转Pi-d2基因水稻对稻瘟病的抗性分析

对由pCB6.3kb、pCB5.3kb和pZH01-2.72kb三个不同表达载体转化的转稻瘟病抗性基因Pi-d2水稻株系进行稻瘟病抗性分析。结果表明,转Pi-d2基因水稻的9个高代株系对来自四川的39个稻瘟病菌株表现不同的抗性,抗病频率最高达91.7%;4个转基因早代纯合株系对来自中国农业科学院的58个菌株中的81.48%以上菌株表现抗性,具有广谱抗性特点。稻瘟病菌粗毒素筛选结果表明,来自转基因植株的幼胚愈伤组织诱导率随培养基中粗毒素浓度提高而降低,粗毒素浓度达到40%时,幼胚愈伤诱导率为49.33%,受体对照幼胚愈伤组织诱导率为5%。田间诱发条件下,转基因株系在大田的穗瘟发病率为0%～50%,抗性较受体对照大幅度提高。     

Genetic Transformation of Rice with Pi-d2 Gene Enhances Resistance to Rice Blast Fungus Magnaporthe oryzae

The gene Pi-d2, conferring gene-for-gene resistance to the Chinese blast strain ZB15, was isolated from a rice variety (Digu) by the map-based cloning strategy. Here, we constructed a control plasmid pZH01-pi-d2tp309 (pZH01-tp309) and three different expression constructs, pCB-Pi-d25.3kb (pCB5.3kb), pCB-Pi-d26.3kb (pCB6.3kb) and pZH01-Pi-d22.72kb (pZH01-2.72kb) of Pi-d2, driven by Pi-d2 gene's own promoter or CaMV35S promoter. These constructs were separately introduced into japonica rice varieties Lijiangxintuanhegu, Taipei 309, Nipponbare and Zhonghua 9 through Agrobacterium- mediated transformation. A total of 150 transgenic rice plants were obtained from the regenerated calli selected on hygromycin. PCR, RT-PCR and Southern-blotting assay showed that the gene of interest had been integrated into rice genome and stably inherited. Thirty-five transgenic lines independently derived from T1 progeny were inoculated with the rice blast strain ZB15. Transformants exhibited resistance to rice blast at various levels. The lesions on the transgenic plant leaves were less severe than those on the controls and the resistance level of transgenic plants harboring the gene of interest from three vectors had no difference. The own promoter of Pi-d2, about 2.2 kb or 3.2 kb, had the similar promoter function as CaMV35S. Field evaluation for three successive years supported the results of artificial trial, and some lines with high resistance to rice leaf blast and neck blast were obtained.     

抗稻瘟病转基因水稻竹转68的选育

应用基因枪法将抗病基因“水稻碱性几丁质酶（RC24）基因”转入籼稻保持系籼B，选育出了具抗稻瘟病特性的转基因水稻品系竹转68。该品系还表现出穗大粒多的高产特性，平均每穗总数粒比竹籼B增加103．6粒，每穗实粒数增加84．4粒，增长率分别达83．8％和86．6％；与竹籼B相比，对稻瘟病的抗性显著增强；稻米品质好；且竹籼A具有恢复能力，而失去了竹籼B的保持特性。     

转Pi9抗稻瘟病基因水稻株系的比较转录组分析

【目的】在转录水平上解析Pi9基因介导的稻瘟病抗性调控机理,为培育抗病水稻品种提供理论依据。【方法】向水稻品种日本晴(NPB)及其转Pi9抗稻瘟病基因株系(NPB/Pi9)接种稻瘟菌。分别于接种后0 h、12 h、24 h、36 h提取叶组织样品,选取12503个水稻基因定制基因芯片,进行水稻基因转录组分析,并通过qRT-PCR对部分差异表达基因进行验证。【结果】NPB/Pi9在接种后12 h、24 h和36 h的基因表达量分别与其接种0 h表达量比较,共检测到7754个差异表达基因;相应地,感病水稻NPB在以上时间点共检测到7385个差异表达基因;在接种后36 h,NPB/Pi9的差异表达基因数目显著多于NPB。比较NPB/Pi9和NPB相同时间点的基因表达量,共获得4065个差异表达基因,其中接种后36 h的差异表达基因显著多于接种后0 h、12 h或24 h。因此,NPB/Pi9的稻瘟病防御反应更强烈。对NPB/Pi9与NPB相同时间点的差异表达基因进行GO和KEGG分析,细胞外区域、植物对刺激应答、转录调控、氧化还原、离子结合、次生代谢和植物激素相关的GO分类在接种后呈显著富集,苯丙氨酸代谢、类黄酮生物合成和植物激素信号途径的KEGG通路在接种后显著富集。与效应分子触发的免疫反应(ETI)相关的水杨酸信号途径、几丁质酶,以及与病原相关分子模式触发的免疫反应(PTI)相关的胞外区域、对刺激的应答、木质素合成等,均在抗感水稻之间差异表达。而且PTI/ETI共有的WRKY转录因子、MAPK激酶、茉莉酸和乙烯信号途径等发生差异表达。综上所述,NPB/Pi9和NPB的差异表达模式与ETI和PTI相关,两者相互联系并在Pi9介导的稻瘟病抗性中发挥作用。【结论】与日本晴比较,抗病基因型NPB/Pi9对稻瘟病防御反应更强烈。转录因子、激酶、NBS-LRR基因、几丁质酶、水杨酸、茉莉酸和乙烯信号途径,以及植物次生代谢在Pi9介导的稻瘟病抗病反应中发挥重要作用。     

水稻稻瘟病抗性基因研究进展

介绍了稻瘟病抗性基因的结构,综述了其定位和克隆的最新研究进展,阐述了在其研究过程中分子标记技术的利用,并提出今后的研究方向。     

转PAL基因水稻抗稻瘟病性和过氧化物酶活性的研究

 用PAL(苯丙氨酸解氨酶）基因正义和反义转化水稻，获得了70株转基因植株。选择正义转化植株(1s)和反义转化植株(4a)进行稻瘟病菌([i]Magnaporthe grisea[/i])接种，针对病原物侵染，1s的PAL活性上升更快，幅度更大。观察水稻叶片超微结构发现，1s的细胞具有更强的抵抗病原菌入侵的能力，其过氧化物酶活性也比对照和4a要高。     

Relationship Between Resistance Gene Analogue and Blast Resistance in Rice

DNA fragments of 43 rice varieties were amplified with 11 pairs of primers designed based on resistance gene analogue(RGA) of plants,and the blast resistance of the varieties was identified by inoculation with 33 isolates of Magnaporthe grisea collected from Yunnan Province,China.Clustering results revealed a significant correlation between the blast resistance and DNA bands with a correlation coefficient of 0.6117(α=0.01) ,indicating that the resistance analysis based on RGA-PCR clustering analysis coincid...     

水稻抗病基因同源序列与抗瘟性的关系

利用11对抗病基因同源序列(RGA)引物扩增43个水稻品种的DNA片段和接种33个稻瘟菌株测定它们的抗病性。结果表明,抗病性聚类结果与DNA扩增条带聚类结果基本一致,两者相关系数达0.6117(α=0.01),即利用RGA分析品种的抗病性具有很好的代表性,但相关性在引物间不同,从0.1701到0.5305。结合多态性分析,5对引物S1/AS3、S1 INV/ S2 INV、XLRR For/XLRR Rev、Pto Kin 1 IN/Pto Kin 2 IN、NLRR For/NLRR Rev可用于水稻品种的抗稻瘟病性分析,它们扩增的DNA条带数据与抗瘟性间达极显著相关。此外,除高感品种丽江新团黑谷和CO39两个品种未被聚类到不同的亚种外,RGA扩增的DNA条带还能区分水稻的籼粳亚种类型。     

水稻稻瘟病抗性基因Pi ta的SNP检测方法

 针对稻瘟病抗性基因Pi ta位点上的抗感基因的编码产物仅有1个氨基酸的差异,利用已经建立的检测该基因的SNP方法以及作者在该基因编码序列构建的四引物扩增受阻突变体系PCR,对62份2012年长江上游国家水稻区域试验材料和97份陕西省水稻区域试验材料进行了Pi ta基因检测,2种分子标记检测结果一致。该方法的检测结果真实可靠,可以用于检测水稻Pi ta基因位点。     

稻瘟病抗性基因研究进展与展望

稻瘟病严重影响水稻的产量和品质,抗病品种的培育和种植是控制该病害最经济有效的措施。当今水稻抗稻瘟病育种的关键是抗性基因的发掘和合理利用。据报道,至少有68个稻瘟病位点的83个主效基因得到定位,其中23个被成功克隆。概述了稻瘟病的抗性遗传、稻瘟病抗性基因定位与克隆及抗性基因在育种上的应用研究进展,并对稻瘟病抗性育种面临的问题和应用前景进行了讨论和展望。     

转抗菌肽B基因和bar基因籼稻植株的再生

 运用基因枪法将含有bar基因和cecropin B基因的质粒pCB1导入籼稻品种特青的幼胚细胞,筛选后得到3株转基因植株。PCR检测和Southern杂交结果表明,外源基因已整合到水稻基因组中。转化当代植株表现出对Basta很强的抗性,同时也增强了对水稻白叶枯病的抗性。     

Relationship Between Blast Resistance Phenotypes and Resistance Gene Analogue Profiles in Rice

A total of 21 rice varieties were assayed based on RGA-PCR using six pairs of RGA primers and evaluated for leaf blast resistance in the nursery as well. Cluster analysis showed that the varieties could be classified into five groups either at the similarity threshold of 0.72 for RGA profiles or at 0.80 for leaf blast severities. Although there did not exist a complete parallel relationship between RGA-based groups and blast resistance-based groups, five out of six varieties with broad spectrum or durable resistance repeatedly fell into same group. This result suggested that application of three primer pairs, viz. RGA1 and RGA2 （both designed from the LRR region of rice Xa21 gene） and RGA3 （designed from the LRR region of tobacco N gene） contributed to better evaluation of the germplasms for their resistance responses to rice blast.     

Resistance of Antimicrobial Peptide Gene Transgenic Rice to Bacterial Blight

Antimicrobial peptide is a polypeptide with antimicrobial activity. Antimicrobial peptide genes Np3 and Np5 from Chinese shrimp (Fenneropenaeus Chinensis) were integrated into Oryza sativa L. subsp. japonica cv. Aichi ashahi by Agrobacterium mediated transformation system. PCR analysis showed that the positive ratios of Np3 and Np5 were 36% and 45% in T0 generation, respectively. RT-PCR analysis showed that the antimicrobial peptide genes were expressed in T1 generation, and there was no obvious difference in agronomic traits between transgenic plants and non-transgenic plants. Four Np3 and Np5 transgenic lines in T1 generation were inoculated with Xanthomonas oryzae pv. oryzae strain CR4, and all the four transgenic lines had significantly enhanced resistance to bacterial blight caused by the strain CR4. The Np5 transgenic lines also showed higher resistance to bacterial blight caused by strains JS97-2, Zhe 173 and OS-225. It is suggested that transgenic lines with Np5 gene might possess broad spectrum resistance to rice bacterial blight.     

水稻抗稻瘟病基因Pi-ta共显性分子标记的建立

 根据抗病基因Pi-ta和感病等位基因pi-ta在DNA序列上的单核苷酸长度多态性（single nucleotide length polymorphisms，简称SNLP）设计了3个特异性引物YL155、YL183和YL200，并分别用蓝色和绿色染料将YL155和YL183进行标记，而YL200不标记。在一个PCR反应中同时加入这3对引物，并应用ABI自动分析仪对PCR产物进行分析，结果发现，抗病基因Pi-ta纯合的样品获得一个峰值为181 bp的图谱，感病等位基因pi-ta纯合的样品获得一个峰值为183 bp的图谱，而抗病基因Pi-ta处于杂合状态的样品出现两个峰。由此建立了水稻抗稻瘟病基因Pi-ta的共显性分子标记。以杂交组合Katy/RU9101001的12个F2单株和15个水稻品种为材料，利用已建立的显性分子标记和稻瘟病菌人工接种试验对共显性标记的正确性进行了验证，结果发现三者的实验结果完全吻合，因此该共显性标记可应用于水稻抗病基因Pi-ta的分子标记辅助选择。     

根癌农杆菌介导的水稻转化系统的优化及转反义Wx基因植株的获得

以具不同直链淀粉含量的籼粳稻品种为受体材料,试验了适宜于根癌农杆菌介导转化的水稻愈伤组织诱导培养基及其诱导培养的时间。结果表明,一种基于MS基本培养基的商品培养基较适合作为籼粳稻幼胚愈伤组织的诱导培养基;对于籼型杂交稻亲本协青早B,转化前幼胚较适合的愈伤组织诱导培养天数为8 d左右。在此基础上,将反义蜡质（Wx）基因分别导入上述受体亲本中,获得了一批转基因水稻植株。PCR和Southern杂交分析表明反义Wx基因已经整合进了转基因水稻的基因组中。遗传分析表明外源基因在转基因水稻后代中的分离符合3∶1的理论比例。     

国际稻瘟病圃种质稻瘟病抗性的鉴定与评价

分别采用苗期喷雾接种和成株期注射、涂抹接种对375份国际稻瘟病圃材料进行稻瘟病抗性人工接种鉴定,发现苗瘟与叶瘟抗性间具有极显著的相关性,而苗瘟、叶瘟与穗颈瘟间相关性不显著,结果筛选出一批抗性较好的材料,可供育种部门在选配亲本时应用。     

水稻稻瘟病抗性基因的定位及克隆研究进展

发掘利用抗稻瘟病基因是防治稻瘟病的基本策略之一。综述了抗稻瘟病基因的发掘、定位和克隆的相关研究情况,初步统计了目前已精细定位和克隆的主效抗稻瘟病基因。     

水稻品种抗瘟性表型与抗病基因同源序列相似性关系

 用6对RGA引物，即RGA1(XLRR for/XLRR rev)、RGA2(XLRR inv1/XLRR inv2)、RGA3（NLRR for/NLRR rev）、RGA4(NLRR inv1/NLRR inv2)、RGA5(Pto kin1/Pto kin2)和RGA6(Pto kin3/Pto kin4)对21个品种进行RGA PCR的DNA指纹分析。以相似系数0.72和田间叶瘟严重度阈值0.84分别聚类，21个水稻品种均可以分成5类。尽管类与类之间没有一一对应关系，但抗谱广、抗性稳定或具持久抗瘟性的品种，能较好地聚为一类，如湘资3150、IR156、株两优02、ZR02。在6对引物中，RGA1和RGA2来自于水稻抗白叶枯病基因Xa21含 LRR结构，RGA3来自于烟草N基因含LRR结构，上述3对引物比较适宜用于水稻稻瘟病抗性基因遗传背景分析。