【作者】 潘凤英；

【导师】 周瑞阳；

【作者基本信息】 广西大学 ， 作物栽培学与耕作学， 2013， 博士

【摘要】 本研究以19个水稻光温敏核不育系为材料,对南方水稻黑条矮缩病毒(Southern rice black-streaked dwarf virus, SRBSDV)的鉴定方法、不育系对SRBSDV的抗性、受SRBSDV侵染后的生理变化、抗性遗传规律、NBS类抗病基因进行研究,得出如下主要结果：1、SRBSDV的鉴定方法及19个水稻不育系对SRBSDV的抗性评价：根据已公布的SRBSDV S10(EU523360)基因序列,设计1对检测SRBSDV的特异引物,利用分子生物学与田间调查相结合的方法,参照广东省生物防治实验站关于南方水稻黑条矮缩病发生规律与防治技术调查研究方案中的标准进行病株分级,对生产上常用的19个水稻不育系进行株高调查、病级划分,统计计算其发病率、病情指数、相对发病率。以病情指数类平均法(UPGMA)聚类分析,聚类结果将19个不育系划分为4个抗性等级：(1)高抗：164S；(2)中抗：福3S、蜀光1268S、长广占63S；(3)中感：马S、95S、6201S；(4)高感：华莫m207S、华莫103S、中华38S、安S、FSA、6303S、25S、116S、华师wy-2S、安徽195S、培矮64S、春S。尚未发现免疫的材料。2、SRBSDV侵染水稻的生理生化变化：(1) SRBSDV感染的再生稻根系形态和根系活力变化以经鉴定为高抗、中抗、高感的水稻光温敏核不育系各1个为材料,每种不育系选取发病等级为0级、1级、2级、3级的材料,采取水培的方法进行再生繁殖,研究SRBSDV感染对不育系再生稻根系生长的形态变化和根系活力变化。结果表明,在没有SRBSDV侵染(0级)时,高抗和中抗不育系的根长分别是感病不育系的1.53和1.43倍,根表面积分别是感病不育系的1.66和1.27倍,根体积分别是1.78倍和1.13倍,根系活力分别是1.45倍和2.27倍,但是三者的根尖数没有显著差异。高抗与高感不育系的上述指标之间差异显著,高抗和中抗不育系差异不显著。受到SRBSDV侵染时,不育系内不同发病等级间随着发病程度的增高,各指标呈现明显下降趋势,除了高抗不育系的根尖数,其余各基因型内各指标均随着发病等级的增加而表现出显著差异。SRBSDV感染对根系的影响主要表现为根系的粗细不同。(2)水稻光温敏不育系对SRBSDV的抗性与内源激素的关系分析利用人工接种SRBSDV和采用高效液相色谱技术(High Performance Liquid Chromatography, HPLC)测定内源激素的研究方法,对SRBSDV胁迫下高抗SRBSDV164S和高感SRBSDV培矮64S两个不同抗性不育系水稻幼苗植株内源激素赤霉素(GA3)、生长素(IAA)、水杨酸(SA)和脱落酸(ABA)的动态变化进行了研究。结果表明：1)人工接种SRBSDV后,促进生长明类的激素GA3、IAA在培矮64S处理植株中含量平均值显著低于其对照植株,在第5和第6天感病症状开始出现时达到最低值；在164S处理植株中IAA含量平均值显著低于其对照植株,GA3含量平均值也低于对照植株,但未达到5%显著水平。2)抗御信号分子SA在处理植株中含量平均值显著高于对照植株,在感病症状开始出现时,其含量都达到最高值,但培矮64S在最高值后开始较快下降,而164S的SA含量则保持显著高于对照植株的水平。3)与衰老、死亡有关的内源激素ABA在处理植株中含量高于对照植株,培矮64S的ABA含量显著高于164S,与其对照植株差异显著；164S与其对照差异不显著。4)SRBSDV胁迫改变了植株体内IAA/ABA和GA3/ABA的平衡。(3) SRBSDV实时荧光定量PCR检测方法的建立及其各病级的病毒含量针对SRBSDV CP基因设计了1对特异性引物,以水稻UBC为内参基因,建立SRBSDV CP两步法real time PCR绝对定量检测方法。对感SRBSDV的0级、1级、2级、3级水稻植株叶片SRBSDV CP基因的拷贝数进行绝对定量,结果显示,通过内参UBC校正后,病级之间SRBSDV CP基因拷贝数差异显著。3、不育系对SRBSDV的抗性遗传分析：以经鉴定为中抗和高抗SRBSDV的水稻光温敏核不育系及感病对照TN1为材料,应用经典遗传学分析方法研究4个抗SRBSDV亲本抗病基因的对数。结果显示,164S与TN1杂交的F2代表现为15：1的抗感分离比例,表明供试亲本对SRBSDV的抗性均受2对显性基因控制。长广占63S、蜀光1268S、福3S这3个与TN1杂交的F2代表现为3：1的抗感分离比例,表明它们对SRBSDV的抗性均受1对显性主效基因控制。4、水稻系列不育系抗病基因同源序列的克隆与测序分析：本研究根据NBS-LRR类保守氨基酸序列设计引物,对水稻系列不育系进行抗病基因同源序列进行克隆与分析,结果显示,18个水稻不育系亲本及抗感病对照恩恢58、TN1,共获得了9类NBS-LRR类抗病基因同源序列,这9类抗病基因同源序列氨基酸相似性为49.5%-83.6%,与已知的9个NBS抗病基因氨基酸序列一致性为26.2%-100.0%。更多还原

【Abstract】 19rice photo thermo sensitive genic male sterile lines (PTGMS) were used to study the SRBSDV (Southern rice black streaked dwarf virus) identification method, their resistance to SRBSDV, the physiological change by SRBSDV infection, the inheritance, the NBS resistance genes. The main results were summarized as follows:1. The identification method of SRBSDV and resistantance evaluation of19rice sterile lines to SRBSDV:SRBSDV S10(EU523360) was used to design a pair of specific primers to detect SRBSDV, using molecular biology and the field investigation method, with reference to Guangdong province biological control experiment station’s grade standards about occurrence regularity and control technology research of southern black rice dwarf virus disease, we measured19rice sterile lines’plant height and disease grades, computing their average incidence, their disease index and their relative incidence. Clustal analysis with main component average incidence,19resistance of sterile lines are divided into four levels:(1) high resistant (HR):164S;(2) moderate resistant (MR):Fu3S, Shuguang1268S, Guangzhan63S;(3)moderate sensitive (MS):Ma S,6201S and95S;(4) high sensitive (HS):Huamo m207S, Huamo103S, An S, FSA,25S,116S,6303S, Huashi wy-2S, Chunghua38S, Anhui195S, Peiai64S and Chun S. Haven’t found any immune material.2. Physiological changes of rice by SRBSDV infection:(1) Analysis of the morphology and activities of ratooning rice root systems infected by SRBSDV:Three rice photo thermo sensitive genic male sterile lines which had been identified to be high resistant, moderate resistant and high sensitive to SRBSDV were used as materials and four disease grades (0,1,2and3grade) were set to study the influence of SRBSDV infection on sterile lines ratooning rice root morphology and activities by the method of hydroponic regeneration. The results showed that while there was no SRBSDV infection (grade0), root indexes reached significant differences between high resistant and high sensitive sterile lines, but there was no significant difference between high resistant and moderate resistant sterile lines. While sterile lines infected by SRBSDV (grade1,2,3), the root indexes indicated a downward trend as disease grades increased within the same sterile line. Indicators reached significant differences as the grades increased except the tips number of the high resistant sterile line. The influence of SRBSDV on root system mainly performed on different size roots.(2) Relationship analysis of endogenous hormone and PTGMS resistance to SRBSDVArtificial inoculating SRBSDV and HPLC (High Performance Liquid Chromatography) technique were used to measure the GA3, IAA, SA and ABA content of high resistant PTGMS line164S and high sensitive PTGMS line Peiai64S in response to SRBSDV infection. The results showed that:1) Peiai64S’GA3and IAA average content were significantly lower in SRBSDV infected plants than that in healthy plants. The lowest value appeared in the5th and6th day after SRBSDV infected when disease symptoms began to appear.164S’IAA average content was significantly lower in SRBSDV infected plants than those in healthy plants; GA3average content was lower in the infected plants than those in healthy plants, but didn’t reach the5%significant level.2) Resistance signal molecule SA content was higher in SRBSDV infected plants than in healthy plants. Both reached the highest level when disease symptoms initialised. Peiai64S’ SA content declined faster than that of164S’ after the highest value and164S’ SA content remained significantly higher than that in healthy plants.3) Endogenous hormone ABA which relevants to aging and death content was higher in SRBSDV infected plants than that in healthy plants. Peiai64S’content significantly higher than164S’ and significantly higher than their healthy plants;164S didn’t show significant difference with its healthy plants.4) SRBSDV infection changed plants’ IAA/ABA and GA3/ABA balance. Conclusion: SRBSDV stress changed rice endogenous hormone content, but this change was direct infection or indirect damage of the virus still remains elusive.(3) Establishment and implication of SRBSDV real-time qPCRDesign a pair of specific primers to amplify SRBSDV CP gene fragment, UBC was used as referance gene, establish SRBSDV CP two-step real time absolute quantitative PCR detection method. Absolute quantitative the grade0, grade1, grade2, grade3rice leaf SRBSDV CP copies number, through UBC regulation, results show that SRBSDV CP copies number significantly difference between grades.3. SRBSDV resistance genetic analysis of rice sterile lines: 3moderate resistant and1high resistant rice photo thermo sensitive genic male sterile line to SRBSDV and sensitive check TN1were used as materials in this study. Classical genetic analysis was applied to study this five SRBSDV resistant parent genetic and logarithm of resistance genes.164S hybrided with TN1, their F2hybrids showed resistant to sensitive15:1separation ratio. The result indicates164S’ resistance to SRBSDV is controlled by2dominant genes. Four sterile lines Guangzhan63S, Shuguang1268S, Fu3S hybrided with TN1, their F2hybrids showed resistant to sensitive15:1separation ratio. The results showed that their resistances to SRBSDV are controlled by1dominant gene.4. Rice sterile lines resistance genes homologous cloning and sequence analysis:Design a pair of degenerate primers according to the NBS-LRR conserved amino acid sequences, homologous cloning and analysis the rice sterile lines’ disease resistant gene sequences. The results showed that18rice sterile lines, Enhui58and TN1, received9types of NBS-LRR resistance gene homologous sequences total. These9types of resistance genes homologous amino acid sequence similarities are between49.5%～83.6%. Contrast with the9had been known NBS resistance genes amino acid sequences similarities are26.2%～100.0%.更多还原