| Part 1: Resistant germplasm resources are valuable for developing resistant varieties in agricultural production. However, recessive resistant germplasm is usually overlooked in hybrid breeding. Compared with dominant traits however, it may confer resistance to different pathogenic races or pest biotypes with different mechanisms of action. The recessive rice bacterial blight resistance gene xa13, also involved in pollen development, has been cloned and its resistance mechanism has been recently characterized. This report describes the conversion of bacterial blight resistance mediated by the recessive xa13 gene into a dominant trait to facilitate its use in a breeding program. This was achieved by knockdown of the corresponding dominant allele Xa13 in transgenic rice using recently developed artificial micro RNA technology. Tissue-specific promoters were used to exclude most of the expression of artificial micro RNA in the anther to ensure that Xa13 functioned normally during pollen development. A battery of highly bacterial blight resistant transgenic plants with normal seed fertility were acquired, indicating that highly specific gene silencing had been achieved. Our success with xa13 provides a paradigm that can be adapted to other recessive resistant germplasm.The main results of this research as follows:1) Four final expression vectors were introduced into indica rice variety Minghui63 via Agrobaterium-mediated transformation. PCR screening in T0 transformants was conducted to remove negative regenerated plants. 31 independent positive T0 transformants of Osrbcsp+ami A, 27 of Atrbcsp+ami A, 38 of Osrbcsp+ami B and 25 of Atrbcsp+ami B were acquired.2) Severe infection of wild-type Minghui63 with POX99 was observed 14 d after inoculation, with average leaf lesion length and lesion area of 22.75±2.12 cm and 88.5±7.0%, respectively. By contrast, transgenic plants expressing ami B showed significantly enhanced resistance, 32 of 38(84.2%) transgenic plants harboring Osrbcsp+ami B and 20 of 25(80.0%) harboring Atrbcsp+ami B showed significant reductions of both lesion length and lesion area. Among these, 19(50.0%) plants with Osrbcsp+ami B and eight(32.0%) with Atrbscp+ami B exhibited high resistance(lesion length less than 3 cm). Transgenic plants expressing ami A showed weaker resistance than those expressing ami B.3) Small RNA northern analysis showed that both ami A and ami B were expressed in rice. Cleavage site mapping was conducted to confirm that the degradation of Xa13 m RNA was due to ami RNA-guided cleavage. Sequencing analysis of the isolated 5’ end of spliced Xa13 m RNA from transgenic plants expressing ami B showed that 100%(12/12) of cleavages occurred at the predicted targeting site of ami B between base pairs 10 and 11 in Xa13 m RNA. Because the ami A targeting site was located at the end of 3’-UTR of Xa13 m RNA, and the m RNA was not long enough to design appropriate primers for 5’-RACE, cleavage site mapping of transgenic plants expressing ami A failed after several attempts.4) We measured ami RNA in both leaves and anthers by stem–loop RT-PCR analysis to confirm the expression specificity of Osrbcsp and Atrbcsp. RT-PCR showed that the amount of ami RNA in anthers was much lower than that in leaves. We analyzed the relative expression of ami RNA in leaves and anthers, and found that expression in leaves was 162–1221-fold greater than that in anthers from the same transgenic plant.5) T1 progeny of two selected lines with a single copy insertion were grown for the investigation of inheritance and segregation. All T1 seedlings were inoculated with PXO99 at the 4–5 leaf stage and RT-PCR was performed to detect the presence of ami RNA in each T1 plant. The T1 progeny of these two selected lines showed enhanced resistance, and the inheritance fit 3:1 Mendelian segregation of a dominant trait according to the chi-square test. RT-PCR was conducted for each T1 progeny to detect the presence of ami B, which cosegregated 100% with the trait of disease resistance.Part 2: The brown planthopper(BPH, Nilaparvata lugens) is a destructive and piercing-sucking insect pest of rice, and is insensitive to BT insecticidal protein. Therefore, looking for the resistance genes in BPH-resistance rice cultivars is very important for prevention and control of BPH. RH(Rathu Heenati), an indica rice cultivar from Sri Lanka, is highly resistant to several different biotypes of brown planthopper. At least, 4 BPH-resistance QTLs has been reported by different research groups. So we chose RH as research material, and TN1 as a BPH-susceptive control. We want to try analyzing why RH has a broad-spectrum of durable BPH-resistance molecular biological basis by c DNA microarray technology, and to identify some candidate BPH-resistance related genes. Through the results of microarray analysis and the posting of BPH-resistance QTLs in RH, we have determined 13 BPH-resistance candidate genes within BPH-resistance QTLs; by the gene expression analysis, we also have determined 22 BPH-resistance candidate genes outside of the QTLs. Previous studies have shown that plant hormone JA and SA signaling pathways play an important role in the plants-pest interactions. We have analyzed the JA and SA synthesis and signaling pathways related gene expression patterns under BPH-infestation condition in RH and TN1, also have measured the hormone SA and JA concentration changes in them.The main results of this research as follows:1) Reference gene is an important decisive factor to confirm if gene expression test is accurate, and to choose a stable reference is very important for the differential gene identification in q RT-PCR. We have extracted the signal value of 8 frequently-used reference genes in 12 chip samples. Based on this result, the TBP and ubiquitin are suitable for use in gene expression test under the BPH-infestation condition. Our research choose ubiquitin as reference genes in all the q RT-PCR test. We also find two new stable references, they are Os.1322.1.S1_at and Os.145.1.S1_a_at.2) The Affymetrix microarray we used has detected expression of 57194 probes. Chip test results show that, under acupuncture treatment, the differential probes in RH are significantly less than the differential probes in TN1. The number of differential probes for acupuncture in RH at timing of 6 hours is 92, and 211 for TN1. This shows that TN1 reacts more quickly than RH under simple physical damage. In addition, most of the differential probes have recovered at 24 hours, and the differential probes for acupuncture treatment at 24 hours for RH is 27, and 38 for TN1.3) In contrast to the results of acupuncture, at 6 hours timing after BPH-infestation, the number of differential probes in RH(209) is more than TN1(173), and the number of differential probes under changing multiples for FC > 5(35) in RH is significantly more than that in TN1(12). The results shows that the number of induced expression gene in RH is more than TN1 at the beginning of the BPH-infestation. This proves that RH reacts more quickly for BPH-infestation and start of resistance reaction. But at the 24 hours timing, the number of differential probes in RH rose to 614, where the number of differential probe for FC > 5 increased from 35 to 73, and t; when the number of differential probe for TN1 rose to 3356, where the number of differential probe for FC > 5 increased from 12 to 349.4) We have respectively conducted Venny diagram analysis for differential genes under acupuncture and inoculation treatment for RH and TN1 at the timing of 6 hours and 24 hours. Eventually we get the number of BPH-specific-induced probes is 192, 74 for six hours and 152 for 24 hours, see. We firmly believe that the 228 BPH-specific-induced probes in RH include potential resistance related genes.5) In order to explain the functions of differential genes in BPH-resistance reaction, we have selected differential genes for the groups of T6N/T6 C, T6P/T6 C, T24N/T24 P, T24P/T24 C, R6N/R6 C, R6P/R6 C, R24P/R24 N and R24P/24 C to conduct GO analysis by GOEAST software. For the results of GO analysis, including BP(biology process), CC(cellular component), MF(molecular function) and GO specific information. In BP project, the first level projects in RH and TN1 P value with significant differences are catalytic activity, response to stimulus and metabolic process, and the project with significant difference is localization project. In the MF project, the first level project with extremely significant difference is the catalytic activity projects with significant difference are binding and enzyme regulator activity.6) 17 differential genes expression patterns that appear in JA pathway are up-regulated, and differential genes of signal transduction pathway in RH and TN1 are all JAZ transcription factors. They are negative regulatory factor in JA pathway, it implies that the rice JA pathway is repressed by BPH-infestation. All the differential genes in ET pathway appear in synthetic metabolic pathways, and they are mainly down-regulated. All the differential genes in SA pathway appear in synthetic metabolic pathways, and except for one common gene in ET pathway which is down-regulated expression, the rest of 5 differential genes are all up-regulated.7) In addition, at different time points in BPH-infestation(6 hours, 24 hours and 48 hours), we measured SA and JA(free) content in RH and TN1. Results show that the after BPH-infestation 6 h, SA content showed obvious increase in RH, but it was no significant change in TN1 until 24 hours, it means that RH SA hormone change is faster and stronger than TN1 under the BPH-infestation. On the contrary, JA content were obviously down-regulated from 24 hours in two varieties. |
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