently in different transgenic rice lines. The highest one reached to 19.12% and the enhancement reached to 17.82%; (3) The other quality was changed as their amylose content increased. GC was changed to harder in the transgenic rice lines as their amylose content was increased. But the variance of GT was complex, i.e. some of them were increased and some of them were decreased; (4) There were also some variance in RVA profile in different transgenic lines as their amylose content was increased, such as PKV, HPV and CPV of the transgenic rice lines were higher than that of the wild type. (5) The starch granules were filled better when their amylose content was increased obviously in the transgenic rice; (6)The 1000-grain brown rice weight was increased in the transgenic rice lines when their amylose content was increased notably, and the enhancement reached to 8.56 %.