【作者】 石军；

【导师】 黄富； 王文明；

【作者基本信息】 四川农业大学 ， 植物病理学， 2015， 博士

【摘要】 水稻作为全球最重要的粮食作物之一,其高产稳产对于全球粮食安全起着至关重要的作用。由于受到非生物/生物的胁迫,每年水稻产量损失严重。稻瘟病是危害水稻产量的重要真菌病害,通过杂交选育抗稻瘟病品种是最经济、环保和持久有效的防治手段。但是稻瘟菌快速的遗传进化导致选育初期还具有抗性的品种,在大面积种植几年后抗性随之丧失。因此,抗病选育需要持续投入以丰富种质资源,从而战胜稻瘟病,实现粮食稳产高产。迄今为止,超过101个抗稻瘟病基因被鉴定,23个抗稻瘟病基因被克隆。目前克隆的抗稻瘟病基因绝大多数都属于NBS-LRR类型,其中有许多是等位基因或者是直系同源基因。雅恢2115是四川农业大学农学院植物病理系选育的优良恢复系。其配组的三系杂交稻宜香优2115于2011年,2012年分别通过四川省和国家新品种审定。通过多年多点鉴定,雅恢2115及其与抗病、中抗、高感类型的不育系所配杂交稻组合均表现为抗稻瘟病,雅恢2115的稻瘟病抗性受显性基因控制。本文研究目的是确定控制雅恢2115抗稻瘟病的基因。通过田间抗性鉴定、室内抗谱测定并结合分子生物学技术,最终推导雅恢2115的主效抗稻瘟病基因为广谱抗稻瘟病基因Pi2,实验主要结果如下：1,探明雅恢2115具有稻瘟病广谱抗病性通过田间抗性鉴定、室内抗谱测定结果表明：雅恢2115对92个供试菌株的抗病频率为100%,平均叶瘟率为0.22%,平均病情指数为0.05。分别含Pi2、Pi9、Pizt、Pi1、Pi5、Pikm单基因系的抗病频率分别为96.63%、90.57%、68.97%、28.74%、23.17%、93.87%；平均叶瘟率为0.58%、0.60%、56.48%、4.66%、65.41%、0.50%；平均病情指数分别为0.10、0.07、23.60、0.59、36.03、0.08。本研究表明,在四川还表现抗稻瘟病病基因有Pi2、Pi9、Pikm、Pil,可加强其在四川抗病育种利用。其他已克隆的部分抗病基因在四川已经或者正在丧失田间抗性。雅恢2115与抗病、中抗、高感类型的不育系所配杂交稻组合,其叶瘟,穗颈瘟抗病级别介于0-3级,抗性频率介于88.15%-100%,病情指数介于0.01-0.25,组合均表现较强的稻瘟病抗性。2,荧光定量PCR表达量分析推测Pi2为雅恢2115主效稻瘟病抗性基因通过设计已克隆的抗稻瘟病基因荧光定量PCR引物,利用荧光定量PCR技术检测其在雅恢2115中的表达情况,结果表明：Pi2、Pib和Pia-RGA4在雅恢2115中表达量较高。而田间抗稻瘟病基因单基因系已经证实Pib、Pia-RGA4在四川丧失了田间抗性。因此,Pi2极有可能是雅恢2115抗稻瘟病的主效抗病基因。3,稻瘟病抗病基因在雅恢2115中的等位克隆证实包含Pi2,Pi25无突变全长通过已克隆的抗稻瘟病基因序列设计全长引物,以雅恢2115 DNA或cDNA为模板扩增后测序,在雅恢2115中得到Pi2、Pit、Pid2、Pi25、Pib、Pita、Pia基因全长。其中Pi2,Pi25无任何突变。4,Pi2连锁标记AP22对LTH/雅恢2115构建的F2群体抗感单株具有有效的区分,证实雅恢2115中含有Pi2以LTH为母本,雅恢2115为父本杂交后自交得到F1代,通过人工接种稻瘟病菌外加病圃内自然诱发,调查F2代的抗、感单株,利用Pi2的特异性连锁标记AP22对感病群体和抗病群体进行连锁分析。通过连锁标记分析,AP22能有效的对抗病和感病群体进行区分,进一步支持雅恢2115中含有抗病功能基因Pi2的推测。5,对雅恢2115中Pi2进行RNAi,转基因阳性单株Pi2表达量显著降低,植株更加感病,证实雅恢2115是由Pi2提供主效稻瘟病抗性为了进一步证明,本研究对雅恢2115中的Pi2进行RNA干涉。通过构建Pi2基因RNA干涉表达载体pANDA-Pi2-RNAi,对阳性转基因株系进行了Pi2表达量分析,与雅恢2115相比,其Pi2的表达量降低范围介于55%-95%。表明干涉载体很好的抑制了Pi2的表达。通过对表达量降低最多的转基因水稻离体接种22个单孢菌株和菌的相对生长量分析,发病后通过病斑大小测定,转基因水稻病斑面积总体大于雅恢2115,其菌的相对生长量约为雅恢2115的140%。证明Pi2的干涉单株抗稻瘟病能力得到显著降低。从而确定Pi2为雅恢2115主效稻瘟病抗性基因。更多还原

【Abstract】 Rice is one of the most major food crops in the word.The stable yield of rice is very important to the safety of world.Because of biologic and abiotic stress, the loss of yield is serious.Rice blast damage is a significant one causing by fungi.Developing resistant cultivars is the most effective, economical, and eco-friendly approach for disease control.But by the rapid genetic evolution of rice blast, the resistant varieties will lost resistance when wildly planted in several years.So, hybrid rice breeding program must take durable effort in order to rich germplasm resources,overcome rice blast,and achieve the high yield aim of rice.So far, more than 101 major blast resistant genes (R genes) have been identified and 23 R genes have been cloned. Most of these cloned blast R genes belong to the nucleotide binding-site leucine-rich repeat (NBS-LRR) family, and some of then are alleles.YaHui 2115(YH2115) is an elit restore, and breeded by agricultural college, Sichuan Agricultural University. Hybrid rice combinations YiXiangYou2115, combined by YaHui2115, have passed new variety certification both in Sichuan and China in 2011 and 2012, respectively.According to our several years and locations identifying,the resistance of YaHui2115 is controlled by dominant gene.We exploited quite a few hybrid rice combinations, that the female parents belong to resistant,middle resistant and susceptible types.The F1 all showed high resistance to rice blast.So we focused on study the reason of blast resistance in YaHui2115.We designed field resistance investigation and molecular biology resaech,finally we verified that the major blast resistant gene is broad spectrum blast gene Pi2. The result is as followed:1.YaHui2115 has a broad broad-spectrum resistance to rice blastThe field blast resistanceYaHui2115 and monogenic lines of blast resistance genes are investgated and it showed:The resistance frequency, average leaf blast, average disease index of YaHui2115 is 100%,0.22%,0.05, respectively; The monogenic lines of Pi2, Pi9, Pizt, Pi1, Pi5, Pikm, the resistance frequency, average leaf blast, average disease index are 96.63%,90.57%,68.97%,28.74%,23.17%, 93.87%; 0.58%,0.60%,56.48%,4.66%,65.41%,0.50%; 0.10,0.07,23.60, 0.59,36.03,0.08, respectively. Pi2, Pi9, Pikm, Pil already have strong resistance in Sichuan Province and should enhance hybrid breeding. Other R genes now have partial resistance or even lose resistance. YaHui2115 has the most powerful resistance and widest resistant spectrum. We exploited quite a few hybrid rice combinations, that the female parents belong to resistant,middle resistant and susceptible types. The leaf blast and neck blast level is between 0-3, resistance frequency is between 88.15%-100%, disease index is between 0.01-0.25, The F1 all showed high resistance to rice blast.2. RT-PCR analysis infers Pi2 is the major R gene of YaHui2115 to rice blastTo identify the functional blast resistance genes in YH2115, RT-PCR was carried out monitoring the expression levels of reported blast R genes in YH2115 infected with M. oryzae. it showed:Pi2,Pib and Pia-RGA4 had much higher expression in YH2115. But Pib and Pia-RGA4 lost blast resistance in the field. So,Pi2 should be a major contributor of resistance in YH2115.3. The allele clone of R genes in YaHui2115 Confirm full length of Pi2, Pi25 existWe amplified the full length of cloned R genes from YH2115. By sequencing the amplified fragments,we got the full length of Pi2, Pit, Pid2, Pi25, Pib, Pita, Pia.We found that the sequence of Pi2,Pi25 in YH2115 were identical to the resistance allele reported4. LTH/YaHui2115 F2 group is developed, Pi2 linked marker can effective distinguish resistant and susceptive plants, It verifies Pi2 existing inYaHui2115.We built up a F2 population of YH2115 crossed with LTH,based on natural infection and artificial inoculation in the field, we employedthe previously reported Pi2linkage marker AP22 to genotype these F2 plants.These data indicated that the Pi2 linkage marker AP22 could effectively distinguish susceptible and resistant plants from F2 population generated from YH2115 and LTH; and that YH2115 has a functional blast resistance gene Pi2.5. RNAi experiment was performed for interfere Pi2, the positive plants show lower expression of Pi2, more susceptible. It confirms Pi2 is the major R gene in YaHui2115.To confirm the role of Pi2 in YH2115’s blast resistance, RNAi experiment was performed. We constructed a RNAi expression vector "pANDA-Pi2-RNAi"for Pi2.Fourteen YH2115 transgenic lines were obtained, with Pi2 expression level reduced by 55%-95%. The transgenic plants were challenged with M. oryzae using punch inoculation method. The results showed that the Pi2 knocking-down plants were more susceptible to blast disease, and the lesion size was significantly larger than that in YH2115 in response to different M. oryzae strains. And transgenic plants showed up to about 140% relative mycelial growth biomass, more number of spores than in YaHui2115.These results further confirmed that Pi2 contributed to YH2115’s blast resistance.更多还原