来自中国云南野生稻的抗稻瘟病遗传资源

与Pi-ta^＋等位基因相比,含有Pi-ta^＋等位基因的栽培稻具有抗稻瘟病特性。本研究用基因序列分析的方法检测了来自云南的不同栽培稻品种以及不同类型和来源的普通野生稻种和非洲长雄蕊野生稻种中的Pi-ta^＋基因,发现Pi-ta地基因在稻属植物中高度保守,但Pi-ta^＋等位基因的存在极其稀有。在所检测的栽培稻和野生稻中仅有来源于云南景洪的直立型普通野生稻中含有Pi-ta^＋等位基因。而与Pi-ta基因相比,另一个水稻抗稻瘟病基因Pib,经部分同源序列克隆及测序发现该基因在不同野生稻中的变异较大。在所克隆的不同野生稻Pib基因同源序列中,也只有来源于直立型普通野生稻的序列能按该基因的开放阅读框进行正常翻译。对不同类型普通野生稻的抗稻瘟病能力的初步鉴定结果表明,直立型普通野生稻对供试的本地稻瘟病生理小种具有较强抗性,其抗性可能源于所含的Pi=ta^＋等位基因及可能有功能的Pib基因。由于普通野生稻与栽培稻同属AA基因组型,因此,云南直立型普通野生稻可通过杂交育种或基因工程途径用于栽培稻的抗稻瘟病性能改良。     

云南景洪直立型普通野生稻抗稻瘟病Pi-ta+等位基因的克隆与分析

用PCR法从景洪直立紫杆普通野生稻中克隆了抗稻瘟病基因Pi-ta⁺ 的4 672 bp序列, 该序列包含完整的编码框、内含子和终止密码子下游的331 bp。所克隆的直立型紫杆普通野生稻Pi-ta基因序列的编码区与已报道的日本栽培稻社糯(Yashiro-mochi)和元江普通野生稻相应序列间的同源性分别为99.86%和98.78%。与社糯的Pi-ta基因相比, 其编码区有4个核苷酸的差异并导致3个氨基酸残基的改变, 而内含子区域有6个核苷酸差异。对该序列进一步分析发现, 其推导的氨基酸残基的918位为丙氨酸, 属于稀有的抗稻瘟病的Pi-ta⁺ 等位基因。景洪直立型普通野生稻Pi-ta⁺ 基因因其编码序列和推导的氨基酸序列与社糯有所不同, 推测其抗病能力大小和抗菌谱可能与社糯的Pi-ta基因不同。直立型普通野生稻中Pi-ta⁺ 等位基因的克隆为进一步利用该基因改良栽培稻抗病能力提供了前期物质基础。     

抗稻瘟病基因Pi-ta、Pi-b、Pi54和Pikm在黄淮稻区水稻品种资源中的抗性分布

为了探明黄淮稻区4个抗稻瘟病基因Pi-ta、Pi-b、Pi54和Pikm在品种资源中的分布及其不同基因型组合的抗病效性。本研究利用上述4个抗病基因的功能标记,对88个黄淮稻区育成品种进行抗稻瘟病基因型检测,结果表明:检出最多的抗稻瘟病基因组合是Pib+Pi54,占总检测材料的60.2%。经过2016-2017年连续两年的人工接种鉴定和田间自然鉴定,稻瘟病感病材料比例分别达到87.5%、77.3%,感病材料的比例增高说明抗性基因的抗性作用在逐渐降低。在检测品种中,抗性较好的4个基因型或基因型组合分别是Pi-ta、Pi-ta+Pi-b、Pi-ta+Pi-b+Pi54和Pi-ta+Pi-b+Pi54+Pikm,但检出率很低。本研究结果说明,黄淮稻区品种中,携带Pi-ta、Pi-ta+Pi-b、Pi-ta+Pi-b+Pi54和Pi-ta+Pi-b+Pi54+Pikm基因型的抗病性较好,应得到加强利用。本研究结果对育成品种抗稻瘟病基因或基因组合跟踪检测和抗病性评价是抗稻瘟病育种的有效途径。     

云南疣粒野生稻稻瘟病抗性

野生稻(Oryza rufipogo)保存有许多栽培稻(O. sativa)不具备或已经消失的优异基因资源, 是扩大栽培稻遗传背景、改良产量与品质、提高抗病虫害及抗逆境能力的重要基因库。疣粒野生稻(O. meyeriana)是中国3种野生稻资源之一, 主要分布在云南。为进一步了解其稻瘟病抗性, 首先利用来自不同稻作区的稻瘟病菌株, 通过注射接种法对疣粒野生稻进行系统的稻瘟病抗性鉴定, 发现疣粒野生稻对接种的所有稻瘟病菌株都感病。进一步采用3'/5' RACE方法, 从疣粒野生稻中克隆了水稻同源基因Pid2和Pid3, 并构建过表达转基因株系对基因功能进行了研究。结果表明, Pid2和Pid3与疣粒野生稻中同源基因间在DNA和氨基酸水平上有较大的序列差异, 过表达转基因的日本晴植株对稻瘟病菌的敏感性与对照相似。推测疣粒野生稻在自然接种条件下, 表现出的抗稻瘟病表型很可能是其旱生叶片结构特征形成了对稻瘟病菌侵染的天然屏障。对控制疣粒野生稻这一类性状基因资源的挖掘和利用, 有利于优良抗性水稻品种的培育。研究结果为疣粒野生稻的研究利用提供了新信息和新思路。     

水稻蛋白激酶基因Ptil的克隆与分析

植物Ptil基因编码丝氨酸／苏氨酸蛋白激酶,是重要的抗病相关基因。在水稻抗褐飞虱基因Qtil所在的染色体区段存在一个与番茄Ptil基因高度同源的序列片段。从抗虫水稻B5中分离了Ptil基因的全长cDNA克隆,测定了基因组序列。分析发现,水稻中Ptil基因长度有5644bp,含有7个内含子,编码368个氨基酸的激酶蛋白。其蛋白质的C末端在不同植物之间具有高度的保守性,而N末端的变异则相对较大。对不同水稻材料Ptij基因的序列进行了比较,发现药用野生稻与栽培稻之间存在较大的差异,而栽培稻各品种之间的差异较小。讨论了Ptil基因在抗虫防卫反应中可能的作用。     

水稻蛋白激酶基因Pti1的克隆与分析

植物Pti1基因编码丝氨酸/苏氨酸蛋白激酶,是重要的抗病相关基因。在水稻抗褐飞虱基因Qbp1所在的染色体区段存在一个与番茄Pti1基因高度同源的序列片段。从抗虫水稻B5中分离了Pti1基因的全长cDNA克隆,测定了基因组序列。分析发现,水稻中Pti1基因长度有5644bp,含有7个内含子,编码368个氨基酸的激酶蛋白。其蛋白质的C末端在不同植物之间具有高度的保守性,而N末端的变异则相对较大。对不同水稻材料Pti1基因的序列进行了比较,发现药用野生稻与栽培稻之间存在较大的差异,而栽培稻各品种之间的差异较小。讨论了Pti1基因在抗虫防卫反应中可能的作用。     

海南黎族聚居区山栏稻的起源演化研究

以14份海南黎族聚居区的山栏稻为研究材料、以原产于中国的69份亚洲栽培稻和110份普通野生稻为对照组,分别对核中SSⅡ基因、ITS基因和Ehd1基因、叶绿体中ndhC-trnV基因以及线粒体中cox3基因等5段序列进行测序,分析基因序列多样性和单倍型,并揭示海南黎族聚居区山栏稻的起源地和驯化过程。结果表明,黎族聚居区山栏稻的基因多样性低于亚洲栽培稻,而亚洲栽培稻的基因多样性低于普通野生稻;85%左右的山栏稻为偏粳型;山栏稻与广东和湖南的普通野生稻亲缘关系较近,而与海南的普通野生稻的亲缘关系较远,推测黎族的山栏稻可能起源于广东和湖南的普通野生稻。     

云南野生稻中Xa21基因外显子II的分离及序列分析

Xa21是已经分离克隆的一个具有广谱抗性的水稻白叶枯病抗性基因,根据已克隆的白叶枯病抗性基因Xa21外显子II序列设计特异性引物对云南三种野生稻及其它稻种进行PCR扩增。结果表明只有普通野生稻（景洪普通野生稻和元江普通野生稻）及长雄野生稻中扩增到了长400 bp的目的片段,而疣粒野生稻和药用野生稻及栽培稻中均没有扩增到目的片段。通过序列比较发现所克隆的序列同长雄野生稻的氨基酸序列变化是随机的。     

云南野生稻中Xa21基因外显子Ⅱ的分离及序列分析

Xa21是已经分离克隆的一个具有广谱抗性的水稻白叶枯病抗性基因,根据已克隆的白叶枯病抗性基因Xa21外显子Ⅱ序列设计特异性引物对云南3种野生稻及其他稻种进行PCR扩增.结果表明,只有普通野生稻(景洪普通野生稻和元江普通野生稻)及长雄野生稻中扩增到了长400bp的目的片段,而疣粒野生稻和药用野生稻及栽培稻中均没有扩增到目的片段.通过序列比较发现所克隆的序列同长雄野生稻的氨基酸序列变化是随机的.     

水稻(Oryza sativa L.)核基因组存在叶绿体psbA基因的同源片段

序列比较说明,重复DNA顺序pRRD9与水稻叶绿体基因组中编码QB蛋白的psbA基因存在高度的同源。用pRRD9亚克隆片段pRRD9R和片段pRRD9L对水稻的叶绿体和核DNA进行Southern杂交分析,揭示了psbA基因同源片段在某个进化时期由叶绿体基因组转移到水稻核基因组,而且两者在水稻进化过程中的变异程度存在明显的差异。利用它们对野生稻和栽培稻总DNA的Southern杂交分析,显示亚洲栽培稻与AA基因组型的野生稻有较近的亲缘关系,以及在部分野生稻产生特异的杂交带谱,说明它可以作为一种分子探针来研究水稻的进化问题。     

A rice blast-resistance genetic resource from wild rice in Yunnan, China.

Compared to Pi-ta(-) alleles, Pi-ta(+) alleles can cause blast resistance response. In this work, Pi-ta gene in multiple rice materials, including local rice cultivars, different types of O. rufipogon and O. longistaminata was detected by molecular cloning and sequence analysis. Results indicated that Pi-ta(+) alleles were rare alleles, because in all the tested materials, only the 'Erect' type of O. rufipogon (ETOR) from Jinghong county in Yunnan province contains a Pi-ta(+) allele. Another rice blast resistance gene, Pib, confers resistance to the Japanese strain of M. grisea, was also confirmed to be functional in this type of O. rufipogon. The results of pathogen inoculation test show that ETOR is more strongly resistant to the tested blast pathogen races than other types of O. rufipogon. The resistance of ETOR may at least partially depend upon the functioning of Pi-ta and Pib gene. As O. rufipogon has the same type of genome with the cultivated rice (O. sativa), Pi-ta(+) and Pib gene in Erect type of O. rufipogon can be used to improve the tolerance of cultivated rice to blast, either by traditional hybridization or by genetic engineering.     

Molecular evolution of the Pi-ta gene resistant to rice blast in wild rice (Oryza rufipogon)

Rice blast disease resistance to the fungal pathogen Magnaporthe grisea is triggered by a physical interaction between the protein products of the host R (resistance) gene, Pi-ta, and the pathogen Avr (avirulence) gene, AVR-pita. The genotype variation and resistant/susceptible phenotype at the Pi-ta locus of wild rice (Oryza rufipogon), the ancestor of cultivated rice (O. sativa), was surveyed in 36 locations worldwide to study the molecular evolution and functional adaptation of the Pi-ta gene. The low nucleotide polymorphism of the Pi-ta gene of O. rufipogon was similar to that of O. sativa, but greatly differed from what has been reported for other O. rufipogon genes. The haplotypes can be subdivided into two divergent haplogroups named H1 and H2. H1 is derived from H2, with nearly no variation and at a low frequency. H2 is common and is the ancestral form. The leucine-rich repeat (LRR) domain has a high pi(non)/pi(syn) ratio, and the low polymorphism of the Pi-ta gene might have primarily been caused by recurrent selective sweep and constraint by other putative physiological functions. Meanwhile, we provide data to show that the amino acid Ala-918 of H1 in the LRR domain has a close relationship with the resistant phenotype. H1 might have recently arisen during rice domestication and may be associated with the scenario of a blast pathogen-host shift from Italian millet to rice.     

Molecular evolution of the rice blast resistance gene Pi-ta in invasive weedy rice in the USA

The Pi-ta gene in rice has been effectively used to control rice blast disease caused by Magnaporthe oryzae worldwide. Despite a number of studies that reported the Pi-ta gene in domesticated rice and wild species, little is known about how the Pi-ta gene has evolved in US weedy rice, a major weed of rice. To investigate the genome organization of the Pi-ta gene in weedy rice and its relationship to gene flow between cultivated and weedy rice in the US, we analyzed nucleotide sequence variation at the Pi-ta gene and its surrounding 2 Mb region in 156 weedy, domesticated and wild rice relatives. We found that the region at and around the Pi-ta gene shows very low genetic diversity in US weedy rice. The patterns of molecular diversity in weeds are more similar to cultivated rice (indica and aus), which have never been cultivated in the US, rather than the wild rice species, Oryza rufipogon. In addition, the resistant Pi-ta allele (Pi-ta) found in the majority of US weedy rice belongs to the weedy group strawhull awnless (SH), suggesting a single source of origin for Pi-ta. Weeds with Pi-ta were resistant to two M. oryzae races, IC17 and IB49, except for three accessions, suggesting that component(s) required for the Pi-ta mediated resistance may be missing in these accessions. Signatures of flanking sequences of the Pi-ta gene and SSR markers on chromosome 12 suggest that the susceptible pi-ta allele (pi-ta), not Pi-ta, has been introgressed from cultivated to weedy rice by out-crossing.     

重复DNA顺序pRRD9在稻属中的拷贝数测定

本文应用狭缝印渍杂交方法,把水稻基因组总DNA和含水稻中度重复顺序片段的质粒(pRRD9)DNA分别转移到尼龙膜上形成狭缝印渍、然后用~(32)P标记的 pRRD9插入片段进行杂交、根据各狭缝印渍的放射性强度,测定水稻(Oryza)一些栽培种和野生种基因组中重复DNA顺序的拷贝数,并就拷贝数与水稻进化关系及基因组型的联系进行讨论.     

重复DNA顺序pRRD9在稻属中的拷贝数测定

本文应用狭缝印渍杂交方法,把水稻基因组总DNA和含水稻中度重复顺序片段的质粒(pRRD9)DNA分别转移到尼龙膜上形成狭缝印渍、然后用³²P标记的 pRRD9插入片段进行杂交、根据各狭缝印渍的放射性强度,测定水稻(Oryza)一些栽培种和野生种基因组中重复DNA顺序的拷贝数,并就拷贝数与水稻进化关系及基因组型的联系进行讨论.     

Genetic control of a transition from black to straw-white seed hull in rice domestication

The genetic mechanism involved in a transition from the black-colored seed hull of the ancestral wild rice (Oryza rufipogon and Oryza nivara) to the straw-white seed hull of cultivated rice (Oryza sativa) during grain ripening remains unknown. We report that the black hull of O. rufipogon was controlled by the Black hull4 (Bh4) gene, which was fine-mapped to an 8.8-kb region on rice chromosome 4 using a cross between O. rufipogon W1943 (black hull) and O. sativa indica cv Guangluai 4 (straw-white hull). Bh4 encodes an amino acid transporter. A 22-bp deletion within exon 3 of the bh4 variant disrupted the Bh4 function, leading to the straw-white hull in cultivated rice. Transgenic study indicated that Bh4 could restore the black pigment on hulls in cv Guangluai 4 and Kasalath. Bh4 sequence alignment of all taxa with the outgroup Oryza barthii showed that the wild rice maintained comparable levels of nucleotide diversity that were about 70 times higher than those in the cultivated rice. The results from the maximum likelihood Hudson-Kreitman-Aguade test suggested that the significant reduction in nucleotide diversity in rice cultivars could be caused by artificial selection. We propose that the straw-white hull was selected as an important visual phenotype of nonshattered grains during rice domestication.     

稻属中一个高度保守和组成型表达酪氨酸／丝氨酸－酸酸双特性蛋白激酶基因的克隆与分析

从水稻中克隆了一个在稻属植物中高度保守和组成型表达的丝氨酸／苏氨酸蛋白激酶基因（OsSTK）。该基因包含两个外显子和一个114bp的小内含子序列,预测编码一个419个氨基酸的蛋白质。该基因推导的氨基酸序列与其它已知序列的一致性均低于52％。利用从不同种和类型的野生稻克隆的部分该基因序列构建的系统树与野生稻的分类和进化关系相一致。OSPKN-端拥有一段富含丝氨酸、碱性氨基酸和带电荷氨基酸的特异性导肽序列,其中包含“GDGDGDGDG”短重复序列。由于该基因蛋白激酶结构域中的VIb,VIII和XI亚结构域中同时具有酪氨酸蛋白激酶和丝氨酸／苏氨酸蛋白激酶的特性,推测该基因可能同时具有催化酪氨酸和丝氨酸、苏氨酸磷酸化的双重功能。     

tA single amino acid difference distinguishes resistant and susceptible alleles of the rice blast resistance gene Pi-ta

The rice blast resistance (R) gene Pi-ta mediates gene-for-gene resistance against strains of the fungus Magnaporthe grisea that express avirulent alleles of AVR-Pita. Using a map-based cloning strategy, we cloned Pi-ta, which is linked to the centromere of chromosome 12. Pi-ta encodes a predicted 928-amino acid cytoplasmic receptor with a centrally localized nucleotide binding site. A single-copy gene, Pi-ta shows low constitutive expression in both resistant and susceptible rice. Susceptible rice varieties contain pi-ta(-) alleles encoding predicted proteins that share a single amino acid difference relative to the Pi-ta resistance protein: serine instead of alanine at position 918. Transient expression in rice cells of a Pi-ta(+) R gene together with AVR-Pita(+) induces a resistance response. No resistance response is induced in transient assays that use a naturally occurring pi-ta(-) allele differing only by the serine at position 918. Rice varieties reported to have the linked Pi-ta(2) gene contain Pi-ta plus at least one other R gene, potentially explaining the broadened resistance spectrum of Pi-ta(2) relative to Pi-ta. Molecular cloning of the AVR-Pita and Pi-ta genes will aid in deployment of R genes for effective genetic control of rice blast disease.     

Diversification of the rice Waxy gene by insertion of mobile DNA elements into introns.

The waxy (wx) gene of Oryza glaberrima was cloned, and its nucleotide sequence was determined. A waxy mutant of O. glaberrima showing a glutinous phenotype was found to contain a substitution mutation generating a termination codon in the coding region of the wx gene. The Wx sequence of O. glaberrima was different from that of Oryza sativa by substitutions and insertions/deletions, among which only a few substitutions occurred in several exons not to severely alter the amino acid sequence of the Wx protein. The most striking difference observed in introns was a 139-bp deletion (or insertion) in intron 10 of O. glaberrima (or O. sativa). In O. sativa, 125 bp of the 139-bp sequence was flanked by direct repeats of a 14-bp sequence. A sequence homologous to the 125-bp sequence was found in the region preceding exon 2; this sequence was also flanked by direct repeats of another 14-bp sequence. This result and the observation that the 125-bp sequence was interspersed in rice genomes indicate that they are SINEs (short interspersed elements) in the plant system. We also identified a DNA sequence with long terminal inverted repeats in intron 13 of both O. glaberrima and O. sativa. This sequence was present in multiple copies in rice genomes, suggesting that it is a transposable element. These results obtained suggest that mobile DNA elements have diversified the rice Waxy gene by inserting into introns, each of which may originally have a length of about 100 bp.